ANTIMICROBIAL AGENTS, DIAGNOSTIC REAGENTS, AND VACCINES BASED ON UNIQUE APOCOMPLEXIAN PARASITE COMPONENTS
Inventors: Rima L.W. McLeod et al.
The U.S. government may have rights in this patent by means of partial support under: NIH NIAID TMP R01 Al 16945; NIH NIAID TMP R01 Al 27530, and NIH ROI Al 43228. This invention relates uses of components of plant-like metabolic pathways not including psbA or PPi phosphorfructokinase and not generally operative in animals or encoded by the plastic DNA, to develop compositions that interfere with Apicomplexan growth and survival. Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoters of these nucleotide sequences to which antibodies, antisense molecules and other inhibitors are directed. Diagnostic and therapeutic reagents and vaccines are developed based on the components and their inhibitors. A cDNA sequence that encodes chorismate synthase expressed at an early state of Apicomplexan development, is disclosed and may be altered to produce a "knockout" organism useful in vaccine production.
BACKGROUND
Apicomplexan parasites cause the serious diseases malaria, toxoplasmosis, sryptosporidiosis, and eimeriosis. Malaria kills more than 2 million children each year. Toxoplasmosis is the major opportunistic brain infection in AIDS patients, causes loss of life, sight, hearing, cognitive and motor function in congenitally infected infants, and considerable morbidity and mortality in patients immunocompromised by cancer, transplantation, autoimmune disease and their attendant therapies. Cryptosporidiosis is an unbeatable cause of diarrhea in AIDS patients and a cause of epidemics of gastrointestinal disease in immunocompetent hosts Eunciia infections of poultry lead to billions of dollars in losses to agricultural industries each year Other Apicomplexan infections, such as babesiosis, also cause substantial morbidity and mortality Although there are some methods for diagnosis and treatment of Apicomplexan caused diseases, some of these treatments are ineffective and often toxic to the subject being treated
The tests available to diagnose Apicomplexan infections include assays which isolate the parasite, or utilize light, phase, or fluorescence microscopy, ELISAs, agglutination of parasites or parasite components to detect antibodies to parasites, or polymerase chain reaction (PCR) to detect a parasite gene Most of the assays utilize whole organisms or extracts of whole organisms rather than recombinant proteins or purified parasite components In many instances, the available assays have limited ability to differentiate whether an infection v. as acquired remotely or recently, and are limited in their capacity to diagnose infection at the outpatient or field setting The primary antimicrobial agents used to treat toxopljsmosis are pyπmethamine (a DHPR inhibitor) and sulfadiazme (a PAJBA antagonist) The use of pyπmethamine is limited by bone marro toxicity which can be partially corrected by the concomitant administration of folinic acid T goiidn cannot utilize folinic acid but mammalian cells can Another problem is that pyπmethamine is potentially teratogenic in the first trimester of pregnancy The use of sulfonamides is limited by allergy, ga_.tioinie_.un:. I intolerance, kidney stone formation and Ste\ ens-Johnson syndrome
1 lieu . i o a -.mail number of antimicrobial agents utilized less (l equentK to tieat
cm spiramvcm, cιn clanihroim cm and atovaquone Usefulness of these medicines for treatment of toxoplasmosis is limited by toxicities including allergy and antibiotic-associated diarrhea, (especially Clostndium difficile toxin associated colitis with clindamycin use) Lesser or uncertain efficacy of macrolides such as spiramycin, azithromycin, and clanthromycin also limits use of these antimicrobial agents Atovaquone treatment of toxoplasmosis may be associated with lack of efficacy and/or recrudescent disease There are no medicines known to eradicate the latent, bradyzoite stage of T. gondu, which is very important in the pathogenesis of toxoplasmosis in immunocompromised individuals or those with recurrent eye disease Medicines used to treat malaria include quinine sulfate, pyπmethamine, sulfadoxine, tetracycline, clindamycin, chloroquine, mefloquine, halofaπtrine, quinidine gluconate, quinidine dihydrochloride, quinine, primaquine and proguanil Emergence of resistance to these medicines and treatment failures due to resistant parasites pose major problems in the care of patients with malaria Toxicities of mefloquine include nausea, vomiting, diarrhea, dizziness, disturbed sense of balance, toxic psychosis and seizures Melfoquine is teratogenic in animals With halofantrene treatment, there is consistent, dose-related lengthening of the PR and Qt intervals in the electrocardiogram Halofantrene has caused first degree heart block It cannot be used for patients with cardiac conduction defects Quinidine gluconate or dihydrochloride also can be hazardous Parenteral quinine ma\ lead to severe hypogl\ cemιa Pi imaquinc can cause hemolvtic anemia especially in patients whose red blood cells aie deficient in glucose (-"-phosphate deh . dro enase Uπfortunatel . there ai e no medicines know n lo be efie ti . e m the tieaimeni of cr. piospondio -.i -. To more effectively treat Apicomplexan infections, there is an urgent need for discovery and development of new antimicrobial agents which are less toxic than those currently available, have novel modes of action to treat drug resistant parasites that have been selected by exposure to existing medicines, and which are effective against presently untreatable parasite life cycle stages (e.g., Toxoplasma gondii bradyzoites) and presently untreatable Apicomplexan parasites (e.g., Ciyptosporidnmi parvitm) Improved diagnostic reagents and vaccines to prevent these infections are also needed Information available on Apicomplexan parasites has not yet provided keys to solutions to health problems associated with the parasites. Analogies to other organisms could provide valuable insights into the operations of the parasite. There are reports of Apicomplexan parasites having plastids, as well as the nuclear encoded proteins, tubulin, calmodulin, PPi phosphofructokinase and enolase, which are reported to be similar in part to, or hom*ologous with, counterparts in plant-like, lower life forms and higher plants There are reports of a plastid genome and components of a protein synthetic system in a plastid-ϋke organeUe of Apicomplexans Plasmodntm andT. gondii plastid DNA sequences were reported to have hom*ologies to algal plastid DNA sequences. The plastid membrane of T. gondii was reported to be composed of multiple membranes that appear morphologically similar to those of plant/algal chloroplasts. except for the presence of two additional membranes in the T. gondii plastid, suggesting that it may have been an ancient algal endosymbiont Some of these Apicomplexan proteins such as tubulin calmodulin and enolase with certain plant-like feai i es also are found in animals, and therefore
appear in the host as ell as the parasite. A hom*ologue to a gene, psbA encoding a plant protein important for photosynthesis, also was said to be present in Apicomplexans. .Certain herbicides have been reported to inhibit the growth of Apicomplexans. The herbicides which affect growth of Apicomplexans are known to affect plant microtubules or a plant photosynthetic protein. In addition, a compound, salicylhydroxamic acid, (SHAM), had been found to inhibit Plasmodi m falciparum (malaria) and Babesia microti.
Techniques of medicinal chemistry and rational drug design are developed sufficiently to optimize rational construction of medicines and their delivery to sites where Apicomplexan infections occur, but such strategies have not yet resulted in medicines effective against Apicomplexans. Rational development of antimicrobial agents has been based on modified or alternative substrate competition, product competition, change in enzyme secondary structure, and direct interference with enzyme transport, or active site. Antisense, ribozymes , catalytic antibodies, disruption of cellular processes using targeting sequences, and conjugation of cell molecules to toxic molecules are newly discovered strategies employed to interrupt cellular functions and can be utilized to rationally develop novel antimicrobial compounds, but they have not yet been utilized to design medicines effective against Apicomplexans. Large scale screening of available compounds with recombinant enzymes is used to identify potentially effective anti-microbial agents
Reagents to diagnose Apicomplexan parasite infections have been developed targeting components of Apicomplexans or immune responses to the parasites, using LϊLlSΛ. western blot, and PCR technologies, but improved diagnostic reagents. especially those that establish duration of infection or that can be used in outpatient settings are needed to diagnose Apicomplexan infections No vaccines to prevent Apicomplexan infections are available for humans and only a live vaccine prepared for prevention of toxoplasmosis in sheep is available for livestock To summarize, Apicomplexan parasites cause substantial morbidity and mortality, and treatments against the parasites are suboptimal or non-existent Improved antimicrobial compounds that attack Apicomplexan parasites are needed Because the diseases Apicomplexan parasites cause in some instances are due to recrudescence of latent parasites, an especially pressing clinical problem is that there are no effective antimicrobial agents effective for treatment of these latent parasite life cycle stages, especially in sequestered sites such as the brain or eye New approaches and drug targets are required Better in vitro and //; vivo assays for candidate compounds are also needed Better diagnostic and therapeutic methods, reagents and vaccines to prevent these infections are needed
SUMMARY OF THE INVENTION
This invention relates uses of components of plant-like metabolic pathways (not usually associated with animals, not encoded in the plastid genome, and not including psbA or PPi phosphofructokinase) to de . elop compositions that interfere with Apicomplexan growth and survival Components of the pathways include enzymes, transit peptides and nucleotide sequences encoding the enzymes and peptides, or promoiei s of these nucleotide sequences to w ich antibodies antisense molecules and oihei inhibitoiϊ aie dnected Diagnostic and thci pentic rcasient and vaccines aie developed ba>ed on the components and their inhibitors Attenuation of live parasites through disruption of any ot these components or the components themselves provide vaccines protective against Apicomplexans
Transit peptides are used to identify other proteins and their organeUe targeting sequences that enter and exit from unique Apicomplexan organeUes The identified components are potential for production of medicines, reagents and assays, and vaccines The protein which includes the transit peptide is not necessaπly an enzyme in a biochemical pathway
The methods and compositions of the present invention arise from the inventors' discover)' that metabolic pathways, and targeting signals similar to those found in plants and algae, especially, but not exclusively those encoded withm the nucleus, are present in Apicomplexan parasites These plant-like pathways in Apicomplexan parasites are targetable b inhibitors, as measured by determining whether the inhibitors, either singly or in combination, are effective in inhibiting or killing Apicomplexan parasite,, m vitto and/or /// vivo
The present invention includes new methods and compositions to treat, diagnose and prevent human and veteπnarv disease due to Apicomplexan infections The invention is based on applications and manipulations of components of algal and higher plant-like metabolic pathways discov red in Apicomplexan parasites "Plant- like ' means that products ol the pathwa . s enz . mes and nucleotide sequences encoding enzv mes in uu pathwa\ s are hom*ologous or similar to products enz\ mes and nucleotide s<_ iiccs know n in plants lurein plants include algae As used herem plant-like -•■ udes mαaboln. pathw a . s. generall\ operative in oi identical to those in animals and pathways involving psbA or phosphofructokinase and those encoded by the plastid genome The limits of a "pathway" are defined as they are generally known to those of skill in the art Methods to detect plant counterparts in Apicomplexan include a) immunoassays using antibodies directed to products and enzymes known in plants, b) hybridization assays using nucleotide probes that hybridize to specific sequences in plants, c) determining hom*ologies of Apicomplexan nucleotide or protein sequences with plant nucleotide or protein sequences, and/or d) substrate tests for specific enzymatic activity
The "plant-like" pathways of the present invention are identified by: a) identification of metabolic pathways characteristic of plants but not generally present in animals, b) identification and characterization of Apicomplexan enzymes, nucleic acids and transit sequences as components similar or hom*ologous to those in a), c) identification and development of compounds (inhibitors) which abrogate the effect of the components of the pathways in vitro and m vivo, singly or in a plurality, against one or more types of Apicomplexan parasites and in conjoint Apicomplexan, bacterial and fungal infections
The identified pathways are then used for a) rational design or selection of compounds more active than the known compounds (inhibitors), with good absorption follo ing oral administration, with appropnate tissue distribution and w ithout toxicity or cai cmogenicity, b) testing ol such rationallv designed compounds alone and together foi safety, etlicacv and appropnate absυi ption and tissue disii ibutioii /// vin o and /// vn o c) development and testing of diagnostic reagents and assays; d) development and testing of live attenuated and component based vaccines By locating new targets in Apicomplexan pathways, doors now are open for development of more effective antimicrobial agents to treat Apicomplexan parasites in humans and agricultural animals. In addition, enzymes in these plant-like pathways provide improved diagnostic tests for diseases caused by Apicomplexans. Vaccines against infectious diseases caused by Apicomplexan parasites are derived from the novel compositions of the invention.
A method for inhibiting an Apicomplexan parasite, includes selecting the metabolic pathway of the present invention and interfering with the operation of the pathway in the parasite. The Apicomplexan parasite is preferably selected from the group that includes Toxoplasma, Plasmodium, Qyptosporidia, Ei eria, Babesta and Theilerict The pathway may utilize a component encoded by an Apicomplexan nuclear
"ene Suitable metabolic pathways or components include a) synthesis of heme from glutamate and tR A glu by the plant-like, heme synthesis (5 carbon) pathway (hereinafter the "heme synthesis pathway"), b) synthesis of C4 acids (succinate) by the breakdown of lipids into fatty acids and then acetyl CoA, and their use in the glyoxylate cycle (hereinafter the "giv oxylate cycle"). c) sv nthesis of chorismate from phosphoenolpyaivate and erythrose 4 phosphate bv the shikimate pathway ( hei emafter the "shikimate pathw ay ') d) S IU O -S of teit aiiydi olblate from choi ismate by the shikimate pathw ay. e) SMithesis of ubiquinone from chorismate by the shikimate pathway, f) electron transport through the alternative pathway with use of the alternative oxidase (hereinafter the "alternative oxidase pathway"), g) transport of proteins into or out of organeUes through the use of transit sequences, h) synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) from chorismate by the shikimate pathway, i) synthesis of the menaquinone, enterobactin and vitamin Kl from chorismate by the shikimate pathway, j) synthesis of the branched chain amino acids (valine, leucine and isoleucine) from pyruvate and ketobutyrate by the plant-like branched chain amino acid synthesis pathway, k) synthesis of the "essential" (i e . not synthesized by animals) amino acids, histidine threonine, lysine and methionine by the use of plant-like amino acid synthases,
1) synthesis of hnoleneic and liπoleic acid, m) synthesis of amylose and am lopectin with starch synthases and Q
(branching) enzymes and their degradation, n ) sv ntliesis of auxin growth regulators from indoleacetic acid derived from chorismate, o) s ntliesis of isoprenoids (diterpenes 5 carbon units with some properties of lipi } . such as giberellms and rhscidic acid b\ the mevalo ic acid to gilv ellm pathwav The interfering compositions are selected from the group consisting of enzyme inhibitors including competitors; inhibitors and competitive or toxic analogues of substrates, transition state analogues, and products; antibodies to components of the pathways; toxin conjugated antibodies or components of the pathways; antisense molecules; and inhibitors of transit peptides in an enzyme. In particular, the interfering compositions include gabaculine, 3-NPA, SHAM, 8-OH-quinoline, NPMG. Interfering with the operation of the metabolic pathway is also accomplished by introducing a plurality of compositions to the pathway, wherein each of the compositions singly interferes with the operation of the metabolic pathway. In certain instances, the plurality of compositions inhibits the parasite to a degree greater than the sum of the compositions used singly, that is, exhibits a synergistic effect. Embodiments of a plurality of compositions include gabaculine and sulfadiazine; NPMG and sulfadiazine; SHAM and gabaculine; NPMG and pyrimethamine; NPMG and cycloguanil (which inhibits Apicomplexan DHFR [TS]), and other inhibitors and competitors of interrelated cascades of plant-like enzymes. Wherein the effect of inhibitors together is greater than the sum of the effects of each alone, the synergistic combination retards the selection of emergence of resistant organisms and is more effective than the individual components alone.
In various embodiments, the interfering composition acts on a latent bradyzoite form of the parasite, or multiple infecting Apicomplexan parasites simultaneously, or on conjoint infections with other pathogenic microorganisms which also utilize the plantlike metabolic pathway. A method of determining the effectiveness of a composition in reducing the deleterious effects of an Apicomplexan m an animal, include a) identifying a composition that inhibits growth or survival of an Apicomplexan parasite /// vitio by interfering with a plant-like metabolic pathway and b) determining a concentration of the composition in an animal model that is non-toxic and effective in reducing the survival of the parasite in the animal host and/or the deleterious effects of the parasite in the animal
Developing a lead compound that inhibits an Apicomplexan parasite is accomplished by a) identifying a plant-like metabolic pathway in an Apicomplexan parasite and b) identifying a composition that interferes with the operation of the pathway as a lead compound
A composition which inhibits a specific life cycle stage of an Apicomplexan parasite by interfering with a plant-like metabolic pathway that utilizes a component encoded by a nuclear gene includes gabaculine, a composition including an enzyme in a metabolic pathway in an Apicomplexan parasite that is selectively operative in a life- cycle stage of the parasite includes the enzymes alternative oxidase, and UDP glucose starch glycos l transferase A composition comprising SHAM and 8-OH-quιnolιne inhibits the alternative oxidase in the latent bradyzoite form of an Apicomplexan parasite λ method to identify a plant-like gene encoding a component of a plant-like metabolic pathw av in an Apicomplexan parasite is a) obtaining a strain of /J coli that is deficient for a component of the metabolic pathway, said deficiency causing the strain lo icquii e supplemented media for giυv tli b) complementing the L toll with a gene or portion of the gene encoding a component of the metabolic pathway in the Apicomplexan parasite, and c) determining whether the complemented E colt is able to grow in uπ iipplemented media, to identify the gene
Another method for identifying a plant-like gene product of a metabolic pathway in an λpicomplexan parasite is a) contacting the parasite with a gene probe, and b) determining whether the probe has complexed with the parasite from which the identity of the gene product is inferred
A method for identifying a plant-like gene product of a metabolic pathway in an Apicomplexan parasite also includes a) cloning and sequencing the gene, and b) determining hether the gene is hom*ologous to a plant gene which encodes a plant enzyme with the same function
A method for identifying a plant-like gene product in a metabolic pathway in an Apicomplexan parasite is a) contacting the parasite or its enzyme with a substrate for the plant-like enzyme, b) measuring enz me activity, c) determining whether the enzyme is operative, and d) inhibiting activity of the enzyme in vitro with an inhibitor
Identify ing a gene or gene product in an Apicomplexan parasite which possesses an organeUe transit sequence which transports a protein, wherein the protein is not necessaruv an enzyme in a metabolic pathway, but is identified because it has a characteristic crganelle transit sequence is also within the scope of the invention The invention also relates to a diagnostic reagent for identifying the presence ot an ApicompleOii parasite in a subject, w here the subject includes a domestic or livestock an'r ,al oi a human The reagent may include all or a portion of a component ol the plam-'.ke pathw av, an aπiibodv. specific lor an en/ me that is a component ol a plant- ke metabolic pathway in the parasite, or all or part of a nucleotide sequence that hybridizes to a nucleic acid encoding a component of the pathway A diagnostic assay that identifies the presence of an Apicomplexan parasite or specific life-cycle stage of the parasite may use the diagnostic reagents defined herein A diagnostic reagent for identifying the presence of an Apicomplexan parasite, includes an antibody specific for an enzyme that is part of a plant-like metabolic pathway.
A diagnostic assay for the presence of an Apicomplexan parasite in a biological sample includes a) contacting the sample with an antibody selective for a product of a plant-like metabolic pathway that operates in an Apicomplexan parasite, and b) determining whether the antibody has complexed with the sample, from which the presence of the parasite is inferred Alternatively, the assay is directed towards a nucleotide sequence In both these cases, appropriate antibody or nucleotide sequences are selected to distinguish infections by different Apicomplexans An aspect of the invention is a vaccine for protecting livestock animals, domestic animals or a human against infection or adverse consequences of infection by an Apicomplexan parasite The vaccine may be produced for an Apicomplexan parasite in w hich a gene encoding a component of a plant-like metabolic pathway in the parasite is manipulated, for example, deleted or modified When the gene is deleted oi modified in the live vaccine, the component of the pathway may be replaced bv the pi esence of the product of an enzv matic reaction in tissue culture medium The v accine sti ain ..an then be cultiv ated in itrς> to make the vaccine A vaccine for protecting animals against infection by an Apicomplexan parasite is based on an Apicomplexan parasite in which the parasite or a component of a metabolic pathway in the parasite is used.
The accine may use a component of the pathway that is operative at a particular life stage of the parasite. A suitable component is the AroC gene from T. gondii or P. falciparum.
A method of treatment for an infection in a subject by an Apicomplexan parasite includes the following steps: a) obtaining an inhibitor of a plant-like metabolic pathway in an Apicomplexan parasite, and b) administering an effective amount of the inhibitor to the subject.
BRIEF DESCRIPTIONS OF DRAWINGS FIG. 1A-C illustrates the heme synthesis pathway and the effect of GSAT in T gondii FIG. 1A diagrams the heme synthesis pathway FIGS. IB and IC show that uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by gabaculine, an inhibitor of GS λ aminotransferase P/S = pyrimethamine and sulfadiazine Note that ALA synthase is also present in T gondii and constitutes an alternative pathway for heme syπthesi.- FIG. 2 Λ-I3 shows unique lipid degradation in the glyo ylatc cycle in 7 gυndii
ITG. 2Λ IS a schematic representation of the glyoxylatc c\ cle FIG. 2 shows uptake of irn._a.ed uracil bv tachyzoites (RH strain) is inhibited bv 3-NPA (0 00s R, . mg G/ML) Note this inhibitor also effects succinate dehydrogenase, so its inhibitory effect does not unequivocally support presence of the glyoxylatp pathway
FIG. 3 A is a schematic representation of a pathway which demonstrates alternative oxidase as an alternative pathway for generation of energy in Apicomplexan parasites FIG. 3B shows that uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by SHAM
FIG. 4A is a schematic representation of the pathway for conversion of shikimate to chorismate in T. gondii.. The inhibitor of EPSP synthase is NMPG FIG. 4B shows uptake of tritiated uracil by tachyzoites (RH strain) is inhibited by NPMG Toxicity of NPMG was assessed by its ability to prevent growth of human foreskin fibroblasts (HFF) after 4 days, as measured by tritiated thymidine uptake and microscopic evaluation FIG. 4C shows product rescue of NPMG's inhibitory effect on EPSP svnthase by PABA The effect of PABA on sulfadiazine is similar, but the effect on pv πmethamine, as predicted reduces the enzyme to the levels that were present when media alone was utilized, as measured by the uracil uptake
S = sulfadiazine
PYR = pyrimethamine
PABA = para amino benzoic acid
FIG. 4D show s functional and enzymatic evidence for the shikimate pathway in / gυndii with inhibition of EPSP synthase enzj mc activ ity by I mM glyosate Squares without glv phosate Circles, w ith glyphosate FIG. 4C show s evidence for the shikimate path . av in /' fakipai ii with functional ev idence for the shikimate pathwav in /' luk tpai iini Glv phosate inhibition of in \ ιιι o growth of asexual erythrocv tic forms and PABA and folate antagonism of growth inhibition. Effect of NPMG on C. parvum was not abrogated by PABA. This suggests that either uptake of PAPA by C. parvum differs or effect of NPMG is on a different branch from the shikimate pathway in C. parvum. FIG. 5 is a schematic representation of interrelationships of metabolic pathways in Apicomplexan parasites.
FIG. 6 shows inhibitory effects of NPMG, gabaculine, SHAM 8-OH-quinoline on Cryptosporidia. 3NPA also inhibited Cryptosporidia.
FIG. 7 shows the effects of gabaculine (20 mM) on growth of tachyzoites/bradyzoites (R5) in human foreskin fibroplasts, over 8 days as determined by uracil uptake. Note increased uptake of uracil by the 8th day.
FIG. 8 shows the effect of NPMG, pyrimethamine, and pyrimethamine plus NPMG on survival of mice following intraperitoneal infection with 500 tachyzoites of the RH strain of T. gondii. Dosage of NPMG was 200mg/kg/day and pyrimethamine was 12.5 mg/kg/day).
FIG. 9 shows nucleotide and deduced amino acid sequences of T. gondii chorismate synthase cDNA. The asterisk indicates the stop codon.
FIG. 10 shows results of CLUSTAL X alignments of the deduced amino acid sequences if the putative T. gondii, chorismate synthase with the corresponding sequences from Synechocystites, S. cerevisiae, S. lypocersicum, N. crassa and
H. influenza. Dashes were introduced maximize alignment. Amino acids which are identical in all 6 organisms are underlined. The percent identity of the chorismate synthase from each organism with the T. gondii protein was calculated to be as follows Synediocystts (51.4%), S. cerevisia (49.6%), S. lycυpersicmn (47.2%), N. crassa (45.0%) and H. influenza (44.5%). The large internal regions ip the T. gondii sequence which have no counterparts in the chorismate synthases of other organisms, were not included in this calculation. FIG. 11 shows the transit sequences of Zea mays and T. gondii chorismate synthases. The sequences of the transit peptide directing the transport of the wx+ protein into maize amyloplasts and chloroplasts and the portion of the T. gondii chorismate synthase sequence which is hom*ologous are aligned. The amino acid sequence is given in one letter code. * indicates an identical amino acid in the Wx Zea mays and T. gondii sequences. • indicates hom*ologous amino acids in the Wx Zea mays and T. gondii sequences.
The transit sequence in the Wx Zea mays protein (UDP-glucose-starch-glycosyl transferase) begins at amino acid number I and ends at amino acid number
RECTIFIED SHEET (RULE 91) 72. The portion (amino acids 359 to 430) of P. falciparum AroC which corresponds to the novel internal sequence of the T. gondii AroC which includes the amino acids hom*ologous to the maize protein, is as follows:
1PVENMSTKKESDLLYDDKGEC10^MSYHST_Q>WEDQI NSTKGFMPPKNDK^ The T. gondii portion of the AroC (chorismate synthase) sequence which demonstrates 30% hom*ology begins at amino acid number 330 and ends at amino acid number 374. The first (single) arrow indicates the processing site of Zeamays UDP glucose Gylcosyl transferase transit peptide and the second (double) arrow indicates the location at which the mature protein begins. FIG. 12 shows P. falciparum, chorismate synthase cDNA and deduced amino acid sequences.
FIG. 13 shows a genomic sequence of T. gondii chorismate synthase. FIG. 14 shows (A) a T. gondii cDNA chorismate synthase DNA construct which is useful to produce antibody or a vaccine; (B) a Western blot (arrows mark chorismate synthase).
FIG. 15 shows green fluorescent (gfp) protein expression in a stably transfected tachyzoite; this tachyzoite has a reporter construct, a chorismate synthase- gfp; gfp is cytoplasmic (grey) and a defined structure in the area of the plastid is the white dot; the nucleus is the larger red area; gfp is in the cytoplasm. FIG. 16 shows life cycle stage associated expression and localization of chorismate synthase in T. gondii.
(A) Tachyzoites: (1) 15 days - - Double stained with tachyzoite surface antigen 1 (SAG1) (perimeter raised, top and bottom left) and DNA stain (DAPI) (bottom right) and chorismate synthase (top right, white); (2) Double stained with dense granule protein 4 (granular stain, top left), chorismate synthase (white); p30, lower right panel, (perimeter raised) rhoptry probe (raised grey, rhop); (3) Double stained chorismate synthase-punctate white, SAG1 (P30, perimeter raised). (Note discrete punctate white area of chorismate synthase staining in perinuclear area, the customary subcellular location of the plastid). (B) Bradyzoites: (1) Abbreviations are the same as in A; Note diffuse granular appearing cytoplasmic staining of bradyzoite chorismate synthase (top
RECTIFIED SHEET (RULE 91) right); (2) Immunoperoxidase stain with antibody to recombinant chorismate synthase shows diffuse cytoplasmic darker staining.
(C) Microgametes (Mi), Macrogametes (Ma); Note darker immunoperoxidase staining of these forms but not schizonts (s) in cat intestine. (D) Chorismate synthase mRNA production in tachyzoites and with bradyzoites; Note SAG1 message for a tachyzoite protein, BAG 1-5 message for a bradyzoite protein and constitutively expressed mRNA for tubulin.
FIG. 17 shows: (a) schematic illustration of gly oxy late cycle, (b) inhibitors of isocitrate lyase (ICL), (c) T. gondii isocitrate lyase enzyme activity, (d) inhibition of ICL enzyme activity by 3NPA, and (e) inhibition of tachyzoites in tissue culture. FIG. 18 shows a T. gondii isocitrate lyase (ICL) cDNA sequence. FIG. 19 shows a T. gondii isocitrate lyase (ICL) amino acid sequence. FIG. 20 shows (a) T. gondii isocitrate lyase (ICL) binding pocket and active site inside box, and (b) comparison with the published sequence of yeast isocitrate lyase with mutated lysine (K) which inactivated the enzyme (arrows).
FIG. 21 shows a T. gondii isocitrate lyase genomic DNA sequence (ICL). FIG. 22 shows T. gondii isocitrate lyase in bradyzoites; Note brown areas in immunoperoxidase stain preparation.
FIG. 23 shows isocitrate lyase (a) in a western blot of tachyzoites (b) during stage conversion, and (c) mRNA during stage conversion. (Abbreviations are the same as in FIG. 16A and D legends).
FIG. 24 shows enzymatic, genetic, functional activity of Apicomplexan parasites and its inhibition and show T. gondii acetyl coA carboxylase is inhibited by - fop herbicides: (A) Acetyl coA carboxylase enzyme activity is inhibited by -fop herbicides;
(B) T. gondii growth in tissue culture inhibited by compounds that inhibit acetyl coA carboxylases;
(Note the inhibitor activity is parallel to that in FIG. 24A. Clodinafop is a lead compound. T. gondii uptake of 3H uracil is inhibited by fop herbicides.)
(C) Effect of clodinafop on T. gondii with 4 days in culture then removal of the herbicide for 2 days. Note plaques (A) and (C) higher view of
RECTIFIED SHEET (RULE 91)
replicating parasites in these plaque controls and complete eradication of parasites in clodinafop (10(M) treated cultures;
Related sequences of Apicomplexan acetyl coA carboxylases; sequences of acetyl coA carboxylase biotin carboxylase domains from apicomplexan parasites are as in Genbank Accession Numbers AF
157612-16. Also, a domain swap yeast with the T. gondii active site and recombinant enzymes made from a fragment of the T. gondii gene are amenable to high throughput screens;
Phylogeny of biotin carboxylase domains of apicomplexan accases; Structures of herbicides that inhibit acetyl coA carboxylases.
RECTIFIED SHEET (RULE 91) DESCRIPTION OF THE PREFERRED EMBODIMENT This invention uses components of plant-like interrelated metabolic pathways that are essential for growth or survival of Apicomplexan parasites The pathways are generally not operative in animals and do not include psbA or PPi phosphofructokinase and are not encoded in the plastid. Components include enzymes, products, targetting. peptides, nucleotide sequences encoding the enzymes or peptides, and promoters, as targets for specific inhibitors. Use of these pathways provide a rational and novel framework to discover, characterize and develop medicines, diagnostic reagents and vaccines for Apicomplexan parasites.
Medicines, diagnostic reagents and vaccines are based upon interrelated plantlike enzyme cascades involved in the synthesis or metabolism or catabolism of Apicomplexan nucleic acids, amino acids, proteins, carbohydrates or lipids, energy transfer and unique plant-like properties of these enzymes which are shared with, and provide a basis for, discovery of other parasite proteins which have unique organeUe targeting signals or unique promoter regions of the genes which encode the proteins Synergistic combinations of inhibitors of the enzymes or proteins or nucleic acids which encode them arc particularly useful in medicines To select pathways for use in the invention a) plant
and the published lneiaiuie arc icv ie .ed for propcπιe> duι _'.ueι ι_.tιc rJ lants but enei.illv not animals, databases such as Genban oi the Apicomplexan ESTs arc re iewed to identify hom*ologous Apicomplexan and plant-like genes, and b) Western, northern and southern analyses, PCR, and ELISAs are used to recognize, or are based upon, for example, plant proteins and genes, to determine whether components of the pathways are present in Apicomplexans, c) cloning, isolation and sequencing of genes and creation of gene constructs are used to identify Apicomplexan plant-like genes and their functions, d) assays of enzyme activity are used to determined the operation of plantlike systems, e) functions of parasite enzymes or part of a parasite enzyme are demonstrated by complementation of a yeast or bacteria deficient in the enzyme, or product rescue, or other methods to demonstrate enzyme activity, f) activity of compounds, (i e , inhibitors) known to abrogate effect of the plant-like etizv me, protein, or nucleic acid which encodes them in vitro and /// vivo. are tested singlv or in a plurality, for ability to abrogate the enzyme activity and against Apicomplexan parasites alone or together, and in conjoint Apicomplexan, bacterial and fungal infections.The general compositions of this invention are A. inhibitor} compounds based on a) targeting proteins bv i i ) substrate competition and transition state analogues ι i) product competition (jii) alteration of active site directly or by modification of secondary structure or otherwise altering function of the active site
(iv) interfering with protein function with antibody (v) targeting an organeUe or protein within an organeUe using a toxic compound linked to a targeting sequence. b) targeting nucleic acids encoding proteins (antisense, ribozymes) c) targeting a component of the protein or nucleic acid (as above)
B. Diagnostic reagents (genes, proteins, antibodies) in ELISAs, western blots, DNA, RNA assays C. Vaccines (live knockout, live mutated, components - genes, proteins, peptides, parts of genes constructs, etc.)
Specific examples of components of plant-like Apicomplexan pathways are in Table 1. Compounds known to inhibit these enzymes or properties in Apicomplexans and/or other microorganisms are listed in Table 1 , as are novel ways to target them in Apicomplexans.
Table 1 A Apicomplexan plant-like metabolic pathways, components and inhibitors
Key S, mcd-fied substrate competitor, AS, antisense, R, πbozyme, Directed at active site, D, None known,
"EPSP synthase inhibitor 4 refers to 3-(phosρhonooxy)-4-hydroxy-5-[N-(phosphonomethyl-2- oxoethyl)amιnc-l -cyclohexene-1-carboxylιc acid (3α, 4α, 5(5), compound with diethyl ethaπamide EPSP synthase inhibitor 5 refers to shortened R pnosphonate
"A new aromatic analogue of the EPSP synthase enzyme reaction intermediate 1 has been identified, which contains a 3- hydroxymalonate moiety in place of the usual 3-phosphate group This simplified inhibitor was readily prepared in five steps from ethyl 3,4-dιhydroxybeπzoate The resulting tetrahedral intermediate mimic is an effective, competitive inhibitor versus S3P with an apparent K(ι) of 0 57 +/- 0 06 muM This result demonstrates that 3- hydroxymalonates exhibit potencies comparable to aromatic inhibitors containing the previously identified 3-malonate ether replacements and can thus function as suitsole 3-phosphate mimics in this system These new compounds provide another example in which a simple benzene ring can be used effectively in place of the more complex shikimate ring in the design of EPSP synthase inhibitors Furthermore the greater potency of the tetraheral intermediate mimic versus the glycolaie derivative and the 5- deoxy analog, again confirms the requirement for multiple anionic charges a: ;he dihydroxybenzoate 5-posιtιon in order to attain effective inhibition of this enzyme
The following were identified inhibition of Toxoplasma gondii ( Tg), Plasmodium falciparum (Pf) and Cryptospondiυm. csrvum (Co) EPSP synthase b; M-phosphonomethylglycine (NPMG), Tg and Pf chorismate synthase [AroC) cDNA and deduced a-r.mo acid sequences a novel sequence in the Tg chorismate synthase gene ( 4roC-fs) a portion of *.nιch is hom*ologous v/ith the plastid transit sequence of Zea mays (s -.ee; corn) The Pf chorismate s π-ase (AroC) also has a corresponding novel and unique internal region Cp Emien.o bows (Eb) geηciic DNA which hybridizes with Tg AroC (chorismate synthase) Inh'cition of Tg in vitro cy ! =MG abrogated by para-aminobenzoate (PABA) Inhibition of Pf ,π ytro by M=r..G abrogated by PA5A and folate Inhibition of Tg EPSP synthase activity by NPMG ιη v..rσ S, nergιsm ol NPMG >-..l pyrimethamine with sulfadiazine and with SHAM for Tg in vitro. Synergy of NPMG with pyrimethamine against Tg in vivo. SHAM and 8-hydroxyquιnohne inhibited Tg Pf Cp in vitro, reactivity of Tg protein of -66Kd with 5 antibodies (monoclonal and polyclonal to VooOoo lily and 7" brucei alternative oxidases) and reduction to monomer similar to VooOoo lily and T brucei alternative oxidases on a reducing gel, Identification of Tg cONA and genomic DNA PCR products using primers based on conserved sequences in other alternative oxidases which are probed and sequenced, Tg, Pf, Cp inhibited by high concentrations of gabaculine Reactivity of Tg protein of -40Kd with 3 antibodies to GSAT (polyclonal α soybean, barley and synechococcus GSATs and not preimmune sera) Reactivity of Cp protein of -40Kd with α barley GSAT Inhibition of Tg, Pf, Cp in vitro by 3NPA, Reactivity of Tg protein with polyclonal antibodies to cotton malate synthase and cotton isocitrate lyase but not preimmune sera In screening 7"c; cDNA library α GSAT antibody reactive clones are identified and are sequenced Tg chorismate synthase and dehydroquinase enzymatic activities are demonstrated
Table I B. Components of Plant-Like Ivletabolic Pathways and Inhibitors
Key S, mod'.fϊed suostrate competitor, AS, antisense, R, nbozyme, D, direct inhibitor, alteration of target These are suitable because they are unique to Apicomplexans Unique to Apicomplexans means that either they do not exist in animals (e g , acetohydroxyacid synthase, linoleic acid synthase starch-amylose cr amylopectin synthase, Q or branching enzyme, UDP glucose, starch glycosyl transferase or have unique antigenic or biochemical properties distinct from those of animals (e g acetylco A carbcyylase)
'Also present in animals
♦Oine' enzymes ι". these pathways unique to Aci omplexans
+ Enzymes invol . d in the synthesis of these essential amino acids include the following Lysine hom*ocitrate synthase hom*ocitrate dehydrase (Euglena fungi) aspartokinase aspartate semialdehyde dehyd'ogenase dihydropicolinate synthase, dihydropicolinate reductase J pipendeine - 2 6 - dicarbowlate Cansferase N - succinyl - ε-keto- α aminopimelate transammase N - succinyl - L L α - ε-diaminopimelate desuccinylase L L α - c diaminopimelate epimerase meso-α c diaminopimelate decarboxylase
Inhibitors of lysine synthesis include +2-4-Amιno-4-carboxybutyl azrιdιne-2-carboxylιc acιd(3) (aziπdiπo-diaminopimelate [DAP], aziDAP), N-Hydroxy DAP4, N-amino DAP5 4 methyelene DAP 6 3,4 didehydro DAP 4 methylene DAP 4 Methioπine L-hom*oseπne acyltransferase, o-succinylhom*oseπne sulfhydrolase, L-hom*ocysteme transferase (to activate methioπine - but not exclusively in plants S-adenosylmethonine [SAM] synthase, SAM-methyltransferase, SAM decarboxylase, S-adenosylhom*ocysteine hydrolase) Threonine L hom*oseπne kinase O-phospho-L-hom*osenne (threonine) synthase Isoleucine, valine L-threonine deammase, acetohydroxy acid synthase, acetohydroxy acid isomeroreductase, dihydroxy acid dehydrase, branched-chain ammo acid glutamate transammase Leucine isopropylmalate synthase, α-isopropylmalate isomerase, S-isopropylmalate dehydrogenase α ketoisocaproate transammase
Histidine phosphoπbulosyl formιmιno-5-amιnolmιdazol-4-carboxamιde πbotide amidocyclase, imidazol glycerol phosphate dehydrase, imidazole acetol phosphate transammase, histidinol phosphate phosphatase, L-histidinol dehydrogenase
Additional herbicides which disrupt cell membranes include Diphenyl ethers [πitro phenyl ethers=] (acifluor en=5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitrobenzoic acid fomeasafen=5-[2- chloro-4-(trιfluoromethyl)phenoxy]-N-(methylsulfonyl)-2-nιtrobenzamιde, lactofen=()-2-ethoxy-1- methyl-2-oxoethyl 5-[2-chloro-4-(trιfluoromethyl)phenoxy]-2-nιtrobenzoate, oxyflurfen=2-chloro-1-(3- ethoxy-4-nιtrophenoxy)-4-(trιfluoromethyl)benzene), Other bentazon=3-(1-methylethyl)-(1H)-2,1 3- benzothιadιazιn-4(3H)-one 2,2-dιoxιde above Additional herbicides which disrupt pigment production include clomazone=2-[(2-chlorophenyl)methyl]-4,4-dιmethyl-3-ιsoxazolιdιnone, amιtrole=1 H-1 ,2 4- trιazol-3-amιne norflurazoπ=4-chloro-5-(methyl amιπo)-2-(3-(trιfluoromethyl) phenyl)-3(2H)- pyndazinone, flurιdone=1-methyl-3-phenyl-5-[3-(trιfluoromethyl)phenyl]-4(1 H)-pyrιdιnone
Enzymes in the heme s>nthesιs [with a default ALA synthase pathway], shikimate pathwav alternative generation of energy and glyowlate cycle are exemplified (Table 1 A) and the others (Table I B) are suitable for the practice of the invention As outlined succinctly above the present invention includes new methods and compositions to treat, diagnose and prevent human and veterinary disease due to Apicomplexan parasites Apicomplexan infections include those due to loxop/αsnici gondn (toxopl ismosis), Plαvnodtα (malaria), Ci )plυspoι idiα (cry ptosporidiosis) Luna tα (ennciO s) Ba/icsia (babesiosis) Iheil na (theileπosis) Neoψoia uiiiiiiuni and others Λ-, \p omplcxan parasite nnplasnia ondn is a rcpi csentati \ c ol i.ihc
loguιeti-.all . rclatcd and have organeUes and enzymes which are critical for their growth and survival. The presence of plant-like pathways/enzymes is confirmed in Apicomplexan.; by a) the effect of known inhibitors of the pathways in plants using /// vitro and in vivo assays; b) Western, Northern and Southern hybridization analyses; c) isolation and comparison of relevant genes; d) demonstration of enzymatic activity; e) demonstration of immunologically reactive proteins which cross-react with proteins in plants; f) complementation of organisms which lack a gene or part of the gene encoding an enzyme with a parasite gene which encodes the enzyme; and/or g) recognition of plantlike transit sequences. //; vitro assays include product rescue (i.e., complete or. partial abrogation of the effect of an inhibitor by providing the product of the reaction and thus bypassing the need for the enzyme which catalyzes the reaction. The assays are based on inhibition of the parasite i.e. restriction of growth, multiplication or survival of the parasite. Another measure of infection is "parasite burden" which refers to the amount (number) of parasites present as measured in vivo in tissues of an infected host. Another measure of infection is destruction of host tissues by the parasites. Inhibitors reduce parasite burden and destruction of host tissues caused by the parasites. Preferably the inhibitors must not be toxic or carcinogenic to the parasites' host and for /'// vitro assays not be toxic to cells in culture.
Enzymes of the newly detected plant-like pathways provide novel, unique and useful targets for antimicrobial therapy. These unique pathways and enzymes are within the plastid, lyoxosomes, cytoplasm or mitochondria In addition, not suggested before for these parasites, some enzymes used in these pathways arc encoded by ge e w ithin the nucleus Plant- ke pathways detected in Apicomplexan parasites include a) the 5-carbon heme biosynthesis pathway that utilizes glutamate as a carbon skeleton for synthesis and requires the unique enzyme glutamate- 1 -semialdehyde ammotransferase, b) the mobilization of lipids in the glyoxylate cycle which is a unique pathway that includes the 5 enzymes isocitrate lyase and malate synthase, c) the generation of energy by an alternative pathway which includes a unique alternative oxidase and/or other unique pathways and enzymes for generating energy in the mitochondria or plastid, and, d) the conversion of shikimate to chorismate utilized in the synthesis of ubiquinone, aromatic amino acids and folate by plants, but not humans. The shikimate pathway includes the 10 enzyme 3-phospho-5-eπolpyruvylshikimate (EPSP) synthase, chorismate synthase, and chorismate lyase, as well as a number of enzymes unique to plants, fungi, bacteria, and mycobacteria, but not to animals. Inhibitors of some of these enzymes also provide information about the functioning and targeting of the enzymes
The heme synthesis pathway invol. es enzymes encoded in the nucleus and 1 imported to the plastid. This pathway is present in Apicomplexans including T. gondii, P. falciparum, and Cryptosporidia parvum. Inhibitors of the enzyme GSAT in the pathway include gabaculine (3-amiπo-2,3-dihydro benzoic acid), 4-amιno-5-hexanoιc acid, and 4-ammo-5-fluropentanoιc acid
The
late cycle, reported to be present in plants, fungi, and algae is also —■-' present in / gondii The cycle uses lipids and converts them to C4 acids through a scries of biochemical reactions One of the last steps in this series of reactions is dependent on the ι>ociU le lyase enzyme and anothei on the malate .svntliase enzs mos liilnbiloi s ol ire>e enzymes include 3-πιtroptopιonιc acid and itaconic acid The alternative respiratory pathway, present in a range of organisms including some bacteria, plants, algae and certain protozoans (trypanosomes), is present in T. gondii, Cryptosporidia parvum, and Plasntodium falciparum (in the latter parasite, two clones designated W2 and D6 were inhibited). The pathway is inhibited by a ranee of compounds including salicylhydroxamic acid, 8-hydroxyquinoIine, Benzyhydroxamic acid (BHAM), m-Chlorohydroxamic acid (m-CLAM), Propylgallate, Disulfliram and others.
Enzymes involved in the synthesis of chorismate, including those which convert shikimate to chorismate, and enzymes which generate folate, aromatic amino acids and ubiquinone from chorismate in plants, are present in T. gondii, Plasntodium falciparum. Ciyptosporidium pan'itm, and Eimeria. Inhibitors include N-(phosphonomethyl) glycine (glyphosate, sulfosate and others). A full-length T. gondii cDNA sequence encoding a chorismate synthase from this pathway and the deduced amino acid sequence provide information useful in developing novel antimicrobial agents. The T. gondii chorismate synthase has features in common with other chorismate synthases and entirely unique features as well. The unique features are novel sequences not shared with chorismate synthases from other organisms but with hom*ology to an amyloplast/chloroplast transit sequence of Zea mays (sweet corn). A P. falciparum cDNA sequence encoding chorismate synthase and its deduced amino acid sequence also provide information useful for developing novel antimicrobial agents.
The genomic sequences provide information about regulation of the gene (e g . unique promoter regions) and such unique regions enable targeting their regulatory elements with antisense A part of the novel internal sequence (i c., SCS FSESAASTI KHERDGSAATLSRE RASDGRTTSRHEEEVEPG) in the 7". gondii AroC (chorismate synthase) gene has hom*ology with the chloroplast/ amyloplast targeting sequence of Zea mays (sweet corn) wx (UDP. glucose-starch-glycosyl transferase) protein (i.e., MAALATSQLVATRAGLGVPDASTΓRRG AAQG RGAPΛs a u-TLSMRTSAΛAAPRHQQQARRGGRFPS wc). This transit sequence provides a novel way to target T. gondii enzymes that move from the cytoplasm into the plastid and is generally applicable to targeting any subcellular organeUe. The P. falciparum AroC (chorismate synthase) has a corresponding novel internal sequence.
Additional pathways found in Apicomplexan parasites include the synthesis of branched chain amino acids (valine, leucine and isoleucine) and acetohydroxy acid synthase is the first enzyme in the branched chain amino acid synthesis pathway, inhibited by sulfonylureas and imidazolinones, as well as the synthesis of other "essential" amino acids, such as histidine, methionine, lysine and threonine. Starch synthesis, including starch synthases, the UDP-glucose-starch glycosyl transferase, and debranching enzymes and enzymes of lipid, terpene, giberellin and auxin synthesis, are part of other pathways in Apicomplexan parasites. Down modulation of the UDP- glucose starch glycosyl transferase pathway leads to a switch from amylose to amylopectin synthesis and thus the bradyzoite phenotype.
Demonstration of presence of one enzyme or the gene that encodes it in a known pathw av implies presence of the full pathway Thus, enzymes in parasite metabolic pathw ays that can be inhibited include glutamyl-tR A synthetase. glutamyl- i NΛ ι educia_-c. pi eμhcnaie dehydrogenase. aromatic acid aminolransferase (aromatic ti nsaminasc). cyclohexadienyl dehydrogenase, tryplophan sv thase alpha subunit. tryptophan synthase beta subunit; indole-3-glycerol phosphate synthase (anthranilate isomerase), (indoleglycerol phosphate synthase), anthranilate . phosphoribosyltransferase, anthranilate synthase component I; phosphoribosyl anthranilate isomerase; anthranilate synthase component II; prephenate dehydratase (phenol 2-monooxygenase); catechol 1,2-deoxygenase (phenol hydroxylase), cyclohexadienyl dehydratase; 4-hydroxybenzoate octaprenyltransferase; 3-oxtaprenyl-4- hydroxybenzoate carboxylyase; dehydroquinate synthase (5-dehydroquinate hydrolase); chorismate synthase (5-enolpyruvyIshikimate 3-phosphate phosph-lyase); dehydroquinate dehydratase; shikimate dehydrogenase; 3-deoxy-d-arabino- . __ heptuloonate 7 phosphate synthase; chorismate mutase (7-phospho-2-dehydro-3- deoxy-arabino-heptulate aldolase); 3-deoxy-d-arabino-heptuloonate 7 phosphate synthase; shikimate 3-phosphotransferase (shikimate kinase); UDP glucose starch glycosyl transferase, Q enzymes; acetohydroxy acid synthase; glutamate- 1- semialdehyde 2, 1-aminotransferase; chorismate lyase. malate synthase; isocitrate lyase. and 3-enolpyruvylshikimate phosphate synthase (3-phosphoshikimate- 1 carboxyvinyltraπsferase).
Recombinant protein produced by constructs with genes encoding these enzymes in E coli or in other expression systems is useful for producing antibodies and obtaining a crystal structure Native enzyme is isolated The expressed and native proteins are used to design and test new inhibitors in enzyme assays Expressed and nativ e (from v aried life-cycle stages) proteins are used and the expressed protein is a souice of ihe euzv inc, and ihe enzyme assay is carried out in the presence and absence of the inhibitor-, eithci alone or in combination and contiols include the buffer for the enzyme alone. The crystal structure is useful for characterizations of enzyme active sitc(s), secondary structure, transit sequence, substrate and product interactions. The design of additional inhibitors is carried out using published methods such as modifyins substrates as had been done with inhibitors of EPSP synthase as well as high through put screening of av ailable compounds.
Certain pathways are shown to be affected by inhibitors which are synergistic //; vitro. Examples of synergistic inhibitors /// vitro are gabaculine, (heme synthesis) and SHAM (alternative energy generation); NPMG and SHAM; NPMG and sulfadiazine; and NPMG and pyrimethamine: Gabaculine and sulfadiazine are an additive . combination /'// vitro.
An aspect of the invention is identifying potential targets for therapeutic intervention by considering nuclear as well as organellar genes as part of the production of enzymes for unique plant-like pathways. For example, the protein synthesis of plantlike proteins that is also demonstrated in Apicomplexan parasites suggests not only conservation of plastid genes but also conservation of nuclear genes which encode enzymes that act inside or outside the plastid, from an ancestor that is common to Apicomplexan parasites and algae. Many vital metabolic pathways of algae (often shared with their evolutionary relatives, higher plants) also are conserved in the Apicomplexan parasites, whether or not the pathways involve the plastid. Consequently. Apicomplexan parasites are sensitive to inhibitors that block several of these unique pathways. Combined attack on multiple targets retards the emergence/selection of resistant organisms Considering nuclear and organellar genes has the dual advantage of rapidly identifying conservation of specific pathways and simultaneously identifying both target sites and lead compounds for therapeutic druς development.
An aspect of the invention is a plurality of inhibitors, singly or in combination, directed against enzymes and/or genes encoding a different metabolic pathway. Examples of inhibitors suitable for practice of the present invention include GSAT,
3NPA, SHAM, 8-0H-quinoline, and NPMG, sulfonylureas, imidazolinones, other inhibitors of EPSP synthase or chorismate synthase which include competitive substrate analogues, transitional state inhibitors and direct active site inhibitors as well as other known compounds (Table I). Some pluralities of inhibitors produce synergistic effects. Improved treatments against Apicomplexan parasites result from a variety of options:
1. some compositions may inhibit the operation of more than one pathway, thereby producing a strong effect and lessening the probability of resistance to the drug emerging because more than one mutation may be required; 2. some compositions may inhibit more than one step in a pathway;
3. some pluralities of compositions may have synergistic effects, producing more effective drugs; and
4. some compositions may target pathways operative exclusively during a life cycle of the parasite, making them more selective e.g. against the latent phase 5 some compositions may inhibit other microorganisms (including other
Apicomplexans )
An additional detail of the invention is that representati e Apicomplexan parasites, notably /'. gondii, are used for assaying candidate inhibitors The inveiuion is directed at effects of inhibitors of the unique plant-like pathways in Apicomplexan, alone and in combination. Organisms used for the assays include T. gondii tachyzoites, bradyzoites and a mutant that expresses 50% tachyzoite and 50% bradyzoite antigens Unique plant enzymes and pathways that were found to be inhibited by compounds shown to inhibit plant pathways in Apicomplexans include: (1) glutamate- 1 semialdehyde amino transferase, an enzyme important in heme synthesis, (2) isocitrate lyase, an enzyme important in utilization of lipids, (3) alternative oxidase enzyme complex, enzymes important in energy production and (4) 3-phospho-5- enolpyruvylshikimate synthase (EPSP synthase), an enzyme important in conversion of shikimate to chorismate which is a precursor for synthesis of folate, ubiquinone, and certain amino acids essential for survival.
The invention provides a rational, conceptual basis for development of novel classes of antimicrobial agents that inhibit Apicomplexan parasites, unique diagnostic reagents, and attenuated vaccines. The inhibitors provide lead compounds for the development of antimicrobial agents. Conserved enzyme active sites or parts of the molecules or genes that encode the protein which are targeted by the inhibitors provide the basis for development of new but related ways to target the enzymes, such as related protein inhibitors, intracellular antibodies, antisense DNA. and ribozymes
Inhibitors are effective against more than one parasite (e.g. T. gondii, P falciparum and C. parvum) and enzymes in these pathways also are present in other bacterial and fungal pathogens such as Pneumocysits cariiin, λ cohacteruim luherciilosi ni Siapliylo occus aiireii.s. and Henuiphilits influenza, but not animals T lui. . inhibitor of these pathways affect susceptible microorganisms which concurrently infect a host. Because enzymes are utilized differentially in different parasite life-cycle stages, stage-specific inhibitors are within the. scope of the invention Genes encoding the enzymes in Apicomplexans are identifiable. The genes encoding the enzymes are effectively knocked out in these parasites by conventional techniques "Knockout" mutants and reconstitution of the missing genes of the parasite demonstrate the importance of gene products to the varying life-cycle stages of the parasite which are identified using antibodies to proteins and ability to form cysts /'// vivo which define the life cycle stages. The parasites in which a gene is knocked out are a useful basis for an attenuated vaccine. The genes encoding the enzymes or parts of them (e.g., a novel targeting sequence) or the proteins themselves alone or with adjuvants comprise a useful basis for a vaccine. The pathways and enzymes of the invention are useful to design related antimicrobial agents. The sequences and definition of the active sites of these enzymes, and pathways, and organeUe (e.g., plastid) targeting sequences provide even more specific novel and unique targets for rational design of antimicrobial agents effective against Apicomplexan parasites For example, proteins which interact with the enzyme and interfere with the function of the enzyme's active site, or are competitive substrates or products or intracellular antibodies (i.e., with a gene encoding the Fab portion of an antibody that targets the protein the antibody recognizes), or antisense nucleic acid or targeted riboz mes that function as inhibitors are useful, novel antimicrobial agents Enzymes of the invention are a novel basis for unique diagnostic tests Because some of these pathwa s are important in dormant parasites, or in selecting the dormant oi active life c cle Mages thcv are especially important as aiuimiαobial agent targets for life cycle stages of the parasite for which no effective anlimicrobial agents are known or as diagnostic reagents which ascertain the duration of infection.
Identification of the pathways in Apicomplexan parasites provides additional enzyme targets present in these pathways which are not present in or are differentially expressed in animal cells. Identification of the interrelatedness of these pathways with each other provides the basis for the development and demonstration of combinations of inhibitors which together have an effect which is .greater than the expected additive effect (i.e., synergistic). The meaning of synergism is that compound A has effect A', compound B has effect B', compounds A+ B have an effect greater, than A'- B'. Synergism is characteristic of inhibitors of these pathways because an initial pathway affected by an inhibitor often provides a product used as a substrate for another pathway so the inhibition of the first enzyme is amplified. These pathways or their products are interrelated. Therefore, the enzymes or DNA which encodes them are targeted by using two or more inhibitors leading to an additive or synergistic effect. Examples include the additive effect of gabaculine and sulfadiazine and the synergistic effects of NPMG and sulfadiazine and NPMG and pyrimethamine. One or more of the inhibitors preferentially affect one of the life cycle stages of Apicomplexan parasites.
Some enzymes are preferentially used by specific stages of the parasites. Detection of an enzyme of this type or a nucleic acid encoding it offers a novel diagnostic les; not only for presence of a parasite, but also for identification of the stage of the parasite
Genes encoding enzymes in pathw ays of ihe present invention are "knocked out" using le.v.πiques known in the an Λ parasite with a gene knocked out is said to be attenuated either because the gene expression of the enzyme is stage specific so the parasite cannot become latent, or because the knocked out enzyme is essential for parasite survival. The importance of an enzyme's functions in various life-cycle stages is determined using a mutant-knockout-complementation system. In the former case, the attenuated parasite is useful as a vaccine because the "knocked out" gene is critical for the parasite to establish latency. Its administration to livestock animals results in immunity without persistence of latent organisms. Mutants with the gene "knocked out" also can be selected because when the parasites are grown /// vitro they are grown in the presence of product of the enzymatic reaction to allow their survival. However, such attenuated parasites do not persist in vivo in the absence of the product and, consequently they are useful as vaccines, for example, in livestock animals. The genes that encode the protein also are used in DNA constructs to produce proteins themselves or the proteins or peptides are used in immunized animals These constructs are used to elicit an immune response and are used for vaccines alone or with adjuvants Specific examples are incorporation of the gene for alternative oxidase or chorismate synthase in a construct which has a CMV promoter and expresses the protein following intramuscular injection (i.e., a DNA vaccine) This type of construct, but with genes not identified or described as plant-like, has been used as in a vaccines that protect against bacterial and protozoal infections Plant-like pathways in Apicomplexans were inhibited /// vitro An
Apicomplexan GSAT enzyme that is pan of a heme synthesis pathway was targeted with inhibilois Λ gene with hom*ologv to ALA svnthase was identified bv analysis of the /. gondii EST:. ( Washington Univ ersity / gondii gene Sequencing project). indicating that 7 gondii has alternative methods for synthesis of ALA An Apicomplexan glvoxylate cycle was analyzed to determine the sensitivity of tachyzoites and bradyzoites to glyoxylate cycle inhibitors Specifically, Apicomplexans have isocitrate lyase and malate synthase which present a unique pathway for lipid metabolism that is targeted by inhibitors Apicomplexan alternative oxidase is targeted, as evidenced by effects of inhibitors of alternative oxidase on this pathway and its expression and immunolocalization in tachyzoites and bradyzoites, Apicomplexan parasites have a metabo cally active EPSP synthase enzyme involved in conversion of shikimate to chorismate These four metabolic pathways, i e , heme synthesis, shikimate pathwav. alternative generation of energy, and the glyoxylate cycle are all exemplified in T. gondii To show that inhibition was specific for key enzymes in these pathways that are generally absent or used only rarely in mammalian cells, product inhibition studies w ere used in vitro For example, growth of 7". gondii is sensitive to NPMG that inhibits the synthesis of folic acid via the shikimate pathway Because mammalian hosts lack the entire shikimate pathway, it is unlikely that the parasites can obtain either PAB λ or its precursor chorismate from the host cells so provision of PABA circumvents the need for the substrate pathway for folate synthesis and rescues the EPSP synthase inhibition bv NPMG
Further proof of the presence of the plant-like pathw avs arises from biochemical assays for an eπz me in analogous plant pathways and isolation of encoding genes Genes ai e identified by search of available expressed sequence tags (ESTs i e . short single pass cDVΛ sequences generated rom randomly selected library clones), bv PCR am lilkaiion using pnmei sequences d -". ed fi om published conserved sequences ol plant genes with parasite genomic DNA or parasite DNA libraries (Chaudhuri et al , 1996), by the screening of Apicomplexan DNA expression libraries with antibodies to previously isolated hom*ologous proteins or the DNA which encodes them and by complementation of E. coli or yeast mutants deficient in an enzyme. Genes isolated by these techniques are sequenced which permits identification of hom*ologies between plant and Apicomplexan genes using sequence databases such as Genbank. These assays confirm that an enzyme and the gene encoding it are present in Apicomplexan parasites. E. coli mutants and yeast deficient in the enzyme are complemented with plasmid DNA from T. gondii cDNA expression libraries or the isolated gene .or a modification (e.g , removing a transit sequence) of the isolated gene which allows the production of a functional protein in the E. coli or yeast, demonstrating that the gene encoding the enzyme is functional. hom*ologous genes in T. gondii, P. malaria, Cryptosporidia. Neospora, and Enneria are identified when relevant plant or T. gondii genes are used as probes to DNA obtained from these organisms and the genes are identified either by cloning and sequencing the DNA recognized by the probe or by using the probe to screen the relevant parasite libraries Genomic DNA is sequenced and identifies unique promoters which are targeted Unique parts of the genes were identified in the sequences and provide additional antimicrobial agent targets, diagnostic reagents and vaccine components or bases for v accines Clade and bootstrap analyses (Kohler et al , 1997) establish the phvlogenetic origin of novel, sequenced, parasite genes and this indicates other related antimicrobial agent targets based on components molecules, and pathways of phv logeπeticallv related organisms Gene products are expies ed and utilized l t enzyme assa . s and lot screening novel inhibitors lot making antibodics for isolation of native protein, for x-ray crystallography which resolves enzyme structures and thus establishes structure-function relationships and enzyme active sites which are useful for the design of novel inhibitors
Immunoelectronmicroscopy using antibodies to enzymes such as chorismate synthase, alternativ e oxidase, malate synthase or isocitrate lyase immunolocahzes the enzymes within the parasite and determines their location, in particular whether they are in plant-like organeUes Apicomplexan transit peptides are identified by their hom*ology to known transit peptides in other species Attachment of reporter proteins to the wild type transit peptide, or deletion or mutations of the transit peptide or portion of the peptide or gene encoding it, and then characterization of targeting of these constructs alone or in association with reporter constructs establishes that the ammo acid sequences of the transit peptide determine intracellular localization and site of function of proteins with this sequence Stage specificity of these enzymes is determined in vino by using antibodies to stage-specific antigens in inhibitor-treated cultures, by Western or Northern analyses (detection), by eπzvme assays using selected parasite life cycle stages, by using RT PCR (Kiπsits, et al 1996) and a DNA competitor as an internal standard to quantitate the amount of mRNA in parasite samples, by ELISA (quantitation) and by determining whether a parasite with the gene knocked out can develop a brcα zoue phenotype m vin in the appropriate bradyzoite inducing cultuie conditions Stage specificity ... VΠY; IS cetermined by observing effects of the inhibiioi s on dilleient I : cvcle stages in aculclv . > chronicallv infected mice and bv determining whethei a p 1 -no w ith the gene knoc- ec out can form cvsts in \ n o Useful techniques to develop diagnostic reagents for detection of these proteins or nucleic acids include ELISAs, Western blots, and specific nucleotides used as probes.
EXΛMI'LES Example 1 : Novel In Vitro Assnv Svstcnis to Assess Antiinicrobinl Effects on 7*. gondii New in vitro and //; vivo assay systems were developed to determine whether plant metabolic pathways are present in Apicomplexans. New elements include use of longer culture times (e.g., extending the duration of the assay to > 6 days is also a unique and useful aspect of this invention, because it allows demonstration of antimicrobial effect for compounds which have to accumulate prior to exerting their effect), use of Me49 PTg and R5 strains /'// vitro, employing synergistic combinations of NPMG and low dosage pyrimethamine in vivo, and assays of parasitemia in vivo using competitive PCR.
Improvements were developed in the assays reported by Mack et al. (19S4) and Holfels et al. ( 1994) to measure T. gondii replication in tissue culture. The improvements are based on microscopic visual inspection of infected and inhibitor treated cultures, and on quantitation of nucleic acid synthesis of the parasite by measuring uptake of Η uracil into the parasite's nucleic acid Uracil is not utilized by mammalian cells Parasites present as tachyzoites (RH, Ptg. a clone derived from the Me49 strain), bradyzoites (Me49), and R5 mutants (mixed tachvzoite/bradyzoites of the Me49 strain that can be stage switched by culture conditions) ( Bohne el al , 1993. Soete et ul , 1994, Tomovo and Boothrovd, 1995, Weiss et al.. 1992) are suitable for assay systems used to study effects of inhibitors Only the RH strain tachyzoites. cultui ed oi up to 72 hours, haό been used in previously reported assays The use of Me49, Pig, and R5 mutant are unique aspects of the methods used in these assays in this invention
Results using the assay systems are shown in FIGS. 4, 6-8 In these assays toxicity of a candidate inhibitor was assessed by its ability to prevent growth of human foreskin fibroblasts (HFF) after 4 days and after S days as measured by tritiated thymidine uptake and microscopic evaluation Confluent monolayers of HFF were infected with tachyzoites or bradyzoites Inhibitor was added one hour later Non- toxic doses were used in parasite growth inhibition assays. Parasite growth was measured by ability to incorporate tritiated uracil during the last 18 hours of culture Example 2: Detection of Plant-like Pathw ays in Apicomplexans
Using assays disclosed herein, some of which were novel, Apicomplexan parasites were found to contain at least four metabolic pathways previously thought to be unique to plants, algae, bacteria, dinoflagellates, and fungi Specifically , the presence of a unique heme synthesis pathway, an alternative oxidase pathway, a glyoxylate cycle and a pathway necessary for the biosynthesis of chorismate and its metabolites were explored Growth of the parasite, T. gondii, depends upon these pathways To examine T. gondii for the presence of plant-like and algal metabolic pathway s, certain inhibitors of metabolic pathw avs are suitable to apply because of their ability to prev ent growth of the parasite in tissue culture Pathway s which arc present in Apicomplexans were analyzed as follows First
/ gondii tach zoites weie tested to see if thev were sensitive in vi o to inhibition by specific inhibitors of target pathwa; s Then braclv zones aie tested Positi e restills lor each pathw av prov ided piesumptivc ev idence thai the inhibitor taigcis w ei e preseni and that their activities are important for parasite survival and growth. The inhibitors effective in vitro were screened for activity /// vivo in mice. An. example of an effective combination in vivo is NPMG and low dosage pyrimethamine.
The presence of an enzyme was further confirmed by product rescue in vitro, in which the product abrogates the need for its synthesis by the enzyme. An example was rescue by PABA for the reaction catalyzed by EPSP synthase. Other methods to demonstrate the presence of an enzyme and thus the pathway include functional enzyme assays, complementation of mutant E. coli strains, PCR, screening of a T. gondii expression library with antibodies or DNA probes, and immunostaiπing of Western blots. For some enzymes, identification of a partial sequence of a gene in an EST library in the gene database led to cloning and sequencing the full length gene. Demonstration of the enzymes also is diagnostic for presence of the parasites. Examples are demonstration of T. gondii and C. parvum GSAT and T. gondii alternative oxidase and 7'. gondii isocitrate lyase and malate synthase by Western analysis and cloning and sequencing of the 7". gondii and P. falciparum chorismate synthase gene Reagents (gene probes and antibodies) obtained during characterization of genes from 7". gondii are used to detect hom*ologous enzymes and pathways in other Apicomplexan parasites Examples were using the T. gondii chorismate synthase gene to probe P. fa/apai urn. Eimeria hovts and Crypiosporidium parvum genomic DNA Other examples are using heterologous plant DNA to detect Apicomplexan GSAT, isociliaie lyase. malate synthase, and alternative oxidase genes Such genes are used as DNA piobes to sci een libraries to clone and sequence the genes (o identify I'CR pi oducts Examplc 3: Effects of lnliibitois In lltro on T. gondii
Using the assays described in Example 1 , five compounds that restrict the growth of 7" gondii in vitio were identified (i) Gabaculine (II) NPA
(in) SHAM (Salicylhydroxamic Acid), (IV) 8-hydroxyquinolιne (v) NPMG Specifically these inhibitors act as follows i. The Effect of Gabaculine, An Inhibitor Of The 5-Carbon Heme
Synthesis Pathway, On the Growth of T. gondii FIG. 1 A compares heme biosynthesis in plants, algae and bacteria with heme biosynthesis in mammals In higher plants and algae, ALA is produced in the plastid by the action of GSA aminotraπsferase on glutamate 1 -semιaldehyde In mammals, ALA is formed through the condensation of glycine and succinyl CoA ALA is subsequently converted to heme In one dinoflagellate and T. gondii both pathways are present
Inhibitors of plant heme synthesis pathway restrict the growth of Toxoplasma gondii in vino As shown in FIG. 1 A, the synthesis of δ-ammolevuhnic acid (ALA) the common precursor for heme bios nthesis occurs in the plastid of plants algae and Apicomplexan parasites by the 5-carbon pathway and ALA synthesis occurs by a difleicnt pathw av in animals The pathw av in animals involves the condensation ol gl cine and succm l CoA The data in TIG. 1 B-C and a Western blot utilizing an antibodv to the hom*olottous sov bean ard barlev and s ncchococcus GSΛTs dcmonstrate that Toxoplasma gondii utilizes the 5-carbon pathway for ALA synthesis and therefore heme biosynthesis 3-amino 2,3-dihydroxybenzoi.c acid (gabaculine) inhibits GSA in the heme synthesis pathway.
First the toxicity of gabaculine was assessed by its ability to prevent growth of human foreskin fibroblasts (HFF) as measured by 3H-thymidine uptake and microscopic evaluation. Non-toxic doses were used in parasite growth inhibition assays. In vitro parasite growth inhibition assays included confluent monolayers of HFF irtfected with tachyzoites (RH) or mutant Me49 (R5). Gabaculine was added 1 hour later. Parasite growth was measured by the ability to incorporate 3H-uracil during the last 1.8 hours of culture. In addition, parasite growth was evaluated microscopically in Giemsa stained slides.
Toxoplasma organisms were grown in human foreskin fibroblasts alone and in the presence of different concentrations of gabaculine (3-amino-2,3-dihydrobenzoic acid). Growth was measured by the ability of T. gondii to incorporate tritiated uracil This compound was effective at inhibiting the growth of T. v ndii at the 20mM concentration. FIG. IB demonstrates the ability of gabaculine (a specific inhibitor of GSA aminolransferase) to restrict the growth of T. gondii in an in vitro assay over a 4 day period 7'. gondii growth is measured by ability of the parasites to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis The X-axis describes how the T. gondii cultures were treated Cultures that were grown in medium (medium) produced a CPM of around 45,000 If no /'. gondii were added to ihe cultures ( ιiκ RH). a C M of around 2,00'J was observed Pyπmeihamine (0 I μiu ml) and suiphadiazinc ( 12 5 uu/ml) added to cultures resulted m a inai kcd reduction in CPM compared with untreated cultures. At a dose of 5 mM gabaculine restricted around 50% of CPM and at a dose of 20 mM it almost completely inhibited parasite growth, with counts of about 5,000 CPM.
FIG. IC demonstrates the ability of gabaculine to inhibit the growth of T. gondii over S days in culture. T. gondii growth is measured by ability of the parasites to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis represents days post infection.. Parasite growth was evident in the cultures where no drug was added (medium) over the entire time course. Parasite growth was restricted in cultures with 20 mM gabaculine (gabaculine) over the 8 day time course. Similarly, parasite growth was restricted in cultures with pyrimethamine and sulphadiazine (P/S) over the 8 day time course. Similar concentrations showed no toxicity to the foreskin fibroblasts indicating the specificity of this compound for T. gondii. Parallel cultures, fixed and stained with Giemsa and examined by microscopy, clearly demonstrated that T. gondii growth was substantially inhibited in the presence of 3-amino-2,3-dihydrobenzoic acid The results in FIGS. I B and I C indicate that T. gondii utilizes the 5-carbon ALA synthesis pathway
FIG. 7 demonstrates the ability of gabaculine to inhibit the growth of the mutant R5 strain of T. gondii over S days in culture This mutant strain is atovaquone resistant and cossesscs certain characteristics of the tachyzoite stage and certain characteristics of the bradyzoite stage T. gondii growth is measured by their ability to mcoi poi te tr.:ιated uracil and is expressed as counts/minute (CPM) on the Y-axis The X-axis represents days post infection Parasite grow th was evident in the cultures w hei e no dru_ '- as added (medium) ov er the enure time cυui se Pai asite gi owih w as restricted in cultures with 20mM gabaculine (gabaculine) over the first 6 days of culture, after w hich a marked increase in parasite growth was detected. Furthermore groups of proliferating organisms which resembled tissue cysts were observed in similarly treated cultures. Parasite growth was restricted in cultures with pyrimethamine and sulphadiazine (P/S) over the entire 8 day time course. Residual R5 organisms in treated cultures at 8 days begin to incorporate uracil again and some of them appeared cyst-like. Therefore, T. gondii cystrlike structures are selected by gabaculine treatment of cultures. Specific immunostainiπg of such cultures treated with gabaculine for tachyzoite and bradyzoite specific antigens demonstrates thatgabaculine selects bradyzoites Table 2 is a schematic representation of experiments designed to test the hypothesis that tachyzoites utilize both conventional oxidase and alternative oxidases, but bradyzoites only use alternative oxidases, therefore interfering with generation of iron sulfated proteins by gabaculine treatment will select bradyzoites The design and predicted results of stage specific immunostaiπing (Kasper ct al., 19S3 ) if the hypothesis were correct are shown in Table 2 and confirm the hypothesis These results suggest that T. gondii has stage-specific utilization of alternative oxidases which are utilized w hen cell cultures are treated with gabaculine because it depletes heme and thus depletes iron sulfated proteins used in conventional respiration.
In summary, 3-amino-2,3-dihydrobenzoic acid (gabaculine) is an inhibitor of t -' 5 carbon heme synthesis pathway present in Apicomplexan parasites Heme synthesis occurs b\ a c.ftei ent pathw ay m mammalian cells and is thercfoi e unaffected by 3 - amino-2, 3-d "• di obenzoic acid Table 2. Gabacul'ne treatment of cultures selects bradyzoites.
Antibody Treatment TachyBradyIFA result on culture day used (or of zoite zoite IFA culture Control Control 0 2 6 α SAGI Media
(expressed on tachyzoites only) Gabaculine
α BSAG edia (expressed on brady- zortos one day after stage Gϊ acuiiπe switch)
α BAGS Madia (expressed on brady- zotles by ri e day Gs aculme alter stage switch in culture)
IFA is immunof.jc-oscent assay. SAG1 is surface antigen 1. BSAG is bradyzoite surface antigen 1. BAGS is bradyzoite antigen 5. A. Hypothesis. B. Design and predicted results of stage specific irrrr nostatning If hypothesis were to be correct. CD Indicates no specific fluorescence of fe crgaπism; Q indicates specific surface lluorescenco of the organism due lo presence of the antigen recognized by the antibody (e.g , αSAGI or αBSAG); C^ Indicates specific Internal f .crescence in the organism duo to presence ol the antigen within the parasite recognized by r . s .'.ibαdy (e g , αBAG5)
ii. Λπ inhibitor of t e glyoxylatc cycle restricts the growth of
T. gondii in vitro.
3-Nitropropionic acid is an inhibitor of isocitrate lyase in the degradation of lipid to C4 and inhibits replication of T. gondii in vitro. FIG. 2A illustrates how the glyoxylatc cycle manufactures C4 acids. Acetyl CoA, a byproduct of lipid breakdown combines with oxaloacetate to form citrate. By the sequential action of a series of enzymes including isocitrate lyase, succinate is formed. Glyoxalate, the byproduct of this reaction is combined with a further molecule of acetyl CoA by the action of malate synthase. Malate is then converted to oxaloacetate, thus completing the cycle. 3-NPA and itaconic acid are inhibitors of this pathway. FIG. 2B demonstrates the ability of 3-NPA (an inhibitor of isocitrate lyase) to restrict the growth of T. gondii in an /'// vitro assay over a 4 day period. This result indicates it is likely that T. gondii degrades lipids using isocitrate lyase. 7*. gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis described how the T. gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM of about 30,000. If no T. gondii were added to the cultures (no RH), a CPM of about 2,000 was observed. Pyrimethamine (0. 1 μg/ml) and sulphadiazine ( 12.5 μg/ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures. A dose of 0.006 mg ml 3-NPA (3-NPA) restricted around 60% of CPM 3-NPA inhibits the glyoxylate cycle (isocitrate lyase) and/or succir.aie dehydrogenase in Apicomplexan parasites iii. :ιιιd i\ . Effect of SHAM and S-hydroxyqiiiiiolitic on alternative ovidasc in T. gondii There is a metabolic pathway found in most plants and algae and in Apicomplexans, but absent in most multicellular animals FIG. 3A describes the electron transpoπ respiratory chain that normally occurs on the inner membrane of mitochondria In animals, NADH and succinate produced by the action of the citric acid cycle diffuse to the electron transport chain. By a series of oxidation reactions mediated in part through the cytochromes, free energy is released This free energy yields the potential for the phosphorylation of ADP to ATP. In plants, in addition to the conventional electron transport chain complexes, there is an alternative pathway of respiration Alternative pathway respiration branches from the conventional pathway at ubiquinone and donates released electrons directly to water in a single four electron step An important feature of this pathway is that it does not contribute to transmembrane potential and thus free energy available for the phosphorylation of ADP to ATP The pathway provides a source of energy and is preferred for conditions with relatively low ATP demands A key enz me in this pathway is an alternative oxidase ' that is cyanide insensitive and does not require heme Toxoplasma gondii utilizes the alternative oxidase for respiration
FIG. 3B demonstrates the ability of SHAM (a specific inhibitor of alternative oxidase) to restrict the growth of T. gondii in an ;// vii/o assay over a 4 day period
The abi tv cf these compounds to inhibit the growth of / gondii was examined bv the assay desert '_ύ in x.imple 1 / gondii growth is measured by their ability to mcoi poKite .Mut d uracil and is as couiits/iiiinutc (CPM) on the Y-a\ιs The X-axis descπbes how the T. gondii cultures were treated Cultures that were grown in medium (medium) produced a CPM of around 54,000 If no 7. gondii were added to the cultures (no RH), a CPM of around 1 ,000 was observed Pyrimethamine (0 1 μg/ml) and sulphadiazine (12.5 μg/ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures A dose of 0 19 μg/ml SHAM (0 19) restricted around 50% of CPM and at a dose of 0 78 μg/ml it essentially inhibited parasite growth, with counts of about 8,000 CPM.
Salicylhydroxamic acid (SHAM) and 8-hydroxyquinoliπe are inhibitors of the alternative oxidase and are also effective against T. gondii, presumably by inhibiting the alternative pathway of respiration Salicylhydroxamic acid and S-hydroxyquinoline inhibit the alternative oxidase of 7". gondii tachyzoites Since alternative oxidative respiration does not occur in mammals, this makes antimicrobial compounds targeting this pathway therapeutic candidates v Effect of NPMG The shikimate pathway is common to plants, fungi and certain other microorganisms and Apicomplexan parasites, but it is not present in mammalian cells FIG. 4A details the events that result in the production of tetrahydrofolate, aromatic ammo acids and ubiquinone in plants, algae, bacteria and fungi In this pathway, chorismate is formed through the sequential action of a number of enzymes including EPSP-synthase and chorismate synthase EPSP-synthase is inhibited by NPMG
Chorismate is t.iπ er pi ocessed to yield tetrah rofolate or ubiquinone bv a further seπe - ol' eιi7v ι- ie reactions T his pathwav has noi been described in mammals w ind ai dependent ■ rι diet loi folate and thereloie loi lurah iofoiaie p duclion 'I his pathway is required for the synthesis of certain aromatic amino acids and aromatic precursors of folic acid and ubiquinone. It is likely that Toxoplasma gondii utilizes the shikimate pathway for synthesis of folic acid, ubiquinone and aromatic amino acids. N-(phosphonomethyl) glycine, an inhibitor of 3-phospho-5- enolpyruvylshikimate (EPSP) synthase and thus an inhibitor of shikimate to chorismate conversion, affects the pathway (Table 1). the ability of this compound to inhibit the growth of T. gondii was examined by the assay described m Example 1.
FIG. 4B demonstrates the ability of NPMG (a specific inhibitor of EPSP-synthase) to restrict the growth of T. gondii in an in vitro assay over a 4 day peπod. T. gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM) on the Y-axis. The X-axis describes how the T. gondii cultures were treated. Cultures that were grown in medium (medium) produced a CPM of around 72,000. If no T. gondii were added to the cultures (no RH), a CPM of around 2,000 was observed. Pyrimethamine (0.1 μg/ml) and sulphadiazine (12.5 μg/ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures. At a dose of 3.12 mM NPMG (3.12) restricted around 60% of CPM and at a dose of 4.5 mM it inhibited parasite growth by around 80%, with counts of about 12,000 CPM. In FIG. 4C the ordinate shows uptake of tritiated uracil mto T. gondii nucleic acids, inhibitory effects of NPMG on nucleic acid synthesis is shown; where PABA at increasing concentrations is added to such cultures, PABA abrogates the inhibitory effects of NPMG on EPSPS synthase restoπng nucleic acid synthesis. vi. Branched Chain Amino Acid Synthesis Imidazolinoncs and sulfonylureas inhibit acetohydroxy acid synthase in Apicomplexan parasites vii. Starch (nniylopcctin) Synthesis and Degradation UDP glucose starch glycosyl transferase is inhibited by substrate competition in
Apicomplexan parasites. viii. Transit Sequences
Antisense, ribozymes, catalytic antibodies, (Pace et al., 1992; Cate et al., 1996, Charbonnier 1997, Askari et al., 1996) conjugation with toxic compounds allow targeting of parasite molecules using transit sequences
Identification of transit sequences in Apicomplexans provides many means for disruption of metabolic pathways. Antisense or ribozymes prevent the production of the transit peptide and associated protein Alternatively production of transit peptide sequences, and the conjugation to toxic molecules, allow disruption of organellar function Catalyiic antibodies also are designed to destroy the transit sequence These antisense compounds or ribozymes or toxic molecules targeted to transit sequences with intracellular antibodies are used as medicines to inhibit the parasite Example 4: Plant-like Pathways and Enzymes in
Parasites Based on the effects of inhibitors of plant-like pathwa s, abrogation of inhibitor effects, and detection of specific enzymes and/or genes, Apicomplexans in general hav e plant-like pa.hwavs Results show n in this example bioaden the obsei v ations ol the presence of plant-like pathways in Apicomplexans beyond the representative parasite T. gondii i. Heme SynthesisGabaculine inhibited the heme synthesis pathway (GSAT) in Apicomplexan parasites (FIGS. IB and IC, T. gondii; FIG. 6, Cryptosporidia) but with modest or no affect on P. falciparum (Table 3, Malaria).
FIG. 6 demonstrates the effect of NPMG, gabaculine, SHAM and 8-hydroxyquinoline and 3-NPA on Cryptosporidia in vitro. C. pan'tim oocysts at 50,000/well were incubated at 37° C (8% C02) on confluent MDBKF5D2 cell monolayers in 96 well microtiter plates with the following concentrations of each drug. The concentrations used were: SHAM (0.2% ETOH was added) 100, 10, 1, 0.1 μg/ml; S-hydroxyquinoline 100, 10. 1. 0.1 μg/ml; NPMG 4.5, 0.45, 0.045 μg/ml; gabaculine 20. 2. 0.2 μg/ml. The level of infection of each well was determined and analyzed by an immunofluorescence assay at 48 hours using an antibody to C parvum sporozoites made in rabbits at a concentration of 0.1%. Fluorescein-conjugated goat anli-rabbit antibody was used at a concentration of 1%. 95% CI count was the mean parasite count per field when 16 fields counted at lOx magnification ± s.d. of the mean The approximate 95% CI counts were as follows' media and ethanol - 1200; paromomycin (PRM) and ethanol - 100. SHAM 100 μg/ml - 400, SHAM 10 μg/ml -- I 100. SHAM 1 μg/ml - 1 100. SHAM 0 I pg/ml - 1200. media alone -
I S00 μg/ml. PRM -200, S-OH-quinolinc 100 μg/ml. -300. S-OH-quinoline 10 μg/ml. - 900, S-OH-quinoline 1 μg/ml - 1 100. 8-OH-qu'moline 0 1 μg/ml ~ 1 00. NPMG 4 5 μg ml - 900, NPMG 0 45 μg/ml - 1200, NPMG 0 045 - 1200, gabaculine 20 μg/ml - 200, gabaculine 2 μg/ml -600, and gabaculine 0 2 μg/ml - 1300 Thus each of these compounds are promising lead compounds as antimicrobial agents effective against Ciyplosporidia. ii. Glyoxylate cycle
3-NPA inhibited the glyoxylate cycle (isocitrate lyase) and/or succinate dehydrogenase in Apicomplexan parasites (FIG. 2B, T. gondii) and also inhibited R. falciparum and C. parvum.
To determine whether there is an Apicomplexan glyoxylate cycle, to analyze the sensitivity of T. gondii tachyzoites and bradyzoites to glyoxylate cycle inhibitors and to determine whether Apicomplexan parasites have isocitrate lyase which presents a unique pathway for lipid metabolism that can be targeted with inhibitors, the following methods are suitable
The inhibitor of isocitrate lyase is 3-nitropropionιc acid (concentration ranging from 0 005 to 5 mg/ml in vitro, and 5 to 50 mg/kg day in vivo) Mutants [Yale Stock Center] used for complementation are as follows E. coli strains, DV 21 A01 (aceA which lacks isocitrate lyase) and DV21 A05 (aceB which lacks malate synthase) Plant gene sequences suitable for comparison are those described by Kahn el al. ( 1977), Maloy ei al ( 19S0), and Maloy el al ( 19S2) A biochemical assay for isocitrate lyase activity is the method of Kahn el al ( 1977) The polyclonal antibodies to cotton malate sy nthase ana coiion isociti ate I se w ich hy bi idize to / gondii proteins of approximately 60 kd are used to identify these enzymes in other Apicomplexan parasites.
iii. Alternative Oxidase
SHAM and 8-hydroxyquinoline inhibited the alternative pathway of respiration, i.e., the alternative oxidase in Apicomplexan parasites [FIG. 3, T. gondii; FIG. 6, Cryptosporidia parvum; Table 3, Plasntodium falciparum (clones W2, D6), pyrimethamine resistant or sensitive clones. Because Cryptosporidia appear to lack mitochondria, the plastid is a likely site for the alternative pathway of respiration
Effect of SHAM on wild type malaria in vitro had been described earlier (Fry and Beesley, 19 I ) However, this observation was presented without knowledge that SHAM affected alternative oxidase function iv. Shikimalc/Chorisiuatc
NPMG inhibited the shikimate pathwav m Apicomplexan paiasites (FIG. B. / goιnlιι.
idiα) Presence of a product of the enzymatic reaction in the pathways of the present invention abrogates the effect of the inhibitor on a specific enzyme because the product no longer has to be made by enzyme catalysis of a substrate Thus, addition of the product proves the specificity of the effect of the inhibitor on the enzyme. The addition of PABA abrogates the exogenous effect of NPMG which is an inhibitor of EPSP synthase (FIG. 4B, T. gondii). Because PABA ablates the effect of the inhibitor NPMG on EPSP synthase, the presence of the shikimate pathway in Apicomplexan parasites is demonstrated.Other specific methods to determine whether Apicomplexan parasites have a metabolically active EPSP synthase enzyme involved in conversion of shikimate to chorismate and further characterize this metabolic pathway in T. gondii are as follows
Use of the inhibitor N-(phosphonomethyl) glycine (concentrations of 3 125mM /// vitro and 100 mg/kg/day /'// vivo) The product rescue assays are performed with PABA The mutants for complementation are as follows. E. coli, AroA, E. coli, AioC. and yeast, AR [Yale Stock Center] Plant gene sequences for comparison are outlined by Klee et al. (19S7) A biochemical assay for EPSP synthase activity in cellular lysates is as described by Mousdale and Coggins ( 1985). Other enzymes in this pathway also are characterized (Nichols and Green, 1992). The full length nucleotide sequence of chorismate sy nthase was obtained follow ing restriction digestion and primer-based sequencing of the Tg EST zyllcOS r l clone obtained from the "Toxoplasma EST Piojcct at Washington University" and of I'. falcipai iim EST czap PFD d2 1 clone obtained fi om the malaria ESI project I) Chakrabarti, Florida The 'oxoplasmu gondii sequence has substantial hom*ology wiih tomato and sev eral othci chorismate synthases and a region of the T. gondii protein has 30% identity and 45% hom*ology with the transit sequence of Zea mays (sweet corn). Other inhibitors of EPSP synthase are Inhibitors 4 and 5, sulfosate (Marzabadi et al., 1996). Other inhibitors of enzymes in this pathway also have been developed by others and provide a paradigm for the rational synthesis of competitive substrate inhibitors of Apicomplexan parasites. v. Branched Chain Amino Acid and Other Essential
Amino Acid Synthesis
Acetohydroxy acid synthase is an enzyme present in plants but not animals and is inhibited by imindazolinones and sulfonylureas in Apicomplexan parasites. Inhibitors of histidine synthesis restrict growth of Apicomplexan parasites. vi. Starch (Amylose/Aniylopcctiu) Synthesis and Degradation
UDP glucose starch glycosyl transferase, starch synthetase and Q (branching) enzymes are inhibited by substrate competitors in Apicomplexan parasites. vii. Lipid Synthesis
The plant-like acetyl coA decarboxylase is inhibited by a number of inhibitors shown in Table I B. Linoleic acid and linoleneic acid synthases are inhibited by newly designed competitive substrates. viii. Auxins and GibcrcIIins The know n auxin mimics and Gibercllin synthesis and Gibcrellin inhibitors inhibit Apicomplexan parasites' growth iv. Glutamiiic/Gliitamate Synthesis
Glufosinate inhibits Apicomplexan glutamine/glutamate. synthesis because the critical enzyme is plant-like. λ. Transit Sequence The transit sequence is conjugated with toxic molecules such as ricins and used to disrupt plastid function in Apicomplexans. Other strategies, such as antisense, ribozymes or the use of catalytic antibodies prevent translation of DNA to protein or catalyze the destruction of mature protein. This interferes with functioning of the molecule and thus the parasite's growth and survival. Example s: The Combined Effects of lnhibitors of Apicomplexan Parasites The effect of enzymes in pathways "in parallel" are additive and in "series" are more than the additive effect of either inhibitor used alone (i.e., synergistic) FIG. 5 demonstrates the inter-relationship of the shikimate pathway and heme synthesis with the electron transport chain The shikimate pathway produces 3,4-dihydroxybenzoate w hich is conv erted to ubiquinone, an essential component of the electron transport chain Thus. NPMG, an inhibitor of EPSP-synthase, indirectly affects ubiquinone production and, thus, the electron transport chain Similarly, heme is required for the production of cytochromes in the electron transport chain Thus, inhibition of heme production
gabaculine also indirectly affects the conventional electron transport chain This scheme allows syncigistic combinations of drugs Thus, NPMG and sulphadiazine ( a competitive PABA analogue) w hich act at different points of the folate sv iuhesis paihw av are pi cdicted to be sv nei gistic, w hereas the effects of gabaculine and ulpludt/me l competitive PABA analogue) winch act on different pathways, ai e prcdicted to be additive. Similarly, gabaculine and SHAM are a predicted synergistic combination of inhibitors. Table 4 demonstrates the additive inhibitory effect of sulphadiazine and gabaculine on the growth of T. gondii over 4 days in culture. T. gondii growth is measured by their ability to incorporate tritiated uracil and is expressed as counts/minute (CPM). Cultures that were grown in medium (medium) produced a CPM of about 36,000. If no T. gondii were added to the cultures (no RU). a CPM of about 2,000 was observed. Pyrimethamine (0.1 μg/ml) and sulphadiazine (12.5 μg ml) added to cultures resulted in a marked reduction in CPM compared with untreated cultures. The growth of T. gondii was inhibited by about 60% in cultures treated with 5 mM gabaculine (gabaculine). The growth of T. gondii in cultures treated with sulphadiazine (1.56 μg/ml) was reduced by approximately 60%. When this dose of sulphadiazine was used in combination with 5 mM gabaculine, as expected, the combined effect of gabaculine plus sulfadiazine is additive because the pathways are in parallel. In contrast, NPMG and sulfadiazine combine in a synergistic manner. Because heme is needed for conventional mitochondrial respiration, it is expected that if both the heme synthesis and alternative oxidase pathways are present, then 3-amino- 2,3-dihydrobenzoic acid and SHAM will demonstrate synergy. Similarly, ubiquinone or end products of the shikimate pathway are needed for mitochondrial respiration and NPMG plus SHAM therefore demonstrate synergy. Table 4 also shows that, the effects of gabaculine and SHAM arc not synergistic as would be predicted by this simple model The likely reason for ihis is that ALA synthase is present in /'. gondii and prov ides a default pathway for the synthesis of δ-aminole ulinic acid Thus, the effects of gabaculine plus SHAM are not synergistic Cycloguanil which affects the plant-like DHFR-TS of 7
'. gondii (McAuley et al, 1994) also is synergistic with NPMG and other inhibitors of enzymes in the shikimate pathway which provides an improved, novel method to treat this infection. Use of synergistic combinations provide an improved strategy for the development of new medicines for the treatment of disease and eradication of the parasite.
Example 6: Effects of lnhibitors In Vivo
Candidate inhibitors are administered to animals by daily intraperitoneal injection or by addition to the drinking water To inhibit EPSP synthase, //; vivo, NPMG is administered at a dose of lOOmg/kg/day a) Survival Five hundred tachyzoites of the RH strain are administered intraperitoneally to BALB/c mice. Cumulative mortality is followed in groups of mice given inhibitor compared to untreated controls b) Formation of Cysts- C3H/HeJ mice that have been infected perorally with the Me49 strain of T. gondii for 30 days are treated with the inhibitor for 30 days Cyst burden and pathology in the brains of inhibitor-treated and control mice are compared using methods described previously (Roberts, Cruickshank and Alexander, 1995, Brown et al., 1995, McLeod, Cohen, Estes, 1984, McLeod et al., 19SS) Cyst numbers present in a suspension of brain are enumerated, or cyst numbers in formalin fixed paraffin embedded sections are quantitated c) Persistence of Cysts C3H/HeJ mice are infected orally with 100 cysts of T. gondii (Me49 strain). Inhibitors are administered to groups of mice from day 30 post infection to day 50 post infection Cyst burden, mortality and pathology are compared in treated and control mice on days 30 and 50 post infection and in mice that receive antibody to gamma mterferon which leads to recrudescence of disease d) S nemy If marked synergistic effect is demonstrated /// viti o bv sho nm that the siibmhibitor. concentrations used touether exert an effect ureater than the additive effects of each used separately, for any combinations, their effect alone and together in vivo is compared. e). New Assays Which Determine the Effects of Antimicrobial Agents on 7". zondn In Vivo Previously reported assay systems measure protection against death following intraperitoneal infection if an animal is infected with the virulent RH strain of 7! gondii Novel aspects of the assay systems in the present invention are using the Me49 (AIDS repository) strain of T. gondii to determine the effect on brain cyst number following acute peroral infection by an Apicomplexan parasite, the effect on the established number of brain cysts during subacute/chronic infection, and use of the Me49 and RH strains to demonstrate synergy of inhibitors of plant-like pathways of the present invention which are "in series," and a novel system to demonstrate reduction of parasitemia which is quantitated using a competitive PCR technique In this competitive PCR method the 7'. gondii B l gene is amplified by PCR in the presence of a construct which produces a product slightly smaller than the wild type B 1 gene The amount of construct can be quantitated to semiquantitate the amount of the competing wild type gene For example, presence of a greater amount of the wild type gene will result in lesser use of the competitor f) Effect of Antimicrobial Agents on Apicomplexan Parasilcs In 1 'ιvo A demonstration of the effect of lnhibitors of plant-like metabolic pathwa in
o ι_, the Nv nei islic effect of NPMG and low dosage py rimethamine NPMG is an inhibitoi of iniection and piomotes su v al of mice infected w ith the v irulent RI I si tain of T. gondii when utilized in conjunction with a low dose of pyrimethamine, whereas neither low dosage pyrimethamine nor NPMG alone are protective. Sulfadiazine reduced manifestations of infection in vivo. SHAM affects parasitemia and number of brain cysts. FIG. 8 demonstrates the ability of NPMG and pyrimethamine administered in combination to protect mice from an otherwise lethal challenge with the virulent RH strain of T. gondii. Mice were infected intraperitoneally with 500 tachyzoites and left untreated (control) or treated by the addition of pyrimethamine (PYR), NPMG (NPMG) or both pyrimethamine and NPMG (PYR/NPMG) to their drinking water. Percent survival is marked on the Y-axis and days post infection on the X-axis.Untreated mice and those treated with either pyrimethamine or NPMG died between day 7 and 9 post infection. In contrast 66 percent of mice treated with pyrimethamine and NPMG survived until day 9 post infection and 33 percent survived until the conclusion of the treatment (day 30 post infection) After the withdrawal of treatment all of these mice survived until the conclusion of the experiment (day 60 post infection i Example 7: Presence of an Enzvnie in a Specific Life Cvclc Stage Predicts
Efficacy of Inhibitors of the Enzvmc on this Stage of the Parasite The effect of candidate inhibitors on different life cycle stages and their effect on stage conversion is of considerable interest and clinical importance. The bradyzoite form of /'. gondii was studied by electron microscopy and was found to have a plastic lntraparasite lmmunolocalization of the enzymes is also performed Gabaculine trcatec culiuies aie >ιaιncd with antibodies to tachyzoites and biadyzoiles Tachyzoites of t!' -- RH strain are grow n in the peritoneum of ND4 mice for 3 days. Tachyzoites are harvested in saline (0.9%) from the peritoneal cavity of euthanized mice and purified by filtration through a 3μm filter. Bradyzoites are isolated as described herein in the Material and Methods. The tachyzoites are pelleted by centrifugation and the pellet is 5 fixed in 2.5% glutaraldehyde. Cysts and bradyzoites are purified from the brains of C57BL10/ScSn mice as described herein in the Materials and Methods and then fixed in 2.5% glutaraldehyde.
Immunoelectronmicroscopy is as described by Sibley and Krahenbuhl ( 1988) using gold particles of different sizes with antibodies to the enzymes to identify the
10 enzyme localization in different organeUes which are identified morphologically.
Immunoelectronmicroscopy localization is accomplished with Amersham Immunogold kit and cryosectioning using standard techniques in the electronmicroscopy facility at the University of Chicago or at Oxford University, Oxford, England. Extracellular organisms are studied as well as tachyzoites and bradyzoites at intervals after invasion.
15 Morphology of the parasites, their ultrastructure and the localization of the intracellular gold particles conjugated to the antibodies is characterized. Invasion is synchronized by placing tachyzoites and bradyzoites with PS 15 cells at 4°C, then placing cultures at 37°C. Intervals to be studied are before I , 5. and 10 minutes and 4 hours after invasion.
-0 Immunostaining and immunoelectronmicroscopy using an antibody to soybean, or synechocoecus. or barley GSAT indicate whether the enzyme is present or absent in both the tachy zoite and bradyzoite life cycle stages and localizes the enzyme in the parasite a) Immunostaiπing for tachyzoites and bradyzoites Immunostaming of tachyzoites and bradyzoites is evaluated with fluorescent microscopy This is performed on cultures of fibroblasts in Labtech slides infected with tachyzoites (RH strain) or bradyzoites and permeabilized using triton, or saponin or methanol, as described by Weiss et al., 1992, Dubremete and Soete, 1996, Bohne el al (1996) Slides are stained 1, 2, 4, 6, and 8 days post infection with anti-BAG (Weiss et ai, 1992) and anti-SAGl (Mineo et α/ , 1993, McLeod el al , 1991 , Roberts and McLeod, 1996) b) Antibodies
Antibodies to the bradyzoite antigens (Weiss et al., 1992, and Bohne el al , 1993) and monoclonal and polyclonal antibodies to SAG1 (Kasper et al 19S3) as a marker for tachyzoite stage specific antigens are used for immunostaming of parasites to establish stage of the parasite Transgenic parasites with bradyzoite genes with reporter genes are also useful for such studies c) Inhibitors and Stage Switching
The effect of inhibitors of conventional (KCN, Rotenone, Antimycin Λ or Mvxothiazol i respiration and alternative respiration on inhibition of growth of tachvzoites and bradyzoites are compared using standard inhibition experiments in coniunαion v uh immunoslaining technique . Tachyzoites use conventional and alternativ e pa hways ol i expiration whcrea . the bradyzoite stage relies on alternativ e rcspiration Inhibitors of conventional respiration favor tachyzoite to bradyzoite switching whereas inhibitors of alternative respiration inhibit tachyzoite and bradyzoite stages d) Svnergy studies, gabaculine treatment Synergy studies with gabaculine are of particular interest because heme is used in the conventional oxidase pathway. If there is synergy, iron influences stage switching. For alternati e oxidase, immunostaining for bradyzoites and tachyzoite antigens is performed using gabaculine treated and control cultures. This is especially informative concerning whether bradyzoites utilize alternative oxidases exclusively, because gabaculine treatment of cultures would limit use of conventional oxidases and thereby select bradyzoites e) Western Blot Analysis, and ELISAs to determine stage specific expression of enzvmes
Brady zoites and tachyzoites also are compared dr eαly for the relative amounts of alternative oxidase, using northern blot analyses, enzyme assays of parasites, isolation of mRNA and RT-PCR, using a competitor construct as an internal standard, and by Western blotting and ELISAs using antibodies to the enzymes (e g , alternative oxidase) UDP-glucose-starch glycosyl Iransferase. chorismate s nthase, isocitrate lyase, GS Λ also arc studied in a similar manner Example S: Pi obing /1f./co..././(_.v._/. DNA w ilh hom*ologous Plant-liUe Genes oi PoU itiallv l loniolouoiis Genes From Other I'.n avilcs The presence of the gsa genes, alternative oxidase genes, EPSP synthase genes chorismate synthase genes, isocitrate lyase genes, and malate synthase genes are identified by probing, and then sequenced. For example, the cDNA clone of soybean gsa is labeled for chemiluminescent detection (ECL) or 32P detection to identify hom*ologous gsa sequences in T. gondii. Probes are used on a membrane containing the genomic DNA of T. gondii and soybean (positive control). When T. gondii genes are isolated,, they are used to probe other Apicomplexan DNA. Thus, the gsa genes of Cryptosporidia, Etmeria, and Malaria are detected in the same manner as the T. gondii gsa. In addition, DNA probes complementary to Trypanosome alternative oxidase
DNA are used to probe the Apicomplexan DNA. The gene for T. gondii alternative oxidase is identified by screening T. gondii cDNA expression libraries using the 7D3 monoclonal antibody or the tobacco alternative oxidase gene used as a probe and thus detecting the gene expressing the relevant protein This gene is used to detect the alternative oxidase genes of other Apicomplexan parasites by Southern analysis and screening other Apicomplexan cDNA libraries
A nucleotide sequence generated from random sequencing of a 7". gondii tachyzoite cDNA library and placed in the Genbank database was found to encode a protein with hom*ology to tomato chorismate synthase The EST was obtained, clone and the full length sequence of the T. gondii chorismate synthase gene and deduced ammo acid sequences were obtained (FIGS. 9 and 10) This provides evidence foi these plant-like pathways and information useful in picpaπng a probe to isolale and scqucπce this full gene from other Apicomplexan parasites as well This gene was used as a probe and identified a chorismate synthase in Eimeria bovts DNA and Cryptosporidium parvum DNA. A P. falciparum EST has also been cloned and sequenced Probes for gsa (soybean) alternative oxidase (soybean and tobacco), isocitrate lyase (cotton), UDP glucose starch glycosyl transferase (sweet corn), and acetohydroxy acid synthase (sweet corn) also are used to screen for clone, and sequence Apicomplexan genes. Large numbers of T. gondii genes from tachyzoite and bradyzoite cDNA libraries are being sequenced and deposited in Genbank Putative hom*ologous genes encoding plant enzymes are used to compare with these sequences to determine whether they are identified in the libraries and if so to determine whether the enzymes are encoded in the nucleus or plastid
Example 9: Identification of Genes Encoding Enzymes of the Plant-Like Biochemical Pathwavs in Apicomplexan Genes are isolated from a cDNA library by hybridization using specific probes to genes know n to encode enzymes in metabolic pathways of plants (see Example 9) Genes are cloned by complementation from a T. gondii cDNA expression library using a scries of E. coli mutants that lack these enzymes and thus depend on the addition of exogenous additives for their optimal growth Transformed bacteria arc used to isolate and sequence plasmid DNA and from those sequences, probes are generated to delcrmme w hether other Apicomplexans have genes hom*ologous to those in T. gondii I j cDNA libiaries A cDNA library a> constructed by Stratagene fi om inRN \ isola d fi om / grind a tachyzoites of the Mc49 strain of 7 gondii usmg the Uni-ZAP XR cDNA library system. The titer of the amplified library is 1-2 X 10l0/ml. Other cDNA libraries also are utilized.
The phagemids were excised with R408 or VCS-M13 helper phage and transduced into XL 1 -Blue Cells. The plasmid DNA was purified using the Qiagen maxiprep system. Other libraries, e.g., early Me49 bradyzoite. in vivo Me49 bradyzoite, and Me49 tachyzoite libraries also are suitable, as are other tachyzoite and bradyzoite libraries prepared by Stratagene..
2) Screening of library for genes. This is done in a standard manner using monoclonal or polyclonal antibodies or a radiolabeled gene probe. 3) cDNA expression libraries are probed with DNA from the genomes of: a) Toxoplasma gondii; b) Plasmodium malar iae; c) Cryplosporidium parvum: d) Eimeria. The existence of plant-like pathways is confirmed in members of the
Apicomplexa by demonstrating the existence of genes encoding the enzymes required for the pathways Genomic DNA is examined by Southern blot analysis for the presence of ihe sequences encoding enzymes required for specific algal or plant metabolic pathways. Genomic DNA is extracted from Apicomplexan parasites by proteinase K digestion and phenol extraction DNA(5- 10μg) is digested with restriction enzymes, clectrophorcscd through 1% Agarose and transferred to a nylon membrane 1 he ECL (Amersham) random prime system is used fo labeling of DNA probes, hybridization and chemiluminescence detection. Alternatively, the Boehringer Mannheim Random Prime DNA labeling kit is used to label the DNA with j2P with unincorporated nucleotides removed using G-50 Sephadex Spin columns. Hybridization with the "P-labeled probe is carried out in [1M NaCl, 20 mM NaH2 P04 pH 7.0, 1% SDS, 40% formamide, 10% dextran sulfate, 5 mg ml dry milk, 100 μg/ml salmon sperm DNA] at 37°C. Washes are optimized for maximum signal and minimum background. Probes are prepared from T. gondii cDNA clones obtained and characterized as described in Example 9. If lack of overall sequence conservation limits ability to detect hom*ology, highly conserved regions are useful. For example, two highly conserved regions of the gsa gene are useful to generate oligonucleotide probes (Matters et al., 1995).
4) PCR: An alternative approach for identifying genes encoding enzymes of the present inveiuion is by using PCR with primers selected on the basis of hom*ologies already demonstrated between plant protein sequences for the relevant gene. For example, for the gsa gene, polymerase chain reaction technology is used to amplify hom*ologous sequences from a T. gondii cDNA library or T. gondii genomic DNA using primers generated from two highly conserved regions of GSAT. The Neurospora crassa alternative oxidase gene has been isolated using degenerate primers designed from conserved regions in alternative oxidase sequences from plant species (Li et al, 1996) These primers are used to detect and clone the alternative oxidase gene from / gondii Candidate PCR products are cloned using the Invitrogen TA cloning kit 5) Sequencing: DNA from candidate cDNA clones is extracted using the Promega Wizard Miniprep System. Clones of interest are purified in large scale using the Maxiprep Protocol (Qiagen) and are sequenced by modified Sanger method with an automated sequencer (ABI Automated Sequencer) by the University of Chicago Cancer Research Center DNA Sequencing Facility.
6) hom*ology Search: to determine whether there is hom*ology of isolated genes with other genes, e.g. gsas, sequences are compared against those in Genbank using the BLASTN (DNA → DNA) and BLASTX (DNA → Protein) programs.
T. gondii sequence data is available in Genbank. Sequences for plasmodia also are available as are some isolated sequences for the other Apicomplexan parasites. T. gondii sequences are searched for hom*ologies to the known plant genes gsa, glutamyl-tRNA reductase, isocitrate lyase, malate synthase, alternative oxidase, EPSP synthase, and chorismate lyase using the BLASTN (DNA— DNA) and TBLASTN (Protein — » Conceptual Translation of DNA Sequence) programs. The conserved plant gene sequences for the shikimate pathway are those described by Kahn et al. (1977) and Maloy et al. 0980; 1982). Conserved plant gene sequences for comparison of hom*ologies are outlined by Klee et al. ( 19S7). Similar libraries and sequence data for Plasmodia also are compared for hom*ologies in the same manner.
7) Complementation: To isolate T. gondii genes or to demonstrate that a gene encodes a functional enzyme product, plasmids from the cDNA library detailed above, or modified constructs, arc used to complement /-J. coli mutant strains GE1 76 or Gil 1 77 ilieml.) and RP523 (lieintt) from the Vale /•_". coli genetic slock center and SASX41 B (liemA from D. Soil. This strategy has been successful for cloning gsa genes from plants and algae (Avissar and Beale, 1990; Elliott et al., 1990; Grimm, 1990; Sangwan and O'Brian, 1993). The hemA gene encodes glutamate-tRNA reductase, an enzyme important in the C5-pathway for heme synthesis. The liemB gene encodes ALA dehydratase, an enzyme common to both heme biosynthesis pathways that should be common to all organisms and is included as a positive control. Mutant bacteria are made competent to take up DNA with CaClj treatment and are transformed with plasmids from the cDNA library. Briefly, chilled bacteria (O.D. 550nm ~0.4-0.5) are centrifuged to a pellet and resuspended in ice-cold 0.1M CaCb and incubated for 30 minutes on ice. Following further centrifugation, the cells are resuspended in 0.1M CaCb, 15% glycerol and frozen at -80°C in transformation-ready aliquots. 0.2ml ice-thawed competent bacteria are incubated on ice for 30 minutes with approximately 50ng plasmid DNA. Cells are placed at 43°C for 2.5 minutes and cooled on ice for 2 minutes. Following the addition of 0.8ml Luria Broth, cells are incubated at 37°C for 1 hour and 0.1 ml is plated onto M9 minimal media plates. The M9 (Ausubel et al., 19S7) medium contains 0.2% glycerol as the carbon source, 1 M MgSO . 0.1 mM CaCI2, 1 mM IPTG, 0.2 mg/ml Ampicillin, and 40 μg ml threonine. leucine, and thiamine Noπselective medium contains 25 μg/ml δ- aminolevulinic acid (lieni and hemA) or 4 μg/ml hemin (lie B). Alternatively, bacteria can take up DNA by elcctroporalion Chilled bacteria arc prepared by a repetition of cciurifugation and resuspcnsion The cells arc w ashed in an equal volume of cold water, a '/: v olume of cold water, a I 5 .■ v olume of cold 10% glycerol, and finally in a 1/500 volume of cold 10% glycerol and frozen in 0 04 ml aliquots at -80°C Cells arc thawed at room temperature and chilled on ice Cells are mixed with the DNA for 0.5-1 minutes and then pulsed at 25μF and 2.5 KV. The cells are rapidly mixed with SOC medium and grown at 37°C for 1 hour. Cells are plated in the same way as for CaCI2 transformation.
Successful complementation of the E. coli mutants with a T. gondii gene is determined by plating the transformed bacteria onto minimal medium which lacks the supplement required for optimal growth of the E. coli mutant. Growth on the selective medium is compared to growth on nonselective medium, which contains 25 :g/m! δ-aminolevulinic acid (hemL or hemA) or 4 μg/ml hemin (hemB). Clones that complement each E. coli mutant are tested for their ability to complement each of the other mutants. Clones of putative T. gondii gsa and glutamate-tRNA reductase should complement only emL and hemA mutants, respectively. Clones that suppress more than one he mutation are candidates for alternative oxidase gene clones. A cDN A clone containing the entire soybean gsa gene was able to transform the
E. coli hemL mutant from auxotrophic to prototrophic for δ-aminolevulinic acid (ALA) Thus the system for obtaining 7". gondii genes that complement E. coli mutants is available
For the glyoxylatc cycle the mutants used for complementation are as follows DV2 I A0 I laceA which lacks isocitrate lyase) and DV2 I A05 (aceli which lacks malate sv nt asel For the shikimate pathway the mutants for complementation are available and used as follows E. coli, AroA and yeast AR
The same procedures are used for Plasntodium falciparum and Plasntodium wwlesii, Ciyptosportdium and Eimeria complementation When transit sequences lead to production of a protein which does not fold in such a manner that the protein can be expressed in E. coli or yeast constructs that lack these sequences are prepared to use for complementation that lack these sequences Example 10: Analysis of Alternative Oxidases in T. gondii
T. gondii bradyzoites use unique alternative oxidases Alternative oxidases are necessary and sufficient for bradyzoite survival Methods to characterize plant alternative oxidases are as described (Hill, 1976, Kumar and Soil, 1992; Lambers, 1994, Li et al. 1996, Mclntosh, 1994)
For in vin o studies, cell lines that lack functional mitochondria are used These cell lines are used to allow the study of inhibitors effective against the conventional or alternative respiratory pathways within the parasite, but independent from their effects on the host cell mitochondria SHAM, an inhibitor of the alternative respiratory pathway is used at concentrations between 0 25 and 2 μg/ml in vitio, and 200 mg/kg/day orally or parenterally /// vivo alone or in conjunction with other inhibitory compounds Other approaches include complementation of alternative oxidase- deficient /." coli mutants to isolate and sequence the alternati e oxidase gene, immunostaming using antibodies for potentiallv hom*ologous eιi7vιncs, enzymatic ssay and the creation of mutant-knockouts for the alternative oxidase gene and studying stage specific antigens in such knockouts.
1) Cell lines: Two cell lines, a human fibroblast cell line (143 B/206) lacking mitochondrial DNA, and the parental strain (143B) which possess functional mitochondria are used. These cell lines have been demonstrated to support the growth of T. gondii (Tomavo and Boothroyd, 1995).
2) Inhibitor studies: Inhibitor studies are carried out as described herein. SHAM concentrations are 0.25 to 2 mg/ml /'// vitro and 200 mg/kg/day in vivo.
3) Immunostaining for tachvzoite and bradyzoites: Immunostaining is performed on cultures of fibroblasts in Labtech slides infected with tachyzoites (RH strain) as described herein. Slides are stained 1, 2, 4, 6 and 8 days post infection with anti-BAG and antiSAGl .
4) RT-PCR is as performed using the protocol of Hill (Chaudhuri et al., 1996) with degenerate primers based on consensus sequences. The product is cloned, sequenced and hom*ology with known alternative oxidases documents its presence.
5) Complementation and alternative oxidase gene cloning: Complementation is used to demonstrate function and is an alternative approach to isolate the gene. Proper function of the complementation system is demonstrated by- using complementation with a plant alternative oxidase gene Mutants suitable for use are hemL, hemA. liemB The alternativ e oxidase gene, AOX. is cloned from a T. gondii cD A expιe>sion library by complementation of the /:. coli hemL mutant HemL mutants of /.. κolι cannot synthesize heme and arc therefore deficient in respiration This cloning strategy has been successful in isolating AOX genes from Aiahidopsis (Kumar and Soil, 1992) The procedure employed for recovering transformants is identical to that used for cloning the T. gondii gsa gene The distinction between the gsa and AOX genes is that the AOX gene should restore function not only to hemL mutants but also to other hem mutants of E. coli. In addition, respiratory growth of E. coli on the alternative oxidase should be antimycin-insensitive and SHAM-sensitive Clones recovered are tested for complementation of hemL, he B and hemA mutants. Growth is tested for inhibitor sensitivity. Sequences of cDNA clones that provide functional alternative oxidase activity by these tests are compared with known AOX gene sequences (Mclntosh, 1994).
The Escherichia coli strain XL 1 -Blue was prepared for infection with the 7°. gondii phage library according to Stratagene manufacturer's protocol The RH tachyzoite library, in the λ-ZAP vector system was titred, and 10° pfu are added to the XL I -Blue preparation Approximately 6 X 10s plaques are plated on agar onto 150 mm2 petri dishes containing NZY medium, and grown at 42°C for 3 5 or 8 hours, depending upon which screening method is employed If antibodies are used for screening, IPTG-soaked nitrocellulose filters are placed on the plates after the short cubation period and the growth of the plaques is allowed to proceed for an equivalent period of time Filters are blocked in BLOTTO o ernight Screening is carried out under the same conditions which had been optimized during Western blotting with that primary antibody, and the appropriate secondary antibodv If DN \ probes ai e used lor sciccnmg the plaques are gi own foi S limT posi-mfection and placed at 45°C for 2 hours to overnight Nitrocellulose filters are placed on the plates, and all subsequent steps for lysis and fixing of the DNA are as specified in the Stratagene protocol Filters are placed into a pre-hybridization solution containing Denhardts, SSC, SDS, and denatured salmon sperm DNA, as directed in Ausubel et al ( 1987) Blots are hybridized to 32P-labeled probe overnight Low stringency washes, containing 5X SSC and 0 1% SDS are performed twice at room temperature, and high stringency washes with 0.2X SSC and 0 1% SDS are performed at a temperature dependent upon the degree of hom*ology between the probe and the T. gondn DNA.
* _»
6) Assays for the presence of genes Evidence for the presence of the genes which encode the novel enzymes is obtained by demonstrating enzyme activity and/or Western blot analysis of Apicomplexan whole cell lysates and/or polymerase chain reaction and'or probing the genomic DNA of the parasite with the hom*ologous DNA Identification of the genes is accomplished by screening an Apicomplexan cDNA library with the antibody to hom*ologous enzymes from plants or other microorganisms or probes which recognize the genes which encode them and/or complementation of mutant bacteria lacking the enzyme with Apicomplexan DNA
7) Mutant-Knockouts The alternative mitochondrial oxidase pathway is the preferred oxidative pathway for bradv zoites and is likely to be important for their survival The genetic system used to examine the function of the gene via targeted gene knock-outs and allelic replacements e-.<entιallv as described (Donald & Roos
1993 1
is not absolutely required for growth when cv tochi ome o - idase can be activ e and mi ants are recov erable The AOX-null strains may be hypersensitive to GSAT inhibitors, both in vitro and in vivo. The ability of the AOX-null strains to switch stages, both in vitro and in vivo is determined. The AOX- null strains are examined for stage specific antigens. Virulence and ability to form cysts are assessed in vivo in C3H/HeJ mice as described herein. Knockouts with a bradyzoite antigen reporter gene are produced and these constructs and organisms with the genes knocked out are cultured under conditions that would ordinarily yield a bradyzoite phenotype. These are used to determine whether expression of the "knocked out" gene is critical for bradyzoite antigen expression and the bradyzoite phenotype. 8) Similar "knockouts" of EPSP synthase or chorismate synthase are produced.9) Similar procedures are used for other Apicomplexan parasites. For example, a similar genetic system is available for P. falciparum. Example I I: Production. Testing, and Use of Vaccines against Apicomplexa "Knock out" organisms (e.g., lacking GSAT, or alternative oxidase or EPSP- synthase or chorismate synthase or UDP-glucose starch glycosyl transferase) are produced as described herein. The knock-out vaccine strain in some cases is cultivated in tissue culture because components which are deficient are provided by a single product or a plurality of products. DNA constructs and proteins are produced and tested as described herein (see Materials and Methods) using unique genes and sequences ar. assay systems and methods which arc known to those of skill in the an and disclose: herein. Briellv. thev arc used to immunize C3H mice, and tissues of immunized and conlrol mice are subsequently examined for persistence of parasites. These immunized mice and controls are challenged perorally with 100 cysts of Me49 strain or intraperitoneally with 500 RH strain tachyzoites. Effect of immunizations on survival, and tissue parasite burden are determined (McLeod et al., 1988). Parasite burden refers to quantitation of numbers of parasites using PCR for the B 1 T. gondii gene, quantitating numbers of cysts in brain tissue, quantitating numbers of parasites by inoculating serial dilutions of tissues into uninfected mice when the RH strain of T. gondii is utilized and assessing survival of recipient mice as 1 parasite of the RH strain of 7". gondii is lethal. Ability to prevent congenital transmission and to treat congenital infections is also a measure of vaccine efficacy. Vaccines are useful to prevent infections of livestock animals and humans. Standard methods of vaccine development are used when substantial prevention of infection is achieved in murine models. Example 12: Nucleotide and Deduced Amino Acid Sequence of T. gondii Chorismate Svnthasc cDNA
Animals and most protista (e.g. Leisli aniά) rely exclusively on exogenous folates. Previous studies which demonstrate the efficacy of anti-folates for the treatment of toxoplasmosis have implied that T. gondii has the enzymes necessary to synthesize folates For this purpose. T. gondii uses PABA. The biochemical events that lead to P BA production in /'. gondii or any other Apicomplexan have not been previously characterized In algae, plants, certain bacteria and fungi, the shikimate pathway facilitates the conversion of shikimate to chorismate, a three step reaction catalyzed by three enzymes, shikimate kinase, 3-phospho-5-enolpyruvyl shikimate synthase (EPSP synthase) and chorismate synthase. Chorismate is then used as a substrate for the synthesis of PABA. In plants, EPSP-synthase and chorismate synthase are encoded in the nucleus. In plants, algae and bacteria, chorismate is not only an essential substrate for the synthesis of folate, but it is required for the synthesis of ubiquinone and certain aromatic amino acids. The shikimate pathway may occur both inside and outside of the plastid: For example, EPSP synthase exists in two forms in' Euglena, one associated with the plastid of those grown in the light and the other found in the cytosol of those grown in the dark. Apicomplexan parasites utilize the shikimate pathway for folate synthesis. An inhibitor of the EPSP synthase, an essential enzyme in this pathway, restricts the growth of 7". gondii. P. falciparum and C. parvum in vitro. This inhibitor, NPMG, synergizes with pyrimethamine and sulfadiazine to prevent T. gondii multiplication. NPMG also synergizes with pyrimethamine to protect mice against challenge with the virulent RH strain of T. gondii. The sequence of a T. gondii gene that encodes a putative chorismate synthase, that has considerable hom*ology with chorismate synthases from other organisms, provides information useful in developing novel antimicrobial agents.
A partial cD A sequence of approximately 250 bases was identified from the "Toxoplasma EST Project at Washington University." This sequence, when translated. had approximately 30% hom*ology with chorismate synthase from a number of organisms Bolh strands of the corresponding clone were sequenced and found to be 23 12 basc_. in length (FIG 9) Analysis revealed a large open reading frame of 160S base pairs which would encode a 536 amino acid protein. hom*ology was determined by the use of CLUSTAL X, a computer program that provides a new window base user interface to the CLUSTAL W multiple alignment program. (Thompson, 1994). The deduced amino acid sequence has considerable identity (44.5 to 51.4%) with chorismate synthases of diverse species (FIG. 10). The putative T. gondii protein differs from other known chorismate synthases in length. Chorismate synthases from other organisms range in length from 357-432 amino acids. The larger size of the T. gondii protein is due to an internal region that has no counterpart in other known chorismate synthases and is novel. The function of this region remains to be -** determined. The T. gondii chorismate synthase sequence was used in a search with the BLAST program. An EST from a Plasntodium falciparum cDNA library was located that has considerable hom*ology with the T. gondii sequence. Chorismate synthase is also present in Mycobaclerium tuberculosis.
The nucleotide sequence of the cDNA which encodes a putative T. gondii chorismate synthase and the amino acid sequence deduced from it is shown in FIG. 9 The deduced amino acid sequence of putative T. gondii chorismate synthase has substantial hom*ologies with chorismate synthases from diverse organisms including Solatium l\ (.oψersιcum (tomato), Synechocyslis species. Hemopliilus influenza. Saccharomyces cerevisiae, and Neurospora crassa (FIG. 10) The Apicomplexan data base in Genbank was searched for hom*ologies to the
7. gondii chorismate synthase gene A hom*ologous /' falciparum EST ( FIG. 1 1 ) was identified. It was sequenced. This provided additional evidence that at least a component of the shikimate pathway also was present in P. falciparum. Sequencing Method
Characterization of Insert and Design of Sequencing Strategy. Clone TgESTzyl lc05.rl was obtained from the Toxoplasma project at
Washington University and supplied in the Bluescript SK vector as a phage stock. Phagemid DNA was excised by simultaneously infecting XL 1 -Blue cells with the phage stock and VCS-M 13 helper phage. Purified phagemids.were used to infect XL 1 -blue cells. Infected XL 1 -Blue cells were grown in LB media and plasmid DNA purified using Qiagen maxi-prep kits. The cDNA insert was excised using EcoR I and Xho I restriction enzymes and found to be approximately 2.4KB. Initial sequencing of the 5 prime end of the insert's plus strand and its translation, revealed 30% hom*ology with previously described chorismate synthases from other organisms. However, sequencing of the 5 prime end of the minus strand yielded a sequence that when translated had little apparent hom*ology with any known protein. A series oi restriction digestion experiments were performed to establish a restriction map of the insert. Restriction fragments were electrophoresed through a 1% agarose gel and fragments visualized by ethidium bromide staining and ultra-violet illumination. Due to the lack of available restriction enzyme sites within the insert, sequencing with the conventional technique of using sub-cloned overlapping restriction fragments as templates would prove to be laborious and time consuming To circumvent this potential problem and facilitate rapid .sequencing, a strategy was designed thai used both conventional sub-cloned ovcrlapping restriction fragments with standard vector annealing primers and the full length clone with custom designed primers. Thus, sequencing was first carried out by using sub-cloned restriction fragments and the information obtained used to custom design unique sequencing primers. These primers allowed efficient sequencing of the internal regions and the external 3 prime end of each strand The customized primers were:
CUSTOMIZED PRIMERS:
CS1 S'TGTCCAAGATGTTCAGCC.T '
CS2 5 ' AGG CTG ATC ATC TTG GAC A 3 ' CS2 5 ' TCG GGT CTG GTT GAT TTT 3 '
CS4 5 ' GAG AGA GCG TCG TGT TCA T 3 '
CS 5 5 ' ATG AAC ACG ACG CTC TCT C 3 '
CS6 5 ' C AT GTC GAG AAG TTG TTC 3 '
CS7 5'GAACAACTTCTCGAC ATG 3' CS8 5" ACTTGTGCATACGGGGTAC3'
CS9 5'GTACCCCGTATGCACAAGT3'
CS 10 5 ' TG A ATG C AA CTG AAC TGC 3 '
CSII 5 'GC A GTT CAG TTG CAT TCA 3'
CS12 5ΑGCCGTTGGGTGTATAATC3' CS13 5' CTACGGCACCAGCTTCAC3'
C 1 5' CGT CCT TCC TCA AC A CAG TG 3'
CS 15 5 ' GI C, AAG CTG GTC} CCG TAG 3 ' CS16 5' CG CTC TGA TTT GGA AGT G 3' CS 17 5' TCT GCC GCA TTC CAC TAG 3' CS18 5' G.λA GCC AAG CAG TTC AGT T 3'
Sub-cloning
Sub-clones were made from restriction fragments isolated by agarose gel electrophoresis and purified using the Qiaex gel extraction kit Qiagen, Chatsworth CA. Double digestions of the plasmid. with Hinc II and Pst I resulted in 4 fragments of 500, 800, 300 and 4000 base pairs. The 800 bp fragment, corresponding- to the base pairs 800-1600 was ligated into the bluescript KS vector. The 1600-2400 base pair portion of the insert was obtained in a similar manner using Pst I and Xho I restriction enzymes and ligated into the bluescript KS vector. Ligatioπs were performed for 12 hours at 18 degrees centigrade on a PTC 100, programmable thermal cycler, MJ Research Inc Watertown. Massachusetts. Plasmids containing the restriction fragments were used to transform DH5α competent cells. Plasmid DNA was purified using Qiagen maxi-prep kits
Prtmei Sequence Design
Pnme-j were designed based on the sequencing information obtained from restriction e zyme fragments To facilitate sequencing of a region on the same strand and 5 prime t : an already sequenced portion of insert, primers were designed from an ai ea appι
200-300 nucleotides 5 prime into the last obtained sequence For sequencing o:• :e complementary strand, primers were designed lo be the complement and reverse of the same region. Primers were designed to be 18-25 nucleotides in length and have a Tm of 55-60 degrees centigrade. G plus C content was 45-55 percent. Primers were designed to have minimal self annealing and to have a low propensity for primer to primer annealing. Primers with the ability to form stable secondary structures were not designed. These criteria for the design of primers were based on theoretical considerations and results of other experiments which found that primers which had Tms of much less than 55 degrees centigrade failed to work or performed poorly, producing ambiguous sequences of low. quality. Sequencing and Assembly of Sequence Information. All sequencing was performed using a Perkin Elmer automated sequencer. The three purified plasmids containing the entire cDNA or a restriction fragment were used as templates for sequencing reactions with the standard M13 and reverse primers. The sequences obtained were used to design primers which allowed sequencing of the internal regions of the inserts. This process was repeated until both strands of the entire clone were sequenced. Chromatograms were critically edited and controlled for quality using Sequencher software. Edited chromatograms of excellent quality were assembled with the same software package and a consensus sequence obtained. The consensus sequence was analyzed for open reading frames using Macvector software package. Kodak International Biotechnology, Inc., New Haven, CT. Example 13: Transit Sequence of T. gondii Chorismate Svnthasc
hom*ology with other peptides was sought using the Genbank database and the unique sequence in the /'. gondii chorismate synthase (amino acids 284 to 435, Figurc 1 I , There was thirty percent identity and forty-five percent hom*ology, with a number of conserved motifs, between this unique sequence of T. gondii chorismate synthase and the amyloplast/chloroplast transit (translocation) sequence of the Waxy protein (UDP-glucose starch glycosyl transferase) of Zea mays (sweet corn). The 5 same methods whereby the Zea mays transit sequence was analyzed (Klosgen and Well, 1991), i.e , construction of the transit sequence with a reporter protein, immunolocalization of the protein, creation of the construct with deletions or mutations of the transit sequence and subcellular immunolocalization using immunoelectronmicroscopy are useful for proving that this is a transit sequence in the
10 T. gondii chorismate synthase. A useful reporter protein for a chimeric construct is β glucoronidase of E. coli, expressed under the control of the 355 promoter of cauliflower mosaic virus The β glucoronidase alone is expressed, in parallel. The transit peptide chimeric construct is found in the plastid The control β glucoronidase is found in the cytoplasm Another useful reporter system is green fluorescent protein
I 5 (gfp) Antibodies to the chorismate synthase protein are also used to detect the presence of the product of the gene (with the transit sequence) in the plastid and the product of a construct in which the transit sequence is not present in the cytoplasm only This is used to immunolocalize proteins in different life-cycle stages Further mutations a:"d deletions are made which identify the minimal transit sequence using the
I ■ same techniques as described above for the entire peptide Antisense, πbozymc or intracellular antibodies diiected against the transit sequence nucleic acid oi translated protein ai e .. -eful as medicines The amino acid or nucleic acid w hich encodes the transit sequence are the bases for diagnostic reagents and vaccine development. This transit sequence is useful for the construction of ribozyme, antisense nucleic acids, intracellular antibodies which target a key parasite protein, and creation of constructs with accompanying molecules which are lethal to the parasites (Roush, 1997; Mahal et al., 1997). This transit sequence also is useful because it provides a general extension of the concept of transit and targeting sequences in Apicomplexan parasites that enable targeting of other parasite. organeUes in addition to plastids:. The transit sequence.of Zea mays. and. T.. gondii. are. shown in Figure 11. Example 14. Nucleotide and Deduced Amino Acid Sequences of f, falciparum Chorismate Svntliasc EST.
Sequencing of P. falciparum chorismate synthase EST followed the same pattern as described above for sequencing the T. gondii chorismate synthase gene with the following exceptions: There was difficulty in obtaining sequence from the 3' region of the cDNA due to an unstable polyA tail. This made it necessary to do all sequencing approaching from the 5' end using gene walking techniques and subcloning of restriction fragments. The AT richness of P. falciparum genes increased the complexity of design of the customized primers. The customized primers utilized were. PFCS 1 AGC TAT TGG GTG GATC PFCS2 TCC ATG TCC TGG TCT AGG PFCS3 ATA A A ACA CAT TGA CTA TTC CTT C PFCS4 GGG GAT TTT TAT TTT CCA ATT CTT TG PFCS5 TTG ,\Λ CGT TGA ATG ATA AGA C PFCS6 TTT TAG ATC AGC AAT CAA ACC PFCS7 AAC TTT TTA TCT CCA TAC TTT G PFCS8 GAA GGA ATA GTC AAT GTG TTT TTA T PCFS9 GTA TTT TAC CAA GAT TAC CAC CC PFCS 10 CCC CCA ACA CTA TGT CG PFCS 1 1 CAG TGG GCA AAA TAA AGA PFCS 12 CCA GTG GGC AAA ATA A PFCS 13 GGA AGA GAA ACA GCC AC PFCS 14 TGC TGC TGG GGC GTG
The gene and deduced amino acid sequences are in Figure 12. Example 15: Southern Blotting Demonstrates Presence of Chorismate Svntliasc (and bv Inference nil of the Shikimate Pathway) in Apicomplexan Parasites Southern blotting using the T. gondii chorismate synthase gene as a "'2P labeled probe demonstrated hom*ology at moderate stringency (e.g 0 2 x SSC, 0. 1% SDS at 42°C) [more stringent conditions define greatest relatedness of genes] with Emieria bo vis and Cnpio.ψoridiiiin parvum DNA
This 7 gondii cDNA also comprises a probe for screening cDNA libraries of all other Apicomplexa to identify their chorismate synthase genes The same principles are applicable to all the othei enzymes in Table I Example 16: Gene Expression. Rccoiiihinant Protein. Production of Antibody and Solving the T. gondii and P. falciparum Crystal Structures of chorismate svnthasc to establish their active site and secondary structure. These are done using standard techniques. The gene construct is placed within a competent E. coli. Recombinant enzyme is identified by hom*ologous antibody reactivity and purified using affinity chromatography. Fusion proteins are useful for isolation of recombinant protein. Protein, is injected into rabbits and antibody specific to the protein is obtained and utilized to purify larger amounts of native protein for a crystal structure. The crystal structure provides information about enzyme active site and facilitates rational drug design (Craig and Eakin, 1997). Recombinant proteins are used for high through put screens to identify new antimicrobial agents. Example 17: Other Uses (e.g. in diagnostic reagents and vaccines) of the
Chorismate Svnthasc Gene as a Representative Example of Uses of Each of the Genes and Enzvmcs in These Pathways That arc not
Present or Rarelv Present in Animals. These uses include T. gondii genes and proteins used as diagnostic reagents and as a vaccine to protect against congenital infection Recombinant protein (all or part of the enzyme) is produced and is used to elicit monoclonal antibodies in mice and polyclonal antibodies in rabbits These antibodies and recombinant protein (c g . to
T-gondii chorismate svnthasc) are used in ELISA (e g antibody to human IgG or IgM. or IgΛ or Igl; attached to ELISA plate - sei m to be tested - antibody conjugated to enzyme - enzyme substrate) The recombinant proteins, pooled human sera from known unmfected individuals (5 individual sera pooled) and infected individuals (5 individuals with acute infection sera pooled, 5 individuals with chronic infection sera pooled) are the controls This test is useful with serum or serum on filter paper Another example of a diagnostic reagent are primers to amplify the target transit sequence or another portion of the chorismate synthase sequence unique to T. gondii PCR with these primers is used with whole blood to detect presence of the parasite Such assays have proven to be useful using the T. gondii B lgeneJKirisits. Mui, Mack, McLeod, 1996) Another example of a diagnostic reagent is useful in outpatient settings such as an obstetrician's office or in underdeveloped areas of the world where malaria is prevalent FABs of monoclonal antibodies (which agglutinate human red cells when ligated) (Kemp, 19SS) are conjugated to antibodies to the target sequence or selected enzyme Antigen conjugated anti-red cell Fab also is used to detect antibody to the component A positive test occurs when the enzyme or antibody is circulating in the patient blood and is defined by agglutination of red cells (in peripheral blood from the patient) mixed with the conjugated antibodies Controls are the same as those specified for the ELISA
Examples of vaccines are protein, peptides. D\A encoding peptides or proteins These are administered alone or in conjunction with adjuvants Mich as 1SCOMS
1 heie v accine preparations are tested first in mice then primates then in clinical it mis Lndpoints ai e induction of pi ieciiv c immune responses, protection measui ed as enhanced survival, reduced parasite burden, and absent or substantial reduction in incidence of congenital infection (McLeod et al., 1988).
Example 18: T. gondii Chorismate Synthase Genomic Sequence
Genomic clones are isolated from commercially available genomic libraries (AIDS repository) using the identified cDNA clones as probes in the screening process.
The genomic library, as λ phage, is isolated onto NZY agar plates using XL 1 -Blue E. coli as the host, resulting in plaques following a 37°C incubation. The cDNA sequence is radiolabeled with 32P and hybridized to nylon membranes to which DNA from the plaques has been covalentiy bound. Plasmids from candidates are excised and their restriction enzyme-digested inserts sequenced. Experimental details are as described in
Ausubel et al. (1987).
Example 19: P. falciparum Chorismate Svnthase Genomic Sequence. This is done with a gene specific subgenomic library as described in
Example 18 (see example 41). Other examples of enzymes and the genes which encode them and which are characterized as outlined above include: glutamyl-tRNA-synthetase; glutamyl-tRNA reductase; prephenate dehydrogenase aromatic acid aminotransferase (aromatic transaminase); cyclohexadienyl dehydrogenase tryptophan svnthase alpha subunit; tryptophan synthase beta subunit; tryptophan synthase beta subunit; indole-3 -glycerol phosphate synthase (anthranilateisomerase), (indoleglycerol phosphate synthase), anthranilate posphoribosyltransferase; anthranilate synthase component I; phosphobiosyl anthranilate isomerase anthranilate synthase component II; prephenate dehdryatase (phenol 2-monooxygcnase) catechol 1,2-deoxygenase (phenol hydroxylase); cyclohexadienyl dehydratase; 4-hydroxybenzoate octaprenyltransferase; 3-oxtaprenyl- 4-hydroxybenzoate carboxylyase dehydroquinate synthase (5-dehydroquinate hydrolase); chorismate synthase (5-enolpyruvylshikimate 3-phosphate phosph-lyase); dehydroquinate dehydratase; shikimate dehydrogenase; 3-deoxy-d-arabino- heptuloonate 7 phosphate synthase; chorismate mutase (7-phospho-2-dehydro-3- deoxy-arabiπo-heptulate aldolase); 3-deoxy-d-arabino-heptuloonate.7 phosphate synthase; shikimate 3-phosphotransferase (shikimate kinase); UDP glucose starch , glycosyl transferase; Q enzymes; acetohydroxy acid synthase; chorismate synthase malate synthase, isocitrate lyase; 3-enolpyruvylshikimate phosphate synthase (3- phosphoshikimate- 1 carboxyvinyltransferase).
Example 20: T. gondii Chorismate Svnthasc. EPSP Svnthase. and Shikimate Kinase Activities were Demonstrated Assay for chorismate synthase, EPSP synthase and shikimate kinase in T. gondii were performed and demonstrated such activity.
Example 21 : T. gondii Dehydroquinate Dehydratase Activity is Demonstrated
An assay for dehydroquinate dehydratase in T. gondii was performed and demonstrated such activity. Example 22: GSAT activity is demonstrated in T. gondii taclivzoitc Ivsntcs An er-.zymatic assay (Sangwan and O'Brian, 1993) demonstrates GSAT activity in /. gondii lysates. The buffer contains 0. 1 M MOPS (3-f N- inorpholinoj rop.inesulfonic acid), pH 6 S. 0.3M glycerol. 1 5 mM MgCI>, I mM dithiothreitol, 20 μM pyridoxal phosphate, 1 mM PMSF (phenylmethylsulfonyl fluoride) The MOPS, glycerol and MgCli are combined and then pH'd. This is important because the glycerol alters the pH, so it must be added first. This is filter sterilized and has a long shelf life. When the buffer is needed, DTT, pyridoxal phosphate and PMSF are added immediately prior to use. The protein extract stock should be -10 mg/ml if possible. The principle of the assay is conversion of substrate which produces a. change in color due to the reactant.
Example 23:. Isocitrate Ivase activity is demonstrated in T. gondii tachvzoite Ivsates An enzymatic assay demonstrates isocitrate lyase activity in 7". gondii isolates prepared by disruption of the parasite membranes using french press or a lysis buffer. Demonstration that the lysis buffer does not alter enzyme activity is carried out by performing the assay with known substrate and enzyme in the lysis buffer and documenting presence of enzyme activity. Example 24: Alternative oxidase activity is demonstrated in T. gondii preparations. 7". gondii tachyzoites and bradyzoites are assayed for alternative oxidase activity and such activity is found to be present in greater amounts in bradyzoites Example 25: Novel Substrate Competitors and Transition State Analogues of
Some inhibitors are competitiv substrates or transition state analogues and they aie utilized in the enzyme assay, in vitro with tachyzoite and bradyzoite preparations and w ith native enzyme, tissues culture assays and in in vivo models as described above These provide a model paradigm for designing inhibitors of any of the enzymes specified above. Briefly, inhibitors are produced as follows: Competitive substrates are produced by designing and synthesizing compounds similar to known compounds but modified very slightly. For example, inhibitors related to glyphosate are known. The structures of glyphosate, sulfosate and the precursor for EPSP have similarities (please see below). Inhibitors are designed by modifying substrates in such a manner that the modification interferes with the enzyme active site. This can be performed using molecular modeling software. Similarly, halogenated substrates for other enzymes have functioned effectively as nontoxic inhibitors. The principles are applicable to the design of inhibitors for any of the unique enzymes with well characterized substrates and active sites.
The approaches to rational design of inhibitors include those standard in the an (Craig and Eakin, 1997; Ott et al., 1996) These methods use information about substrate preference and three-dimensional structure of the target enzyme (e g , chorismate synthase or EPSP synthase)
In one approach, the structure of the target is modeled using the three- dimensional coordinates for amino acids in a related enzyme An example of this is that the crystal structure of GSAT from a plant has been sol ed and its active site is known
In anoihei part of this appioach expicssion of high lev els of recombinani enzyme is piυduced using cDNA (e g . the chonsmate sv hase of 7' gondii oi /' falciparum) and quantities of protein adequate for structural analysis, via either NMR or X-ray crystallography are obtained.
Drug resistant mutants are produced in vitro following mutation with nitrosoguanidine and culture with the inhibitor. The surviving organisms have acquired resistance to the inhibitor. This process is carried out either with the Apicomplexan parasite or with bacteria or yeast complemented with the gene encoding the enzyme or part of the gene (e.g., without the transit sequence). PCR amplifies the relevant cDNA and this cDNA encoding the resistant enzyme is cloned and sequenced. The sequence is compared'with that of the enzyme that is not resistant. With the information about the inhibitor target and three-dimensional structure, the point mutations which cause resistance are analyzed with computer graphic display. This information provides the mechanism for altered binding of the drug, and the inhibitory compound is then modified to produce second generation medicines designed to treat resistant pathogens prior to their development in nature. An example of the use of toxic analogues to kill parasites used by others provides a means whereby there is production of analogues toxic to parasites. Specifically, the purine analogue prodrugs, 6 sulfanyipurinol. 6 thioguanine, 6 thioxanthine and allopurinol interact with hypoxanthine phosphoribosyltransferase which is responsible for salvage of purines used to produce .AMP and GMP. Such toxic analogues are effective against the plant-like enzymes i . the pathways (see Table 1 ) in Apicomplexans Transit state analogues bind with extraordinarily high efficiency to the enzyme active site and are predicted from the three-dimensional structure and kinetic information Analogues that mimic the structural properties and electrostatic surface potentials for the transition state arc designed and synthesized Empirical testing usmg recombinant enzyme demonstrates that these transition state analogues are good leads with high affinity for the active site of the target enzyme
Multisubstrate analogues are useful because they markedly enhance the binding affinity to the enzyme Similarly, if enzymes in a cascade are linked in such a manner that the substrate for one reaction provides the substrate for the next reaction, multisubstrate analogues are more useful
Selective inhibitor design and lead refinement: Co-crystal zation of inhibitors with target enzymes of host and pathogen enable three-dimensional analysis of molecular constructs and atomic interactions between inhibitors and enzymes and redesign of lnhibitors (leads) to enhance their affinity for the pathogen enzyme Iterative crystallography, lead redesign and inhibitor testing /// vili o and in vn o enable design and development of potent selective inhibitors of the target of the pathogen enzyme Recombinant methods for screening large numbers of analogues for those that bind selectiv elv to the enzy mes of specific parasites prov ide justification for inclusion of the analogues which bind best in the design of transition-state or multisubstrate analogues
\ddιtιoιιal examples (included to illustrate principles emplov ed) but aheadv patented bv olhei s n lude Inhibitoi of l.l'SP sv iitli.isc Iπve been designed based on the similarities of the inhibitor to the substrate Based on molecular modeling algorithms additional inhibitors are designed. Phosphoenolpyruvate =
0 o
« / glyphosate = HO-C-CH, N-CH2-P-OH
I \
H OH
Inhibitors that effect components of these pathways are halogenated substrates or analogues which are effective competitors.
Inhibitors of Ubiquinone: Modifications (substitutions) of benzhydroxamic acids produce CoQ (ubiquinone) analogues such as esters of 2, 3 and 3,4 dihydroxybenzoic acid and structurally related compounds
Inhibitor of Isoleucinc/valinc biosynthctic pat .. ay: These are noncompetitive inhibitors as is shown by the lack of relatedness of the inhibitors (e.g., imidazolinones, sulfonylureas) to ihe target enzymes
Inhibitors of GSAT
The follow ing acids (5 amino- 1.3 cyclohcndienyl carboxylic acid, 4 amino 5 hcxynoic acid ( aceiylenic, GAB A), 4 amino 5 hcxonoic acid ( vinyl GAB A) 2 ammo butanoic acid ( v l glycine), 2 amino methoxy-trans-3 butenoic acid. amino 5 lluoiopenlanoic _. id allei catalysis dependent formation of a stable cov lent adduct Inhibitors of ly sine biosynthctic pathway: There are noncompetetive inhibitors of lysine synthesis that target enzymes in this patway (e g , azi DAP, 3, 4 didehydro DAP, 4 methylene DAP4, 4 methylene DAP6) and inhibitors of other plant-like enzymes as in the Table 1 A and B
Example 26: Modifications of Inhibitory Compounds to Improve Oral Absorption Tissue Distribution (especially to brain and eve'). Tissue distribution is characterized using radiolabeled inhibitor administered to mice with its disposition to tissues measured by quantitation of radtolabel in tissues Compounds are modified to improve oral absorption and tissue distribution by standard methods
Example 27. Efficacy of Antimicrobial Compounds Alone. Together and In Conjoii't Infections in Murine Models Inhibitors of plant-like pathways are effective against the Apicomplexan infection alone, together with the bacterial and/or fungal infections and also treat the bacterial and fungal infections alone
Presence of inhibitory activitv of new antimicrobial compounds is tested using Apicomplexans, bacteria and fungi in enz matic assav s //; \ ιtιo, and in vn o assays as described above and known to those of skill in the art Infections arc established in murine models and the influence of an inhibitor or combination ot inhibitoi s on outcomes are determined as follow Infections Infections with Toxoplasma gondii, Pneumocystis carinii, Mycobactertum tuberculosis, Mycobactertum aviiim intracellular and Cryptosporidtum parvum are established alone and together using an immunosuppressed rodent model. Endpoints in these infections are: Survival. Ability of an inhibitor to protect the infected animal is measured as prolonged survival relative to the survival of untreated animals. Parasitemia: Is a measure using isolation of mRNA and RT-PCR. A competitive inhibitor is used for quantitation.
Tissue Parasite Burden: Is determined by quantitating brain and. eye cyst numbers.
Inflammatory Response: This is noted in histopathologic preparations. Representative combinations of inhibitors are NPMG and sulfadiazine, SHAM and atovaquone, NPMG and pyrimethamine, NPMG and SHAM. Example 28. Establishing Efficacy. Safety. Pharniakokinetics. and Therapeutic/Toxic Index: The testing in murine models includes standard Thompson tests Testing of antimicrobial agents for efficacy and safety in primate models for malaria is performed Dosages are selected based on safety information available from data bases of information concerning herbicides and the literature Measurements of serum and tissue levels of antimicrobial compounds are performed using assays which detect inhibitor concentrations and concentrations of then metabolites Representativ e assa . ai e high pei formance liquid chiomaiography and assaying tissues for pei ccniage of radiolabeled compounds administered, using liquid scintillation, and other assays also are used.
Example 29. Determining whether there is Carcinogcnicitv and Teratogenicitv: Standard assays to evaluate carcinogenicity and teratogenicity include administration of medicines as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity (i.e. development of malignancies).
Observation includes general physical examination, autopsy and histopathologic studies which detect any teratogenic or carcinogenic effects of medicines.
Example 30. Constructs to Measure Parasitemia: Portions of genes are deleted and the shorter gene is used as an internal standard in RT PCR assays to measure amount of parasites present (Kirisits, Mui,
McLeod, 1996).
Example 31. Vaccine Constructs and Proteins and their Administration: These are prepared, as described. They include DNA constructs (Ulmer, Donnelly and Liu, 1996) with the appropriate gene or portions of the gene alone or together, with adjuvants. Representative adjuvants include ISCOMS, nonionicsurfactant, vesicles, cytokine genes in the constructs and other commonly used adjuvants Native and recombinant proteins also are used in studies of vaccines
Protection is measured using iminunologic in vitro assays, and assessing enhanced survival, reduction of parasitemia tissue and parasite burden and prevention of congenital infection [McLeod et al . I9S8] I l l -
Example 32: Stage-Specific Expression of Proteins
This is evaluated by enzyme assays, northern or western analysis, ELISA, semi-quantitation of mRNA using RT-PCR with a competitor as internal standard in gene-knockout organisms using culture conditions (e.g alkaline pH, increased temperature, nitric oxide exposure) which ordinarily elicit a bradyzoite phenotype, or engineering a reporter construct and characterizing presence of the reporter in stage specific expression of antigens. Ability to change between life cycle stages or to persist in a particular life cycle stage is affected by presence or absence of particular plant-like genes and by treatment of inhibitors with plant-like processes. Suitable examples of plant-like enzymes which make parasites less able to switch from or persist in a specific life cycle stage include: alternative oxidase, enzymes critical for amylopectin synthesis such as starch synthases, UDP glucose-glucosyl starch transferase and branching (Q) enzymes Exa ple 33. Preparation of Diagnostic Test Reagents and Diagnostic Tests: These assays are as described (Boyer and McLeod, 1996). Sensitivity and specificity are established as is standard in the field. Tests and reagents include ELISAs in which antibodies to the proteins or peptides and recombinant proteins of this in ention such as chorismate synthase {Awe) are used and PCR methodology in which primers to amplify DNA which encodes the enzymes, or parts of this DNA, are used A test useful in an outpatient setting is based on conjugation of a monoclonal a iibodv to human red blood cells with antibody to plant-like peptides or proteins based on an assav described by Kemp et al (Kemp et al , 198S) The red cells are cross linked ia the monoclonal antibody moietv, resulting in agglutination of the red blood cells in the blood sample if the antigen or antibody to the parasite component is present in the blood sample ELISA and PCR can be utihzed with samples collected on filter paper as is standard in Newborn Screening Programs and also facilitates outpatient and field use
Example 34. Development and use of Antisense Oligonucleotides in Design and Use of Medicines to Protect Against Apicomplexans: Antisense ohgonucleotides directed against the nucleic acids which encode the enzymes of the essential parasite metabolic process described herein are effective medicines to treat these infections Antisense oligonucleotides also are directed against transit sequences in the genes Antisense ohgonucleotides are short synthetic stretches of DNA and RNA designed to block the action of the specific genes described abo e for example chorismate svnthase of T gondii or P fa/cipai um, by binding to their RNA transcript They turn oft the genes by binding to stretches of their messenger RNA so that there is breakdown of the mRN A and no translation into protein When possible, antisense do not contain cytosine nucleotides Antisense reagents hav e been found to be active against neoplasms, inflammatory disease of the bowel (Crohn s Disease) and HIV in earlv trials Antisense w ill not contain cvtosme nucleotides followed by guanmes as this generates extreme in mune responses (Roush 1997) Antisense ohgonucleotides with sequence for thymid kinase also is used for regulatable ge le therapy Example 35
Ribozymes are RNA enzymes (Mack, McLeod, 1996) and they and toxic compounds such as πcins (Mahal et al. 1997) are conjugated to antisense oligonucleotides, or intracellular antibodies, and these constructs destroy the enzyme or other molecules Example 36. Intracellular Antibodies to Target Essential Enzymes Proteins and Organdies: Intracellular antibodies are the Fab portions of monoclonal antibodies directed against the enzymes of this invention or portions of them (e.g , anti-transit sequence antibodies) which can be delivered either as proteins or as DNA constructs, a's described under vaccines.Example 37. Development of New Antimicrobial Compounds Based on Lead Compounds: The herbicide inhibitors comprise lead compounds and are modified as is standard Examples are where side chain modifications or substitutions of groups are made to make more active inhibitors (Table 1) Their mode of action and structure as well as the enzyme and substrate structures are useful in designing related compounds which better abrogate the function of the enzymes Examples of such substrate or active site targeting are listed in Table I
Native or recombinant protein used in enzvmatic assays and in vitio assays described abo e are used to test activ uv of the designed newly synthesized compounds Subsequently, they are tested m animals Exainplc 3S. Trials to Demonstrate Efficacv of Novel Antimicrobial Agents for Human Disease:
Trials to demonstrate efficacy for human disease are performed when ... vttio and murine and primate studies indicate highly likely efficacy and safety They are standard Phase I (Safety), Phase II (small efficacy) and Phase III (larger efficacy with outcomes data) trials For medicines effective against T. gondii tachyzoites, resolution of intracerebral Toxoplasma brain lesions in individuals with HIV infection with no other therapeutic options available due to major intolerance to available medicines is the initial strategy for Phase II trials Endpoints for trials of medications effective against 7. gondii bradyzoites include absence of development of toxoplasmic encephalitis in individuals with HIV HIV infected patients who also are seropositive for T. gondii infection are evaluated Evaluation is following a one-month treatment with the nov el anti 7 gondii medicines Observation is during a subsequent 2 year period when the patients peripheral blood CD4 counts are low Effective medicines demonstrate efficacy measured as absence of T gondii encephalitis in all patients Otherwise, 50% of such individuals develop toxoplasmic encephalitis When medications efficacious against bradyzoites and recrudescent toxoplasmic encephalitis in patients w ith AIDS are discov ered and found to be safe, similar trials of efficacy and safety for indi iduals with recurrent toxoplasmic choπoretinuis are performed All such trials are pei tormed with informed consent consistent with Institutional NIH and Helsinki guidelines applicable to treatment trials inv ol ing humans Example 39. Vaccine Trials for Humans
After vaccine efficacy in rodent models to prevent congenital and latent Toxoplasma infection are established, for component vaccines only, trials to establish safety and efficacy in prevention of congenital and latent infection are performed. They follow standard procedures for phase I, II and III trials as outlined above and as is standard for vaccine development.
Endpoints for vaccine effect and efficacy are development of antibody and cell- mediated immunity to T. gondii (effect) and most importantly, prevention of T. gondii congenital infections. After establishing in phase I trials that the vaccine is entirely safe, nonpregnant women of childbearing age will be vaccinated with recombinant vaccine. Assay for efficacy is via a serologic screening program to detect newborn congenital toxoplasmosis (described in Boyer and McLeod, 1996) with usual testing to document whether seropositive infants are infected (described in Boyer and McLeod, 1996) Example 40. Vaccine Efficacy and Safety for Livestock Animals
The efficacy of candidate vaccines is tested in sheep as previously described (Buxton el al.. 1993) Vaccines are live attenuated, genetic constructs or recombinant protein The most cflicious routes and frequency of inoculation is assessed in a serious of experiments as described below Intra-muscular, sub-cutaneous and oral are the preferred routes, although intravenous intraperitoneal and intradcrmal routes may also be used Scottish blackface or/and swaledale ewes foui to
years old are tested for luG antibodies to I oxoplusnta gondii using an ELI SA assay Only scro-negative animals are used for the study Three groups of 10-15 ewes are used for each experiment. Groups 1 are vaccinated, while group 2 and 3 are not Three months later all ewes are synchronized for estrous and mated. At 90 days gestation the ewes in groups 1 and 2 are given 2000 sporulated oocyst of T. gondii. The outcome of pregnancy is monitored in all groups Aborted lambs or those dying soon after birth are examined histologically and by PCR for the B 1 gene or sub- inoculation into mice or tissue culture, for the presence of T. gondii. All placentas are examined histologically and as above for parasites. Lambs are weighed at birth, Pre- colostral serum is taken from each lamb Congenital transmission is assessed by performing ELISA assays on the serum for IgG or IgM. Protection is measured as a decrease in congenital transmission, a decrease in the incidence or severity of congenital disease, or a decrease in abortion■ U 7-
E.xamp_e l : T. yff.tttfrChorismate Svnthase Genomic Se uenrp Used to Produce "Knockouts" (Attenuated Vaccine
Strain).
The genomic sequence of chorismate synthase is in FIG. 13. As with other genomic sequences herein, it provides an example of a gene which is "knocked out" to produce an attenuated vaccine and also can be utilized as described in other parts of this document.
A chorismate synthase knock out parasite was produced as follows: The genomic T. gondii chorismate synthase sequence consists of 9 exons. To prepare the knockout construct, this sequence was digested with EcoN I to remove a 1.8 kb fragment that included exons 2, 3, and 4. The EcoNl digested ends were blunt ended followed by dephosphorylation. A 1.9 kb piece bearing HXGPRT flanked by the 5' promoter region and 3' untranslated region of dhfr (called dhfr HXGPRT) was isolated by digestion of a construct, obtained from J. Boothroyd, and Xbal and hoi.
After blunt ending, the 1.9 kb fragment was cloned into the chorismate synthase construct so that dhfr HXGPRT replaced chorismate synthase exons 2, 3 and 4. This construct was used for knockout of the wild type chorismate synthase gene. The sequence of the construct was verified by PCR. Following transfection into T. gondii (deficient in HXGPRT) and selection in medium containing 25 μg/ml mycophenolic acid and 50 μg/ml Xanthiπe, successful transfection was confirmed by PCR of the chorismate synthase/dhfr HXGPRT junction and sequence the product. Parasites were cloned by limiting dilution and clones were cultured in the presence or absence of folate and other aromatic products in this medium with replica cultures. Aromatic compound deficient medium with 10% AlbuMax® as a serum substitute was prepared. Final concentrations of aromatic compounds in the supplemented medium are 0.1M phenylalanine, tyrosine, tryptophan, PABA, 2,3 dihydroxybenzoate and p- hydoxybenzoate. DNA was extracted from those replicate cultures of parasite clones that grew only in the presence of aromatic compound
RECTIFIED SHEET (RULE 91) supplementation. PCR primers were designed to confirm presence of the knockout construct and demonstrated that hom*ologous recombination occurred resulting in replacement of exons 2-4 with the dhfr HXGPRT sequence. The knockout parasite was passaged in aromatic compound supplemented medium. Whether this selection clearly demonstrates inability of the knockout parasite to grow in aromatic compound deficient medium, but ability to grow in aromatic compound sufficient medium using a uracil assay. Such aro deficient strains of bacteria have been used as vaccines precisely because they are nonpersistent. Complementation with aroC in an episomal vector to prove that the phenotype of the chorismate synthase knockout organisms is due to deletion of the chorismate synthase gene, was also done. This complementation system also allows characterization of the effects of mutations in chorismate synthase or its promoter region on transcription or on enzyme function, importance of the pathway for parasite viability, stage switch and subcellular localization. An episomal vector was obtained from John Boothroyd. Chorismate synthase was cloned within this plasmid under control of a constitutive promoter (e.g., the promoter for tubulin or DHFR). The resulting construct was transfected intc the chorismate synthase knockout parasite described above. Proof that the construct produces mRNA for chorismate synthase is with northern and western blotting. The lack of ability of the knockout and the ability of the complemented parasite to grow in folate and other aromatic compound deficient medium indicates a functional construct. This knockout organism is suitable for use as an attenuated vaccine strain.
Example 42: T. eondii Chorismate Svnthase cDNA Sequence in a
DNA Vaccine Vector Elicits Antibodies. T. gondii chorismate synthase cDNA sequence placed in a DNA vaccine vector with a CMN promoter (Vical, San Diego) and administered intramuscularly to mice elicits serum antibodies to chorismate synthase (FIG. 14 A and B). Antibody production is detected on Western blot and in other immunoassay systems. This is an example of
RECTIFIED SHEET (RULE 91) a recombinant vaccine and a system to produce antibody reagents useful in diagnostic tests without the need to produce recombinant protein.
Example 43: T. gondii Chorismate Svnthase-green Fluorescent
Protein Construct is Made and Used in Parasite Survival Assavs to Test Antimicrobial Agents.
A T. gondii chorismate synthase-green fluorescent protein DNA construct elicits a fusion (reporter) protein detectable with conventional immunofluorescence microscopy and deconvolution microscopy (FIG. 15) and other techniques known in the art to detect fluorescence. This construct accelerates the growth rate of the parasite and is useful for measuring effects of antimicrobial agents on the parasite by detecting relative amounts of the green fluorescent reporter protein. This is useful for testing antimicrobial agents.
Example 44: Chorismate Svnthase and Life Cvcle. Chorismate synthase is differentially located and expressed in different life cycle stages indicating that it can be an antimicrobial agent target in, and reagent to detect, specific stages of the parasite.
Immunostaining This is performed as is standard in the art with tachyzoites, converting organisms, intestinal life cycle stages using specimens produced in vivo and in vitro.
In some tachyzoites, chorismate synthase was concentrated in a small area contiguous to the nucleus in the area of the plastid (FIG. 16A).
In other life cycle stages it was distributed diffusely throughout the cytoplasm (FIG. 16B, C). It was most abundant in bradyzoites and macrogametes. A C-terminal green fluorescent protein reporter alters its localization in tachyzoites (FIG. 15). Unique stage-associated expression and subcellular localization of T. gondii chorismate synthase is identified in tachyzoites, bradyzoites and in the stages of the parasite in the cat intestine including macrogametes, microgametes but not schizonts. Stage-associated expression of T. gondii chorismate synthase (FIG.
16A-C) is an example of the expression and differential subcellular localization of this protein. This stage-associated expression demonstrates
RECTIFIED SHEET (RULE 91) that this protein is present in tachyzoites (A), bradyzoites (B) and microgametes (C) and macrogametes (C). This is an antimicrobial agent target, useful diagnostic reagent and vaccine constituent for infections with these life cycle stages. The differential stage associated subcellular localization demonstrates that organeUe targeting is another way to target these enzymes.
Example 45: Recombinant Chorismate Svnthase is Useful for
Antibody Production and in Enzv e Assavs for Hioh
Throughput Screens. Recombinant chorismate synthase was produced and is useful for high throughput screens, development of diagnostic reagents and a vaccine.
Overexpression of Chorismate Svnthase Chorismate synthase was expressed in E. coli using a pGEX expression system (Pharmacia). Briefly, PCR was used to amplify the coding region and to introduce
BamHI and EcoRl sites to the 5' and 3' ends respectively. Following removal of the 3'adenosine overhangs, the PCR product was first cloned into pUC18 using the Sureclone Ligation Kit (Pharmacia Biotech, Herts,
UK). The pUC18 plasmid containing the insert was digested with EcoRl and BamHI and following purification by electrophoresis, the insert was eluted from an agarose gel and then cloned into pGEX-2T. DNA sequencing confirmed that the nucleotide sequence was in frame and that no PCR errors had been introduced. Following transformation the protein was expressed in BL21. To optimize expression and to test protein for enzymatic activity, expression is increased using BL21 Codon Plus
(Stratagene). This strain of £. coli has been engineered to contain extra copies of tRNAs for codons in E. coli that are rarely used (σrgU, ileY, leuΨ and proL). In some cases the presence of an N-terminal tag can interfere with the ability of a protein to function and that although a GST tag can be removed with thrombin this treatment itself can be too harsh to retain the activity of some proteins. Thus as an alternative approach is to employ the Protein C Epitope Tagging system (Roche). This system
RECTIFIED SHEET (RULE 91) allo s the production of recombinant proteins which have either C- terminal or N-terminal protein C tags. The protein C tag is used to purify protein using an antibody that binds the protein C tag only in the presence of Ca"2. Calcium chelation then provides a gentle means of elutin^ the purified protein from the antibody.
The Pichia Expression System (Invitrogen) is also used. This system offers advantages of bacterial systems such as high-level expression and ability to use large scale cultures. In addition, it offers certain advantages of eukaryotic expression systems that facilitate protein processing, folding and post-translational modifications. The system makes use of the powerful alcohol oxidase promoter (AOX1) to aid high expression levels. Tranformants are selected by Zeocin resistance and inframe C-terminal His tag allows purification by metal-chelating resins and detection through an anti-;πvc antibody. This produces additional recombinant chorismate synthase protein, in order to produce polyclonal antisera to chorismate synthase. Antisera is employed to determine subcellular localization of T. gondii chorismate synthase. Recombinant protein also is used for later crystallography studies and for high throughput screens. Production of anti-chorismate svnthase antibody To produce polyclonal antiserum to the entire protein, mice with 10 ug of recombinant protein emulsified with TiterMax initially and then again 2 weeks later. A commercial source for immunization of rabbits is also suitable. Preimmune sera and sera containing polyclonal antibody, is obtained 7 days after the second immunization. To produce monospecific antibody, anti-peptide antibodies to specific regions of the protein also is produced in rabbits by a commercial laboratory (Alpha Diagnostic, San Antonio, TX). Analysis for B cell epitopes indicates that amino acids 342 to 363, KHERDGCSAATLSRER ASDGRT, and amino acids 35 to 55, SVEDNQPQLNRRRPGQGPLST are peptides that should elicit monospecific antibodies. The advantage of polyclonal antibodies is that
RECTIFIED SHEET (RULE 91) they recognize native folded protein, and of the anti-peptide antibodies is that when they recognize native protein, peptide epitopes are defined. Development of enzvme assav for high throughput assays To measure chorismate synthesis, a phosphate release assay is performed using a malachite green dye and the product is detected spectrophotometrically with a plate reader. This is adapted for large scale screening for high throughput screens. This assay is performed anaerobically (i.e., in a nitrogen environment) using polyethylene bags. Substrate EPSP will be synthesized as described previously. Example 46: Antibody to Recombinant Chorismate Svnthase is
Useful in Diagnostic Assays. Antibody to recombinant chorismate synthase was produced in mice and is useful as an immuno-diagnostic test kit reagent. Example 47: Isocitrate Lvase. T. gondii isocitrate lyase activity was demonstrated and has the same uses as chorismate synthase activity, and other enzymes, e.g., it is useful for high throughput screens of T. gondii, Isocitrate lyase enzyme activity (FIG. 17C, D) and its inhibition by 3 nitropropionic acid (3NPA) (FIG. 17D) was identified. This exemplifies the presence of a key enzyme in the glyoxylate cycle, and provides a method useful for both screens of available libraries of compounds and rational development of combinatorial libraries of compounds based on lead compounds and their interactions with the enzme and analysis of enzyme structure. Use of a knockout microorganism complemented with the parasite ICL gene is another example of a method useful for high throughput screens to idnetify an inhibitor of ICL. sequences Antisense gene sequences to interfere with parasite growth or survival. This is a representative example of inhibition of this enzyme in this pathway. This enzvme is potentially useful in development of antimicrobial agents, diagnostic reagents or vaccines.
RECTIFIED SHEET (RULE 91) Example 4δ: The T. gondii Isocitrate Lvase Binding Pocket anri Active Site Form a Basis for Rational Antimicrohint Agent Development . The T. gondii isocitrate lyase cDNA sequence (FIG. 13), amino acid sequence (FIG. 19), and isocitrate lyase binding pocket and active site
(FIG. 20, box) were identified and have absolute hom*ologies with all other isocitrate lyases and not with other partially hom*ologous enzymes such as CPEP mutase. A yeast with a mutation in a base encoding a lysine (K) only in this area produced an inactive isocitrate lyase. This observation is useful for development of antimicrobial agents as described for other sequences herein.
Example 49: T. gondii Isocitrate Lvase Genomic Sequence is Useful for Vaccine Development A genomic ICL sequence is in FIG. 21 and is useful for vaccine development as described for tho*r genomic sequences.
Example 50: Demonstration of T. gondii Isocitrate Lvase Stage Associated Protein and mRNA. T. gondii isocitrate lyase stage associated protein is present in bradyzoites and is useful as described herein for producing diagnostic reagents, identifying anti-microbial agents and for vaccines. T. gondii, isocitrate lyase stage-associated protein is present in bradyzoites (FIG. 22) and there is stage associated mRNA expression and protein (FIG. 23). This observation is useful in the same manner as other examples of mRNA and protein described herein in for diagnostic reagents, antimicrobial agent and vaccines.
Example 51: Additional Inhibitors of Apicomplexan Isocitrate Lvase are Based on Compounds that Inhibit Isocitrate Lvases of Other Organisms. Additional inhibitors of apicomplexan isocitrate lyases are identified and designed. They are used as lead compounds for designing new inhibitors as described herein and this is useful for development of
RECTIFIED SHEET (RULE 91) diagnostic reagents, antimicrobial agents and vaccines as described for other enzymes herein.
Example 52: Genetic, Eπzvmatic and Functional Evidence and Active Inhibitors of Apicomplexan Acetyl coA Carboxylases Such as Clodinafop Provide a Basis for Development of
Novel Antimicrobial Agents, Diagnostic Reagents and Vaccines. FIG. 24 presents enzymatic, genetic and functional evidence of a wheat-like T. gondii acetyl coA carboxylases. Partial gene sequences were identified for T. gondii, Plasmodia and Cryptosporidia acetyl coA carboxylases. Inhibitors of T. gondii acetyl coA carboxylase inhibited parasite survival in vitro. This is useful for diagnostic reagents, antimicrobial agents and vaccines as described for other sequences herein. Example 53: Synergism of Antimicrobial Agents that Inhibit Apicomplexan Lipid Synthesis.
Other examples of synergistic effects on lipid synthesis pathway are the synergistic effects of clodinofop, thialactomycin, and cerulin. Example 54: Growth of Toxoplasma gondii is Inhibited bv
Aryloxyphenoxy-propionate Herbicides Targeting Acetyl-CoA Carboxylase.
The recently discovered plastid-like organeUes in apicomplexan parasites provide new targets for antimicrobial agents. Aryloxyphenoxypropionates, known inhibitors of the plastid Acetyl-Co Carboxylase (ACC) of grasses, inhibit Toxoplasma gondii ACC by 50% at a concentration of 20 μM Clodinafop, the most effective of the herbicides
RECTIFIED SHEET (RULE 91) tested, inhibits growth of T. gondii in human fibroblasts by 70% at 10 μM and is not toxic to the host cell even at much higher concentrations. Infected fibroblasts treated with Clodinafop for two days show a substantial reduction in the number of T. gondii cells at 10 μM and almost complete removal of parasites at 100 μM. Longer treatments are even more effective. Fragments of genes encoding biotin carboxylase domain of multi-domain ACCs were cloned. One ACC from T. gondii (ACC1) clusters with the putative Cyclotella cryptica chloroplast ACC and Plasmodium ACC, while another (ACC2) clusters with Cryptosporidium ACC, probably the cytoplasmic form. In plants, genes encoding enzymes for fatty acid synthesis, including various subunits of ACC except one, are present in the nuclear genome and their protein products are imported and function in plastids. ACC, catalyzing the first committed step of de novo fatty acid biosynthesis, is a known selective target of aryloxyphenoxypropionate ("fops") and cyclohexanedione ("dims") herbicides in sensitive species. The molecular mechanism of inhibition/resistance of the enzyme is not know but there is a strong correlation between the enzyme structure and its origin. The high molecular weight multi-domain ACC that is localized in plastids of grasses is extremely sensitive to these herbicides. All of the multi-subunit chloroplast enzymes of dicot plants and bacteria as well as other multi-domain cytosolic ACCs, such as those from man, chicken, rat and yeast, are resistant. ACC activity is conveniently measured in vitro by the incorporation of the carboxyl group from bicarbonate into an acid-stable form using crude protein extracts after Sephadex G50 filtration. Substantial, acetyl-CoA dependent activity was observed in extracts from tachyzoites of the RH strain of T. gondii isolated from infected mice, and no ACC activity could be detected in a control extract of macrophages from uninfected mice, the usual minor contaminant of the parasite preparation. Two biotin-containing proteins were revealed with streptavidin following electrophoresis of the extract proteins. One band at 240 kDa corresponded to the expected size for a subunit of ACC, while another at 130 kDA corresponded to the size expected for pyruvate carboxylase (PC).
Structures of fops and dims were tested on the ACC-containing protein extracts of T. gondii described above. Three of the four fops were striking inhibitors of the activity, while none of the dims had any effect against the enzyme. There was 50% inhibition at 20 μM and 90% inhibition at 100 μM by Clodinafop, Quizalofop, and Haloxyfop. Effects of the herbicides on uninfected fibroblasts and on T. gondii growth and replication were tested as previously described by Roberts et al., 1998 using incorporation of radiolabeled thymidine by growing fibroblasts to assess toxicity and incorporation of radiolabeled uracil to measure T. gondii growth and persistence. Anti-parasite activity and toxicity for four fops and one representative dim were determined. Pyrimethamine and sulfadiazine, antimicrobial agents which are known inhibitors of folate synthesis, were included as positive control. The combination of candidates inhibited uracil incorporation by T. gondii by more than 95% without toxicity for fibroblasts. Consistent with the data for ACC activity in vitro, the inhibitory activity of the fops and the dim on T. gondii growth in fibroblasts was in the same concentration range. Clodinafop was even more active in this assay than in the enzyme assay, giving 70% inhibition at 10 μM. With regard to toxicity, fops are mildly toxic at the highest concentration, 400 μM. In separate experiments, the effect of Clodinafop on T. gondii was assessed by light microscopy. Micrographs showed infected fibroblasts treated with Clodinafop at 10 and 100 μM compared with control infected cells without herbicide and uninfected cells. There is substantial reduction of the number of Toxoplasma tachyzoites at 10 μM and almost complete removal at 100 μM. The effectiveness of Clodinafop at 10 μM is greatly enhanced by a 4-day treatment with one change of medium and inhibitor after 2 days. In this experiment, cultures were incubated for 2 more days without the inhibitor. No parasite cells were found in infected fibroblasts treated in this way.
The active form of fops used as herbicides in the field are esters, which are converted to free acids by plant esterases. The true inhibitor of ACC is the free acid. Two esters of Halosyfop, two esters of Quialofop and one ester of
Clodinafop (Topik) have no effort on T gondii ACC activity in crude extracts and were relatively inactive in the uracil incorporation assay except for Topik that was as active as the free acid, suggesting significant level of hydrolysis of this ester. In general, in this assay fop esters are not more effective than free acids.
Single stranded cDNA prepared from total RNA extracted from T. gondii tachyzoites was used as a template for the PCR amplification of a 440- bp fragment encoding the biotin carboxylase (BC) domain of ACC, using primers and conditions described for wheat ACC. Several independent PCRs yielded five different products. Two of them appeared to encode eukaryotic- type multi-subunit ACCs. Genomic clones encoding the entire BC domain were then isolated from a genomic library using the PCR-cloned fragments as probes and these were sequenced. Similarly, sequences of a fragment of the BC domain of ACCs of P. knowlesii, P. falciparum and C. parvum were determined from PCR-cloned gene fragments. A phylogenetic analysis was performed based on amino acid sequence comparisons of the two candidate ACCs from T. gondii with those of other BC domains. Three apicomplexan sequences (T. gondii, P. knowlesii, and P. falciparum) cluster together with Cyclotella cryptica ACC, an enzyme thought to be in the diatom chloroplast. This isozyme, called ACC1 in T. gondii, is likely the plastid form. This assignment awaits cloning and sequencing of the 5'-terminal portion of the cDNA, where a sequence encoding a signal/transit peptide ought to be found. The other ACC, called ACC2 in T. gondii, clusters with the ACC of C. parvum. These two are probably cytosolic forms. The partial genomic sequences revealed differences in intron number and location before ACC1 and ACC2 of T. gondii, and the three ACC genes from the other apicomplexa. One of the other PCR products encoded a BC domain similar to that of pyruvate carboxylases. Deduced amino acid sequences encoded by the remaining two PCR products were similar to the BC domains of rat ACC and prokaryotic-type biotin-dependent carboyxlases, respectively. These fragments were assumed to encode the host mouse ACC and a carboxylase from a bacterial comaminat. Protein gels blotted with streptavidin revealed pyruvate caroxylases (130 kDa) in addition to ACC (240 kDa), but no bacterial-type biotin carboxyl carrier protein (20 kDa) or biotinylated subunit of propionyl-CoA carboxylase (70 kDa).
There is a very strong correlation between the pattern of sensitivity/resistance of the ACC activity and Toxoplasma growth inhibition by the twelve different compounds tested. This result provides important evidence linking the Toxoplasma growth phenotype to the effect of the compounds on the enzyme activity. The basis for the sensitivity of some of the multi-domain ACCs to fops and dims is not known, nor is it known why some, like the T. gondii ACC activity reported here, are sensitive to fops but resistant to dims. Compounds in the fop family differ in their properties as well, with a clear correlation between activity and structure, e.g. relatively low inhibitory activity of Fluazifop.
The target for sensitivity (herbicide binding site) is likely in to a region encompassing the β domain of carboxytransferase, based on experiments using yeast gene replacement strains, in which chimeric genes encoding wheat ACCs replace the yeast ACC1 gene. Such strains are herbicide-sensitive if they contain a gene encoding sensitive ACC. Availability of the genes encoding T. gondii ACCs may clarify which of the isozymes is targeted to the plastid and whether one or both of them are sensitive to fops (the majority of the activity in the protein extracts was inhibited). Inhibition of T. gondii growth in infected fibroblasts by herbicides targeting ACC suggests, based on earlier studies of herbicide action on plants and yeast gene-replacement strains, that inhibition of ACC activity in sensitive species leads to metabolite depletion to a level at which the organism cannot support its needs. This reflects an essential contribution of ACC to the pathway of de novo fatty acid synthesis and is the basis for the use of the ACC inhibitors as herbicides in agriculture and their potential future use in medicines. Example 55: An Apicomplexan Glyoxylate Cycle. To determine whether there are additional plant-like metabolic pathways as potential targets for novel chemotherapeutic agents, because they are not present in animals or differ substantially from those of animals, evidence was sought that the glyoxylate cycle might be operational in apicomplexan parasites, and play an essential role in certain stages of the life- cycle of these organisms.
Evidence was sought for the presence of isocitrate lyase and malate synthase, key enzymes unique to the glyoxylate cycle. Enzymes of the glyoxylate cycle were detected in protein extracts of T. gondii. Polyclonal antibodies to cotton malate syntase and isocitrase lyase were used to detect heterologous apicomplexan proteins by western blot analysis. A protein band of approximately 64 kD was detected using antibodies to cotton isocitrate lyase and malate synthase in lysates of T. gondii tachyzoites. Isocitrate lyase was also sought, and found in western blots of T. gondii bradyzoites. Antibody to cotton isocitrate lyase also was used for immunohistochemistry to study bradyzoites within cysts in brain tissue. Isocitrate lyase was identified in bradyzoites. Whether there was stage related expression of isocitrate lyase in T. gondii was studied by using smaller number of parasites in semiquantitative western blots. There was greater expression of isocitrate lyase in parasites undergoing stage conversion in vitro on the first and second days of culture following pH shock, with loss of detectable isocitrate lyase protein on the third and seventh day with concomitant appearance of increasing levels of the bradyzoite marker BAG 1 as the bradyzoites matured when relatively small numbers of parasites were used. Stage specific expression of the gene was analyzed by RT PCR using mRNA obtained from
Me49 strain T. gondii tachyzoites differentiating in bradyzoites in vitro. Tachyzoites had demonstrable ICL mRNA whereas bradyzoites did not. These results suggest that expression of isocitrate lyase may be developmentally regulated. In other microorganisms, isocitrate lyase is regulated at a number of different steps. For example, in E. coli there is an ace operon comprised of ace B, A, and K encoding malate synthase, isocitrate lyase and isocitrate dehydrogenase kinase phosphatase, respectively. Expression of the ace operon is under the transcriptional control of two genes, the iclR gene and fadR. The fadR is also involved in the regulation of fatty acid degradation. It has been suggested that these genes encode repressor proteins, which act independently or in concert, to repress the ace operon. Moreover, functionally related isoenzymes with distinct roles in the metabolic pathways needed for growth under different minimal conditions also have been described. In addition, different iso forms of the isocitrate lyase enzyme related to the age of the organism have been identified. Interestingly, in germinating seeds, isocitrate lyase plays a time-limited role with decline in isocitrate lyase activity in the senescent endosperms.
Next, evidence for the presence of a functional glyoxylate cycle enzyme and its inhibition in apicomplexan parasites was obtained isocitrate lyase enzyme activity and its inhibition by 3 Nitropropionic acid (NPA) was detected in lysates of T. gondii tachyzoites. Functional evidence for the glyoxylate cycle was sought by examining the effects of inhibitors of isocitrate lyase on growth and survival of apcomplexan parasites in vitro. Uracil incorporation by T. gondii in the presence and absence of inhibitor was measured in tachyzoites. 3 NPA inhibited parasite growth. Similarly, 3NPA inhibited growth of P. falciparum.
Then, genetic evidence for the presence of isocitrate lyase was obtained in T. gondii. First the primary structure of isocitrate lyases from varied organisms (bacteria to higher plants) were compared, and absolutely conserved amino acid sequences were identified across species. A partial complementary DNA sequence was next identified from the WashU-Stanford-PAMF-NIH Toxoplasma EST project (EST TgESTzz53c08.rl; GenBank accession number AA520237; Steve Parmly, PAMF, www.ncbi.nln.nih.gov/Malaria/plasmodiumbl.html). Both strands of the corresponding clone were sequenced. This sequence when translated had an open reading frame (ORF) of 857 base pairs, had over 30% hom*ology with isocitrate lyases from varied organisms (range: 29 - 53% identities; 43 - 67% positives). A T. gondii RH strain genomic Lambda DASH II library (Stratagene) was then screened using TgESTzz53c08.rl as a probe, and a genomic clone was obtained and sequenced (GenBank accession number to be assigned). The binding pocket and catalytic site that are absolutely conserved among other isocitrate lyases was identified in the T. gondii gene. The deduced amino acid sequence also showed partial hom*ology with putative carboxyphosphonoenolpyruvate phosphonomutase from E. coli and Salmonella species. Two regions of isocitrate lyase have been implicated as part of the active site. The motif KKCGHM(L) is conserved in all isocitrate lyases, and it is proposed that the cysteine is a critical active site residue. The absolute identity of the T. gondii sequence in the region of the active site, the binding pocket and other conserved regions to that of all isocitrate lyases, not demonstrated by any carboxyphosphonoenolpyruvate phosphonomutase, makes it highly likely that the gene cloned is an isocitrate lyase gene. Also, interestingly, a single mutation of a K to R at the second lysine in the K CGHM(L) motif (a substitution noted in a number of carboxyphosphonoenolpyruvate phosphonomutase genes) in a yeast and E. coli isocitrate lyase rendered it inactive (Figure 4B)1"1"16. The putative T. gondii isocitrate lyase gene sequenced thus far has predicted 4 exons.
These studies provide protein, enzymatic, functional and genetic evidence for the presence of a glyoxylate cycle in apicomplexan parasites. The presence of the glyoxylate cycle pathway enzymes, but not expression of its mRNA appears to be more abundant in certain life cycle stages of T. gondii in which lipids may be utilized in preference to carbohydrates as an energy source. This pathway provides a novel antimicrobial agent target and an inhibitor of an enzyme in this pathway has been identified.
MATERIALS AND METHODS T. gondii
Swiss Webster mice ( 12-15 mice per assay) were infected intraperitoneally with T. gondii tachyzoites (Rh strain, 2xl07 per mouse) 2 days prior to assay. Tachyzoites were extracted with a peritoneal lavage using 5 ml of sterile saline per mouse.
Alternatively, the PTg strain of 7. gondii was cultured as tachyzoites or tachyzoites induced to become bradyzoites, as described8. Antibodies
Rabbit control preimmune serum was obtained and then antibodies to cotton malate synthase or isocitrate lyase were produced in rabbits SDS PAGE and Western blots
T. gondii tachyzoites or bradyzoites were obtained at indicated time points from host cells by scraping the monolayer, passing the infected cells through a syringe with a 25g needle twice to disrupt them, and then organisms were counted and centrifuged at 2000 rpm for 10 minutes at 4°C to pellet the parasites. The supernatant was discarded and the pellet was suspended in SDS PAGE loading buffer (with 2 mercaptoethanol) at a concentration of l l O5 parasites per μl and boiled for 10 minutes. Unless otherwise
indicated, material from 2x IO6 parasites was utilized per lane. This was electrophoresed in a 12% polyacrylamide gel under reducing conditions and transferred onto nitrocellulose membranes blocked with 5% milk in PBS tween (0.05%), and probed with rabbit preimmune serum or polyclonal antibody to cotton isocitrate lyase or malate synthase, or mouse monoclonal antibody to BAG l antigen, followed by HRP conjugated anti rabbit or anti mouse secondary antibodies 'as appropriate. Bands were visualized using ECL. PCR and Northern blots
Messenger RNA, isolated on oligo dT solid phase matrix columns and reverse transcribed using a random priming method, was used for semi-quantitative PCR analysis of tachyzoite surface antigen (SAG) l, bradyzoite cytosolic antigen (BAG) 1-5, and isocitrate lyase (ICL), relative to beta tubulin (TUB). The primer sets were as follows: SAG1 (5'-CGC? TTG TAT GTC GGT TTC GCT-3' and 5'-TGT TGG GTG AGT ACG CAA GAG TGG-3"), BAG 1-5 (5'-CCC ATC GAC GAT ATG TTC GAG-3' and 5'-CGT AGA ACG CCG TTG TCC ATT G-3'), ICL (5'-TTG CCG TTC TGG AAA GCT AGT AAG A-3' and 5'- GCA AAC GCT GGT CCT CAA TGT-3'), and TUB (5'-GTT TCC AGA TCA CCC ACA GTC TTG G-3' and 5'-GAG CAA ACC CAA TGA GGA AGA AGT G-3"), yielding PCR product sizes of, 346, 225, 574, and 420bp, respectively. The BAG 1-5 primers flank an intron serving as a control for genomic DNA contamination, yielding a PCR product of 784 bp. cDNA from T. gondii tachyzoites of the RH strain and induced bradyzoites from the Me49 strain were used as templates. Immunohistochemistry
Immunoperoxidase staining was performed' as previously described using control preimmune or immune rabbit antisera. Enzvme assays
Parasite lysates were obtained from tachyzoites, suspended in elution buffer ( 100 M KCl. 20% glycerol, 7 mM 2-mercaptoethanol, 20 mM Tris-HCl, pH 7.5, and complete TM protease inhibitor co*cktail [Boehringer Mannheim, I tablet per 50 ml buffer], sonicated 3 times for 3 seconds at 30 sec intervals, and centrifuged at 12,000 g for 15 min. The supernatant collected was applied to a Sephadex® G l OO column (25 ml, Pharmacia) equilibrated with elution buffer, eluted with 15 ml of elution buffer, and = 1.5 ml fractions were collected. Fraction(s) with the peak protein concentrations (protein analysis performed on a spectrophotometer at 280 nm) were selected and used in enzyme assays.
A discontinuous method described by Ko and McFadden17 was employed with minor modifications to measure the ability of isocitrate lyase to convert isocitrate to succinate and glyoxylate. This method utilizes the colorimetric reaction between the phenylhydrozone of glyoxylate and ferricyanide. Reaction mixtures (92 mM MOPS,.5 mM MgCI,, 1 mM DTT, 1% phenylhydrazine, 4.4 mM isocitrate, in 0.5 ml with fractionated parasite lysate) were incubated in a 37 "C water bath for a determined amount of time. After incubation, enzymatic reactions were stopped with concentrated HCl, mixed with 25% (w/v) potassium ferricyanide, and then measured in a spectrophotometer at 520 nm. Culture of parasites in vitro with inhibitors
Parasites were cultured with host cells and inhibitors and the effects analyzed as described1. Identification of T. gondii isocitrate lvase genes:
Library screening, phage DNA purification. Southern blot (cloning and sequencing), host strains and vectors
XL 1 Blue MRA and pBluescript KS* DH5 α were used. Lambda Dash II (Stratagene) was the vector for the genomic library. A 550 bp ECORl-XhoI fragment of
the cDNA EST clone TgZZ13 CO8 r 1 was labeled with α (52P) dCTP and used for initial
screening of the library. For subsequent secondary and tertiary screening to obtain pure phage, a biotinylated, non-radioactive, labeled probe of the entire 857 bp EST clone was prepared and used. The genomic library was screened (Stratagene), phage purified to >99% hom*ogeneity, the clone amplified and DNA extracted (Current Protocols in Molecular Biology). Southern blot
The purified phage DNA was digested with Notl; xhol or EcoRI enzymes, run on a l agarose gel, transferred onto a nylon membrane probed with the biotinylated probe (above). A -4kb band which was identified with the probe and was cloned into pBluescript KS* and sequenced. DNA sequencing and sequence analysis
DNA sequencing was performed using an automated DNA sequencer. This sequence was compared to peptide sequence databases at The National Center for Biotechnology Information (NCBI) using the program TBlastX or BlastP (for derived open reading frames). Gene construction using the sequence obtained was also performed utilizing the Baylor College of Medicine program. Primers for sequencing were made at Integrated DNA Technology. Sequence analysis was carried out by software programs MacNector, ClustalX and MACH Box.
MATERIALS AND METHODS A. Methods to Assay Candidate Inhibitors 1 Inhibitors of Toxoplasma gondii a) Cell lines' Fibroblasts. Human foreskin fibroblasts (HFF) are grown in tissue culture flasks in Iscoves' Modified Dulbecoes Medium (IMDM), containing 10% fetal bovine serum, L-glutamine and penicillin/streptomycin at 37°C in 100% humidity and a 5% C02 environment. Confluent cells are removed by trypsinization and washed in IMDM They are used in a growth phase for toxicity assays or when 100% confluent for parasite inhibition assays b) Tachyzoites: Tachyzoites of the RH and pTg strains of T. gondii are passaged and used for /'// vitro studies (McLeod et al., 1992) The R5 mixed tac yzoite bradyzoite mutant was derived from mutagenesis with nitrosoguanidine in the present of 5 hydroxynapthoquinone These organisms are used for in vitro experiments at a concentration of 2 x 10'", 2 x IO4, or 2 x 10' organisms per ml, dependent upon the planned duration of the experiment (i.e., larger inoculations for shorter duration experiments) c) Bradyzoites Bradyzoites are obtained as described by
De on et al ( 1996b) Specifically, C57B I0 ScSn mice are infected imraperitoneallv with 20 cysts of the Mc49 strain of T gondii Their brains are removed 30 days later and hom*ogenized in PBS by repeated passage through a 21 gauge needle Aliquots containing the equiv alents of 3-4 brains are diluted in PBS and 6 5 mis of 90% percoll added to the niixtui e w ich is allow ed to settle for 30 nuns Z.nls of 90% Percoll ι> t en added as a bottom layer and the mixture centrifuged for 30 mms at 2500xg The cysts are recov ered from the bottom layer and a small portion of the layer above After the removal of Percoll by centrifugation, the contaminating red blood cells are removed by lysis with water followed by the addition of 1 ml of lOxPBS per 9 ml brain suspension in water Bradyzoites are released from the purified cysts by digestion in a 1% pepsin solution for 5 minutes at 37°C This method routinely permits recovery of greater than 90% of the cysts present which yields approximately 100 bradyzoites per cyst. Bradyzoites are used at concentrations of 2x10'', 2xl04 and 2xl05 per ml in parasite growth inhibition assays pH shock is also used to retain organisms fn bradyzoite stage when such pH does not interfere with inhibitor activity d) Inhibitors: Inhibitor compounds are tested over a range of concentrations for toxicity against mammalian cells by assessing their ability to prevent cell growth as measured by tritiated thymidme uptake and inspection of the monolayer using microscopic evaluation A range of concentrations that are non-toxic in this assay are tested for their ability to prevent the growth of 7". gondii and also other Apicomplexans within these cells i ) Heme Synthesis The inhibitor of the heme synthesis pathwav, gabaculine (Grimm. 1990 Elliot et al , 1990, Howe et al , 1995, Mets and Thiel. 19S9, Saiigwan and O'Bπaπ 1993, Matters and Beale, 1995) is used at a concentration of 20 m [which has been demonsu ated to be effective against ta hv zones, of the RH and R5 Nti -iins] Oilier inhibitors of this pathwav include 4 amm -^-hew noic acid and 4-aminofluoropentanoιc acid which provide additional corroborative evidence that this pathway is present ii) Glyoxylate Cycle The inhibitor of isocitrate lyase is 3 nitropropionic acid (ranging from 0 005 to 5 mg/ml m vitro) iii) Alternative Oxidase T. gondii bradyzoites use unique alternative oxidases. Alternative oxidase is necessary and sufficient for bradyzoite survival. Methods to characterize plant alternative oxidases are described (Hill, 1976, Kumar and Soil, 1992, Lambers, 1994; Li et al, 1996, Mclntosh, 1994) For the in vitro studies, cell lines that lack functional mitochondria are used. These cell lines are used to allow the study of inhibitors effective against the conventional or alternative respiratory pathways within the parasite, but independent of their effects on the host cell mitochondria Two cell lines, a human fibroblast cell line ( 143B/206) lacking mitochondrial DNA. and the parental strain ( 143 B) which poses functional mitochondria are used These cell lines have been demonstrated to support the growth of 7". gondii (Tomavo S and Boothroyd JC, 1995) SHAM, an inhibitor of the alternativ e respiratory pathway is used at concentrations between 0 25 and 2 μg/ml in vitro iv) Shikimate Pathway For EPSP synthase, the inhibitor is -(phosphonomethv l) glycine (concentration-- of 3 1 mM in folate deficient media) e) Culture assav s .ic - lor a_. -.e ~- H__ inhibitor effect i) Toxicity assays: Aliquots of cells (HFF) are grown in 96-well tissue culture plates until 10% confluent. Cells are incubated with various concentrations of drug for 1, 2, 4 and 8 days. Cultures are pulsed with tritiated thymidine (2.5 μCi/well) for the last 18 hours of the culture after which the cells are harvested using an automated cell harvester and thymidine uptake measured by liquid scintillation. ii) In vitro parasite growth inhibition assays: Confluent monolayers of HFF cells, grown in 96-well plates are infected with T. gondii tachyzoites of the RH strain and serial dilutions of anti-microbial compound are applied 1 hour later* T. gondii growth is assessed in these cultures by their ability to incorporate tritiated uracil (2.5 μCi/well) added during the last I S hours of culture. After harvesting cells with an automatic cell harvester, uracil incorporation is measured by liquid scintillation. Alternativel , confluent HFF cells are grown in the chambers of Labtech slides and parasite growth is assessed microscopically following fixation in aminoacridine and staining in 10% Giemsa (McLeod et a/., 1992). f) Product rescue assays to evaluate specificity of the inhibitor: To attempt to demonstrate specificity of the site of action of the inhibitor, growth inhibition assays are performed in the presence of varying concentrations of product, e.g., in the case w here gabaculine is the inhibitor, ALA is added simultaneously to determine w hether product rescue occurs. This type of studv is only interpretablc when rescue is demonstrated because ii is possible that exogenous "product" is not traπsported into the 71 gondii within host cells For EPSP synthase, product rescue assay is performed with PABA g) Assays for synergy in vitro. This is an assay in which < 50% inhibitory concentrations of two antimicrobial agents are added alone and together to determine whether there is an additive, synergistic or inhibitory interaction All other aspects of this assay are as described herein.
2. Inhibitors of Cryptosporidia parvum
C. pamim oocysts at 50,000/well were incubated with each drug (PR_M=paromomyciπ which is the positive control, NPMG, gabaculine, SHAM, S- hydroxyquinoline) at 37°C (8% carbon dioxide) on confluent MDBKF5D2 cell monolayers in 96 well microtiter plates. The level of infection of each well was determined and analyzed by an immunofluorescence assay at 4S hours using as an antibody C. parvum sporozoite rabbit anti-serum (0. 1%), and using fluorescein- conjugated goat anti-rabbit antibody ( 1%) Data are expressed as mean parasite count/field w hen 16 fields counted at lOx magnification "s.d of the mean (FIG. 6)
3. Inhibitors of Plasinodiuin falciparum
This assay is performed in folate deficient RPMI 1640 over a 66 hour incubation in plasma as described by Milhous et al. ( 1985) Both the W2 clone DHFR resistant phenotype and the D6 clone are used (Odula et al , 19SS) (Table 3) 4. Inhibitors of Eimeria tenella
Suscer .ibi ty ol" ' imei in tenella in vin o is analyzed bv a method similar to that dcsci ibed bv McLeod el al , I 2 or foi (
idiiim as disclosed liei em. S. In vivo studies, measurement of parasitemia of Toxoplasma gondiiA method to measure the amount of parasitemia in mouse peripheral blood has been developed Briefly, the target for PCR amplification is the 35 fold repetitive B 1 gene of T gondii and the amplification was performed using primers previously 5 reported In order to semiquantitate the PCR product and to avoid false negative results, a competitive internal standard is generated using a linker primer and the original B 1 primers Competitive PCR was performed by spiking individual reactions (containing equal amounts of genomic DNA) with a dilution of the internal standard Since this internal control contains the same primer template sequences, it competes 0 with the B 1 gene of 71 gondii for primer binding and amplification The sensitivity of the PCR reaction in each sample can be monitored Following competitive PCR, the PCR products are distinguished by size and the amount of products generated by the target and internal standard can be compared on a gel The amount of competitor DNA yielding equal amounts of products gives the initial amount of target gene 5 6. Interpretation of Data/Statistical A nalysis of Data:
In vin o studies are performed with triplicate samples for each treatment group and a mean - sd determined as shown in the FIGs All //; vn o studies utilize at least 6 mice per group Statistical analysis performed by Students' t-test or the Mann-Whitnev U-test A p v akie of < 0 05 is considered statistically significant '"> B. Vλ cstcm Blots Dcnionstr.itc Plant-Like Fnzvnics
Wester" analv si foi GS ΛT isocitrate lv se malate s nthasc alternativ e oxidase and EPSP is used to dcmonsti atc the presence of plant-like enzv mcs in main Apicomplexan parasites, e.g., Plasmodia, Toxoplasma, Cryptosporidia, Malaria and Eimerta
Tachyzoites and bradyzoites (McLeod et al. 1984, 1988; Denton et al., 1996a, b), or their mitochondria and plastids are isolated as previously described. Equivalent numbers of tachyzoites and bradyzoites are separately solubilized in 2x sample buffer and boiled for 5 minutes Samples are electrophoresed through a 10 percent SDS- polyacryϋmide gel. Proteins are transferred to a nitrocellulose membrane at 4°C, 32V with 25mM Tris and 192mM glycine, 20% v/v methanol, pH 8.3. Blots are blocked in PBS (pH 7.2) containing 5% powered milk and 0. 1% Tween 20 for 2 hours'at 20°C. After washing in PBS (pH 7.2), 0.1% Tween 20, blots are stained with polyclonal or monoclonal antibodies specific for alternative oxidases in PBS (pH 7.2) containing 0. 1% Tween 20 for I hour at 20°C. Following washing in PBS (pH 7.2) containing 0. 1 %) Tween 20, blots are incubated with an appropriate secondary antibody conjugated to HRP at a dilution to be determined by methods known in the art After further washes, binding is visualized by chemoiilumiπescence (Amersham)
Antibodies to various enzymes, e.g. , soybean GSAT, barley GSAT, synechococcus GSAT, plant and/or trypanosome alternative oxidase, cotton isocitrate lyase, cotton malate synthase, soybean malate synthase, petunia EPSP synthase were used to determine whether hom*ologous enzymes are present in 71 gondii tachyzoites, bradyzoites mitochondrial and plastid enriched preparations Antibodies used include monoclonal antibodies to I panosoina bi nceii and Voo Doo Lily (Chaudhuri el al 1996) altei nativ oxidase and polyclonal antibody to
hi uceii alternati e oxidase The hybridizations with antibodies to plant and related protozoan alternative oxidases demonstrated the relatedness of 71 gondii metabolic pathways to those of plants and other non-Apicomplexan protozoans The products GSAT and alternative oxidase were demonstrated by Western analysis Both polyclonal and monoclonal antibodies w ere reacted with alternative oxidase to confirm this observationC. Probing Other Parasite Genes. The genes isolated from 71 gondii as described herein are used to probe genomic DNA of other Apicomplexan parasites including Plasmodia, Cryptospor odium, and Emieria.
D. Genomic Sequence. Genomic clones are identified and sequenced in the same manner as described above for cDNA except a genomic library is used Analysis of unique promoter regions also provide novel targets
E. Enz . mntic Activity Demonstrates Presence of Plnπt-Likc Enzymes in Metabolic pathways
The presence of the enzymes putatively identified bv inhibitor studies is confirmed bv standard biochemical assays Enzyme activities of GSAT, isocitrate lyase. malate synthase, alternative oxidase, and EPSP synthase, chorismate synthase, chorismate lv ase, TDP-glucose starch glycosyl transferase and other enzymes listed herein are identified using published methods Representative methods are those of Jahn t'/ / , 19v 1 , Wetnstein and Beale, 1995, Kahn el al , 1977 Bass el al . 1990 Mousdale anG Coggins ( 19S5) In addition, enzv me activity is used to determine in w hich of the i h zoite and bradyzoite life cv cle stages each pathway is operativ e Tachv/ones a: J bi adv zoite are pui ilicd as desu ibed ci ein Tiie pai asites aie Iv sed in 50mM HEPES (pH7 4) containing 20% glycerol, 0 25% Triton X- 100 and proteinase inhibitors (5nιM PMSF, 5FM E64, 1 FM pepstatin, 0 2mM 1 , 10-phenanthroiine) This method has proven successful for measurement of phosphofructokinase, pyruvate kinase, lactate dehydrogenase, NAD- and NADH-linked isocitrate dehydrogenases and succinic dehydrogenase activity in tachyzoites and bradyzoites of 71 gondii (Denton et ai, 1996a,b).
1) GSAT: GSAT activity is measured by the method of Jahn et al., (1991), which uses GSA as substrate. GSA is synthesized according to methods of Gough et al. (1989) Heat-inactivated (60°C, 10') lysates are employed as non-enzymatic controls. ALA is quantified following chromatographic separation (Weinstein and
Beale, 1985). This approach allows the definitive detection of GSAT activity in crude extracts.
2) ALA Synthase To determine whether parasites contain ALA synthase, an activity also present in mammalian host cell mitochondria, cell fractions from purified parasites are assayed. (Weinstein and Beale, 19S5) ALA produced from added glycine and succinyl CoA is quantified as for GSAT.
3) Isocitrate Lvase The biochemical assay for isocitrate lyase activity used is the method of Kahn et al. ( 1977)
4) Alternative Oxidase activity is measured in parasite lysates or purified mitochondria or plastids by oxygen uptake using an oxygen electrode described by Bass et al ( 1990) Confirmation of the oxidation being due to alternative
is achieved bv successful inhibition of oxygen uptake in the presence of 0 5mM SHAM, but not in the presence of KCN5) Shikimate Pathwav The biochemical assay for EPSP synthase, chorismate synthase, chorismate lyase, activity in cellular lysates is conducted as
5 descπbed by Mousdale and Coggins (19S5) and Nichols and Green (1992)
6) Branched Amino Acids The biochemical assay for hydroxy acid synthase is as described
7) Amylopectin Synthesis The biochemical assays for starch synthase, Q enzymes, and UDP-glucose starch glycosyl transferase are as described 0 8) Lipid Synthesis Assays for lipid synthases are as described
Some of the additional representative enzyme assays are precisely as described by Mousdale and Coggins(1985) and are as follows
5-Enolpyruvylshιkimate 3-phosphate synthase is assayed in forward and reverse directions as described previously (Mousdale and Coggins 1984) 5 Shikimate NADP oxidoreductase (shikimate dehydrogenase), shikimate kinase,
3-Dehvdroquιnase (DHQase) are assayed Assay mixtures contained in a total volume of 1 ml 100 mM potassium phosphate (pH 7 0) and 0 8 mM ammonium 3-dehydroquιnate 3-Dehydroquιnate synthase is assayed by coupling for forward reaction to the 3-dehydroquιnase reaction, assay mixtures ' contained in a total volume of I ml 10 mM potassium phosphate (pH 7 0), 50 μM \ \D 0 1 mM CoCN 0 5 nkat partiallv -punlled Lsc ei idua t ali DHQase and (to initiate assay) 0 4 mM DAHP The DAHP is prepared from E. coli strain AB2847A and DHQase from E. coli strain ATCC 14948
Assay of DAHP synthase is by a modification of the method of Sprinson et al Assay mixtures contained in a total volume of 0.5 ml. 50 mM 1 ,3-bis [tris(hvdroxymethyl)-methylamino] propane-HCI (pH 7.4), 1 mM erythrose 4- phosphate, 2 mM phosphoenolpyruvate and 1 mM CoCl2. The reaction is initiated by the addition of a 50 to 100 μl sample containing DAHP synthase and terminated after 10 min at 37°C by 100 μl 25% (w/v) trichloroacetic acid The mixture was chilled for 1 h and centrifuged to remove precipitated protein A 200 μl aliquot of the supernatant was mixed with 100 μl 0.2 M Nal04 in 9 M
H3PO. and incubated at 37°C for 10 min; 0.5 ml, 0 8 M NaAS02 and 0.5 M NajSOj in 0 1 M H2S04 in 0 1 m H2SO was then added and the mixture left at 37°C for 15 min, 3 ml 0 6% (w/v) sodium thiobarbiturate and 0 5 M Na:S04 in 5 m NaOH was added and the mixture placed in a boiling-water bath for 10 min After cooling to room temperature the solution was centrifuged (8500 xg
2 min) and the optical density at 549 nm read immediately Appropriate controls assayed in triplicate lack substrates, sample or both " Another representative assay is an assay for chorismate lyase which is as descπbed by Nichols and Green 1992 Chorismate lyase assays are earned out in a v olume of 0 5 ml containing 50 mM
Tnc-HC I ( pl l 7 5), 5 mM ΕDT Λ 10 mM 2-mcrcaptocthanol, 60 μM chorismate and 0 2 to 4 U of chorismate lvase After incubation at 37°C for 30 min, 4-hydroxybenzoate is detected and quantitated by high-pressure liquid chromatography (HPLC). Fifty microliters of each reaction mixture is applied to an HPLC system (Waters 625) equipped with a Nova-Pak C|8 column equilibrated in 5% acetic acid and monitored at 240 nM. The height of the 4-hydroxybenzoate peak is compared with those of standard curves generated by treating known amounts of 4-hydroxybenzoate in a similar manner. One unit of chorismate lyase activity is defined as the amount of enzyme required to produce 1 nmol of 4-hydroxybenzoate in 30 min at 37°C. Assays for 4-aminobenzoate and 4-amino-4-deoxychorismate are performed as described previously." Enzyme Assays: The 5-enolpyruvyIshikimate-3-phosphate (EPSP) synthase assay entailed monitoring the generation of EPSP using HPLC. Reaction components were separated using a Hypersil H3APS2 HPLC column (Hichrom Limited, Reading. UK) and a NaH2P04 elution gradient (50-400 mM). UV spectra (200-320 nm) of the column eluate were collected to identify eluants. Shikimate-3-phosphate and 5-enolpyruvylshikimate-3-phosphate, synthesized enzymatically and purified to at least 95% purity as described (12), eluted after 3.9 and 6.S min, respectively; phosphoenolpyruvate did not interfere with the EPSP detection and eluted after 5.3 min. The peaks at 21 5 nm were integrated; the EPSP produced was quantified using a standard curve of authentic EPSP. Parasite extracts were produced at 4;C by suspension of pure tachyzoites in extraction buffer (50 mM
Tris HCl, pH 7 5. containing complete TM protease inhibitor co*cktail [Boehringer Mannheim, 1 tablet per 50 ml bulVer]). sonicalion 3 times for 3 seconds at 30 second intervals, and cenlrifugation at 12000 g for 15 min. The resulting supernatant was diluted 6-fold with extraction buffer and loaded onto a ResourceQ column (1 ml, Pharmacia) equilibrated with extraction buffer. The bound protein was eluted in a single step using extraction buffer containing 500 mM KCl. The eluted material was used for enzyme assay. The assay mix contained 1 mM phosphoenolpyruvate, 1 mM SP and 50 mM HEPES, pH 7.5. The reaction was started by addition of parasite extract and incubation was at 30°C. Times 10 : 1 aliquots were subjected to HPLC analysis. Protein concentrations of lysates were determined using the Lowry method. (Roberts et al., 1998, In Press) E. Construction and Analysis of Gene "Knock-Outs"
In order to determine whether a gene, e.g., chorismate synthase or alternative oxidase is essential for growth or survival of the organism, gene knockout organisms are generated by the method of Roos et al., 1996. Specifically, the strategy for creating mutants is with hom*ologous recombination and to generate a targeted gene knock-out a sequential positive/negative selection procedure is used (Roos et al., 1996). In this procedure positive and negative selectable markers are both introduced adjacent to, but not within the cloned and suitably mutated locus. This construct is transfected as a circular plasmid Positive selection is applied to yield a single-site hom*ologous recombinant that is distinguished from non-hom*ologous recombinants by molecular screening In the resulting 'pseudodiploid.' mutant and wild-type alleles flank selectable marker and other vector sequences In the next step, parasites are removed from positive selection, which permits recombination betw een the duplicated loci This event appears to occur at a frequency of 2 x 10 ' per cell generation These recombinants are isolated with negative selection Next, they are screened to distinguish those that have recombined in a manner that deletes the mutant locus and yields a wild-type revertant from those that deleted the wild-type gene to leave a perfect allelic replacement.
This 'hit-and-run' approach has the disadvantage of being time-consuming Nonetheless, it offers several distinct advantages over other gene knock-out strategies First, because gene replacement occurs by two sequential single-cross-overs instead of one double cross-over which is a very rare event, it is more likely to be successful Second, because selectable marker(s) are located outside of the targeted gene itself, experiments are not limited to gene knock-outs A variety of more subtle point mutations are introduced as allelic replacements Third, this strategy provides a means of distinguishing essential genes from those which cannot be deleted for purely technical reasons Specifically, if the hit-and-run mutagenesis procedure yields only wild-type reveπants instead of the theoretical 1 1 ratio of wild-type mutant, this provides positive evidence that the locus in question is essential.
An example is a knockout created for the chorismate synthase gene It also can be made more general to include knockout of other genes for attenuated vaccines such as EPSP synthase and alternative oxidase The parasite with the gene of interest to be knocked out is grown ("manufactured") in vitro in presence of product, but when used /// \ ι\ o (he needed pi oduct is not pr esent The pai asite functions as an attenuated v accine s des_.nbed below under vaccines Λ specific example follows Specilicaliv the strategy of product inhibition discussed above is also useful for growing gene knockout parasites (which lack a key gene for their survival) in vitro by providing the essential product and thus bypassing the need for the gene during /// wtrσ propagation of the parasite Such gene knockouts cultivated /// vitro in this manner are useful attenuated organisms that are used as attenuated vaccines
The chorismate synthase cDNA clones are used as hybridization probes for recovering genomic clones from a 71 gondii genomic cosmid library. Coding regions are mapped onto the genomic clones using the cDNA clones as a guide Appropriate sections are sequenced to verify the gene location. Ultimately, full genomic sequences are obtained. Enough of the genomic clones are sequenced to develop a strategy for generating a putative null allele. Segments that can be deleted at the 5 ' end of the coding region to generate an allele that is unlikely to generate a functional gene product are identified A putative neutral allele is generated that can be distinguished from the wild type allele on the basis of an introduced restriction site polv morphism, but that does not differ in encoded protein sequence These putative chorismate synthase-null and chorismate synthase-neutral alleles are cloned into the pminiHXGPRT transfection vector plasmid
The resulting chorismate synthase -null and chorismate synthase-neutral plasmids are transfected into HXGPRT-negative strains of 7 gondii (strains RH(EP)ΗXGPRT [a ME49 derivative] Numerous independent clones are selected for survival on mv cophenolic acid to select for insertion of the pla>mtd These strains are scr eened bv Souther n analysis designed to detect the pi csence oi both the nor ma! and modified copies of the chorismate synthase gene and for tandem location of the two copies (with the vector HXGPRT gene between). This is the structure expected for insertion of the plasmid by hom*ologous recombination at the AroC genomic locus (the "hit" needed for the hit-and-run gene knock-out strategy). The feasibility of recovering these strains is critically dependent upon the ratio of hom*ologous to non-hom*ologous integration following transfection, which will depend upon the length of hom*ologous, genomic DNA in the clone (Donald and Roos, 1994; Roos et al., 1996). Eight kb of hom*ology is sufficient to obtain >50% hom*ologous integration (Roos et al., 1996): HXGPRT clones with verified pseudodiploid structure of the- chorismate : synthase alleles are selected for loss of HXGPRT using 6-thioxanthine (the "run" part of the protocol). Numerous clones are selected. If the loss of HXGPRT is based upon random hom*ologous exchange between the two chorismate synthase pseudodiploid alleles, theoretically half of the events should lead to excision of the modified chorismate synthase allele along with the HXGPRT, leaving the original wild type allele in the chromosome. The other half should excise the wiJ type allele, leaving the modified allele in the chromosome. During selection and grow-out of these clones, the medium is supplemented with chorismate at the concentration determined to best rescue cells from inhibitor toxicity. The purpose of the supplementation is to enhance the chances of recovering chorismate synthase-null strains. The genomic structure of the selected clones is examined by Southern analysis to confirm loss of the vector HXGPRT and of one copy of the chorismate synthase and to identify the remaining allele of chorismate synlhase The ratio of mutant to w ild type is tabulated The chorismate synthase-neutral allele is intended as a positive control to confirm that either allele (wild type or mutant) can be lost in this procedure. If chorismate synthase- neutral strains can be recovered but chorismate synthase-null strains cannot, the conclusion is that the chorismate synthase gene is essential for growth. If it proves possible to recover chorismate synthase-null strains, they are subjected to further phenotypic analysis, first, using immunoblotting of electrophoretically separated cell extracts to confirm absence of chorismate synthase protein, then, determining if these strains sho hypersensitivity to inhibitors of the alternative oxidase or to any of the other potential inhibitors. Sensitivity to chorismate. synthase inhibitors is analyzed to. determine the relative specificity of inhibition. If chorismate synthase is the sole target of the inhibitors, then the null mutants should be insensitive to further inhibition. Sensitivity analysis is conducted /// vitro as described herein. Whether strains show alterations in expression of the alternative oxidase or in any stage-specific antigens is of interest. These analyses are conducted by immunoblotting of electrophoretically separated cell extracts. /// vivo analysis using a mouse model is conducted to determine if these strains are infective and what stages of parasites can be detected following infection. Genetically altered 71 gondii organisms are used to infect C3H/HeJ mice by the intraperitoneal route Mortality is monitored and brains examined for cysts at 30 days post infection Knockouts with bradyzoite reporter genes are useful to determine whether these enzymes influence stage switch Stage switch also is characterized by quantitating relative amounts of parasite mRNA present in each stage of parasite using Northern blotting, isolation of mRNA and RT-PCR using a competitive inhibitor, and enzyme assay.
G. Reagents used for construction of "Knock-Outs" Library
Me49 genomic libraries are used.
Plasmids
/. miniHXGPRT. Contains T. gondii HXGPRT gene under control of DHFR-TS 5' and
3 ' flanking sequences. Functions as either a positive or negative selection marker (using 6-thio.xanthine or mycophenolic acid, respectively) in suitable host strains.
Parasite Strains (obtained from AIDS Repository. Bethesda. Md.)
RH(EP). Wild-type host strain RH (highly pathogenic in mice).
RH(EP)ΗXGPRT HXGPRT knock-out mutant of RH strain. Suitable for positive selection of HXGPRT-containing vectors. P(LK). Wild-type host strain P, (clonal isolate of strain ME49, produces brain cysts in mice).
P(LK)HXGPRT-. HXGPRT-deficient mutant of P strain. Suitable for positive selection of HXGPRT-containing vectors
I I. Antibodies
Antibodies have been raised against hom*ologous plant enzymes by standard techniques for both polyclonal and monoclonal antibodies (Current Protocols in Immunology, 1996). 1) Heme Synthesis
Antibody to soybean, barley and synechococcus GSAT are polyclonal antibodies with preimmune sera the control for the barley and synechococcus antibodies.
2) Glvoxylnte Cycle T. gondii contains a glyoxylate cycle that allows growth using lipids as a carbon source, thus the lipid mobilization pathway of T. gondii is similar to the pathway of plants (Tolbert, 19S0) A similar approach using polyclonal antibodies to isocitrate lyase and to malate synthase and preimmune control sera are used
3) Alternative Energy Generation Monoclonal and polyclonal antibodies to alternative oxidases in plants
(Mclntosh ei a/., 1994) and Trypanosomes (Hill, 1976) are used
4) Shikimate Pntliwnv
To demonstrate that 71 gondii has the same unique enzv mes that permit inierconvcrsion of shikimate to chorismate as plants do, the antibody to shikimate pathway plant EPSP synthase is used 5) Svntliesis of Branched Chain Amino Acids
Antibodies to acetohydroxy acid synthase are used.
6) Amylose and Amylonectin Synthesis and Degradation
Antibodies to starch synthesis, branching (Q) enzymes and UDP glucose starch glycosyl transferase are used.
I. Complementation of Enzvme Deficient E. coli Demonstrates Functional
Product
The E. coli AroC mutant which lacks chorismate synthase (AroC) was obtained from the E. coli genetic stock center. AroC bacteria is made competent to fake up DNA by transformation with CaCI2 treatment. Alternatively, the cells are electroporated to take up DNA. The presence of the plasmid is demonstrated in this system by growth on media which contains ampicillin, as the plasmid contains an ampicillin resistance gene. Complementation is confirmed by demonstrating growth on media lacking the product catalyzed by (i.e., chorismate). Thus, this transformation complementation is used with the T. gondii cDNA librar, system or a construct which contains some or all of the chorismate synthase gene to transform the AroC mutant. Functional enzyme is then demonstrated. J. Imm unizations Of M ice For Polvclon.il Antibody Production:
As an alternative approach if complementation studies are unsuccessful and the monoclonal antibodies to a plant protein are not cross reactive, purified plant protein is used to immunize mice to raise polyclonal antibodies to each enzyme Where necessary, antibodies to the pertinent enzymes are generated in mice, ND4 outbred mice are immunized with 20 μg of enzyme emulsified in Titermax complete adjuvant injected intramuscularly into their gluteal muscle Two weeks later mice are immunized with a further 20 μg of enzyme emulsified in Titermax. After a further 2 weeks mice receive a further boost of enzyme alone in PBS by the intraperitoneal route. Mice are bled and the serum tested for specificity by the standard Western blotting technique. K. Immunofluorescence
Antibodies used to identify enzymes in the Apicomplexan metabolic pathways disclosed here are used for immunofluorescence studies. Examples are demonstration of alternative oxidase in 71 gondii by immunofluorescence assay (IFA). T. gondii alternative oxidase is immunolocalized to mitochondria. L. ELISAs
ELISAs are used for documenting the presence and quantitating the amounts of alternative oxidase
INI. Reporter Constructs To Demonstrate OrganeUe Targeting Are Made And Characterized As Described Using β Glucoronidase Or Other Chimeric
Constructs
Importance of the targeting sequence for localization of the enzyme to an organeUe is demonstrated with immunoelectronmicroscopy OrganeUe targeting sequences in proteins expressed in bacteria which lack the organeUe cause misfolding of proteins and therein impair protein function
A useful reporter pi otem for a chimeric construct is β glucoronidase. expressed in /•.. coli undo: comi ol of ihe 355 promoter of cauliflower mosaic vu us The glucoronidase alone without the transit sequence is expressed in parallel The transit peptide construct is found in the plastid The control glucoronidase is found in the cytoplasm Antibodies to the chorismate synthase protein are also used to detect the presence of the product of the gene (with the transit sequence) in the plastid and the product of a construct (in which the transit sequence is not present) in the cytoplasm only Further mutations and deletions are made which identify the minimal transit sequence using the same techniques as described above for the entire peptide Antisense, πbozyme or intracellular antibodies directed against the transit sequence nucleic acid or translated protein are useful as medicines The amino acid ofnucleic acid which encodes the transit sequences are the bases for development of diagnostic reagents and vaccines N. Modifications of In ibitot-y Compounds to Improve Oral Absorption
Tissue Distribution (especially to brain and eve).
Tissue distribution is characterized using radiolabeled inhibitor administered to mice with its disposition to tissues measured Compounds are modified to improve oral absorption and tissue distribution O. Methods to Demonstrate Protection Against Coniomt Infections
Infections are established and influence of an inhibitor or combination of inhibitoi s on outcomes arc as outlined below Infections Intections with lυxoplas a gondii Pneumocy stis cat mil. M\ cobacleι nun luhei Lidosis \ l\ κ ohu ieι nun cn ium intracellular and Cι \piospoι idiiim parv um ar e estabhshed alone and together using an immunosuppressed rodent model Endpoints in these infections are
Survival Ability of an inhibitor to protect, measured as prolonged survival Parasitemia' This is measured using isolation of mRNA and RT-PCR with a competitive inhibitor for quantitation
Tissue Parasite Burden This is determined by quantitating brain and eye cyst numbers Inflammatory Response This is noted in histopathologic preparations Representative combinations of inhibitors are NPMG and sulfadiazine, SHAM and atovaquone, NPMG and pyrimethamine, NPMG and SHAM P. Testing of Antimicrobial Compounds
Presence of inhibitory activity of new antimicrobial compounds is tested in enzymatic assays, /// viti o, and //; vivo assays as described above and in the literature Q. Efficacy. Safety. Pliarπiakokinetics. and Therapetitic Tovic Index
The testing in murine models includes standard Thompson tests Testing of antimicrobial agents for efficacy and safety in primate models for malaria is performed Dosages are selected based on safety information available from data bases of information concerning herbicides and the literature Measurements of serum and tissue levels of antimicrobial compounds are performed using assays which detect inhibitor concentrations and concentrations of their metabol.tes Representative assays are high performance liquid chromatographv and assaying i.>>ues for percentage of l adiolabeled compounds admmistei cd using liquid scintilla; , ^ and other assav s also are used R. Carcinogenicity and Tcratogcnicitv
Standard assays to evaluate carcinogenicity include administration of medicines as described above to rodents and observation of offspring for teratogenic effects and carcinogenicity. Observation includes general physical examination, autopsy and histopathologic studies which detect any teratogenic or carcinogenic effects of medicines. S. Constructs to Measure Parasitemia
Portions of genes are deleted and the shorter gene is used as an internal standard in RT PCR assays to measure amount of parasites present (Kirisits, Mui, Mack, McLeod, 1996).
T. Vaccine Constructs and Proteins and their Administration
These are prepared, and sensitivity and specificity are established as is standard in the literature and as described above. Tests and reagents include DNA constructs (Tine et al., 1996) with the appropriate gene or portions of the gene alone or together. with adjuvants. Representative adjuvants include ISCOMS, nonionicsurfactant vesicles, cytokine genes in the constructs and other commonly used adjuvants. Native and recombinant proteins also are used in studies of vaccines Protection is measured using immunologic //; vitro assays, and by assessing survival and reduction of parasitemia and tissue parasite burden and prevention of congenital infection (McLeod ei al., 19SS) U. Preparation of Diagnostic Test Reagents and Diagnostic Tests:
These assays are as described (McLeod and Boyer, 1996) They include ELISAs in w hich antibodies to the proteins or peptides and recombinant proteins are used and PCR methodology in which primers to amplify DNA which encodes the enzymes or parts of this DNA are used A test useful in an outpatient setting is based on conjugation of a monoclonal antibody to human red blood cells with antibody to peptides or proteins. The red cells are cross linked if the antibody to the parasite component interacts with the parasite component and agglutinates the red cells in the blood sample ELISA and PCR can be utilized with samples collected on filter paper as is standard in Newborn Screening Programs and also facilitates outpatient and field use. V. Antisense
Antisense oligonucleotides are short synthetic stretches of DNA and RNA designed to block the action of the specific genes described above, for example, chorismate sv nthase of T. gondii or P. falciparum, by binding to their RNA transcript They turn off the genes by binding to stretches of their messenger RNA so that there is breakdown of the mRNA and no translation into protein Antisense reagents have been found to be active against neoplasms, inflammatory disease of the bo el (Crohn's Disease) and HI V m early trials Antisense oligonucleotides directed against the nucleic acid> w hich encode the essential parasite metabolic process described hei ein ai e efleeiiv e medicines to tr eat these infections Antisense o gonucleotides also ai e dn ecicd ,ιg... -t it ansil sequences m the gene-. Antisense w ill not contain cytosine nucleotides followed by guanines as this generates extreme immune responses (Roush, 1997). Antisense oligonucleotides with sequence for thymidine kinase also is used for regulatable gene therapy.
\V. Ribozvmes and Other Toxic Compounds Ribozymes are RNA enzymes (Mack, McLeod. 1996) and they and toxic compounds such as ricins (Mahal et al, 1997) are conjugated to antisense oligonucleotides (see V, DNA), or intracellular antibodies (see X, for proteins), and these constructs destroy the enzyme. X. Intracellular Antibodies Intracellular antibodies are the Fab portions of monoclonal antibodies directed against the enzymes or portions of them (e.g., anti-transit sequence antibodies) which can be delivered either as proteins or as DNA constructs, as described under vaccines.
V. Dev elopment of New Antiniicrobi. l Compounds Based on Lead Compounds
The herbicide inhibitors comprise lead compounds and are modified as is standard For example, side chain modifications or substitutions of groups are made to make more active inhibitors Their mode of action and structure as well as the enzyme and substrate structures are useful in designing related compounds which better abrogate the function of the enzymes. Examples of such substrate or active site targeting are described above
Nativ e or recombinant protein is used in enzymatic assays and //; vitro assays described abov e are used to test activity of the designed newly synthesized compounds Subsequently, they will be tested in animals Z. Trials to Demonstrate EfTicacv for H uman Disease
Trials to demonstrate efficacy for human disease are performed when /// viti o and murine and primate studies indicate highly likely efficacy and safety They are standard Phase 1 (Safety), Phase II (small efficacv ) and Phase III (larger efficacy with outcomes data) trials For medicines effectiv e against 7 gondii tachyzoites, resolution of mtracerebral 1 υxoplasma brain abscess in HlV-infected individuals with no other therapeutic options available due to major intolerance to available medicines is the initial strategv for Phase 11 trials For medications effective against / gondii bradyzoites absence of development of toxoplasmic encephalitis in indiv iduals w ith I I I \ mtection .Mid indiv iduals w ho ai e scropo -uu e for / gondii infection lollow ed altci a one-m -nth t i calinenl for a 2 v eai pen. ; v nen their CID-t .ounl s ar e low Effective medicines demonstrate efficacy, as 50% of such individuals otherwise develop toxoplasmic encephalitis. When medications efficacious against bradyzoites and recrudescent toxoplasmic encephalitis in patients with AIDS are discovered and found to be safe, similar trials of efficacy and safety for individuals with recurrent toxoplasmic chorioretinitis are performed.
DEFIN'ITIONS
3-deox\-d-arabino-hcptuloonate 7 phosphate s nthase: An enzyme which functions in chorismate synthesis
phosphate synthase (3-phosplιoslιikimatc-l- carboxyvinyltransferase): An enzyme which functions in chorismate synthesis3-NPA: An inhibitor of isocitrate lyase in the glyoxylate pathway and also of succinate dehydrogenase.
3-oxtaprenyI-4-hydroxybenzoatc carboxylyase: An enzyme which functions in ubiquinone synthesis. 4-hydroxybenzoate octaprenyltransferase: An enzyme which functions in ubiquinone synthesis.
8-OH-quiπoline: An inhibitor of the alternative oxidase.
Abscissic Acid Metabolism in Plants: A 15-carbon sequiterpenoid synthesized partly in plastids by the mevalonic acid pathway Abscissic acid protects plants against stress and is.a marker of the plant's maturation and activation of transcription, and causes dormancy Inhibits protein synthesis and leads to specific activation and deactivation of genes
Acctohydroλλ acid synthase: Enzyme w hich catalyzes production of acetohydroxy acids (the branched chain amino acids valine. leucine and isoleucine in plants) Alternative ovidase: An enzyme important in the alternative pathway of respiration
There ai e examples of alternativ e oxidases in plants and trypanosomcs ( I'ollakis et al .
199s. Rhoads Λ. Mclntosh. 19'. : Clark son et al 19 ^) Allcrnativc respiration or energy generation: A different pathway for energy generation utilizing the alternative oxidase and election flow in the electron transport chain which is not dependent on conventional cytochromes or heme
Altered gene includes knockouts. Amide: The R portion of the amino group has an amino group connected to a carbonyl carbon. Glutamine and asparagine are amides. Important for nitrogen transport and storage.
Amylopcctin: A branched starch of plants Also found in T gondii bradyzoites.
Amyloplast: Storage granule for starch in plants. Derived from chloroplasts. Amylosc: An unbranched starch of plants
Anabolism: Formation of large molecules such as starch, cellulose, proteins, fats and nucleic acids from small molecules. Requires input of energy.
Anthranilate phosporibolsyltransfcrase: An enzyme which functions in tryptophan synthesis Anthranilate svnthasc component I: An enzyme which functions in tryptophan synthesis
Anthranilate synthase component II: An enzyme which functions in tryptophan synthesis
Antimicrobial agent: A chemical, for e xample a protein or antisense nucleic acid which effectiv ely inhibits or kills a pathogenic microbe There are examples (Schwab ei al . 1994 Strath e/ al . 1993 , Beckers _■. al 1 995 Blais et al.. 1993 , Fichcra et al . 1995, Pfefferkorn &. Borotz, 1994, Pfefferkorn et al. , 1992, Pukivittaykamee et a/. , 1994)
Apicomplcx: The common feature of Apicomplexan parasites including a conoid and rhoptry organeUes and micronemes at the apical end of the parasite. Apicomplexan parasite: A microorganism that belongs to the Apicomplexan group of parasites. These parasites share a number of morphologic features, including a conoid and rhoptry which are organeUes in the cytoplasm at the apical end of the organism and plastids which are multilamellar structures Representative examples of Apicomplexan parasites include Toxoplasma gondii, Plasmodium, Cryptosporidia and Emieria Aromatic acid aminotransfcrase (aromatic transaminase): An enzyme which functions in tyrosine synthesis.
Aspartatc, glutamate and glutamine synthesis: Involve glutamine synthase and glutamate synthetase and are plastid associated in plants Glutamine synthase in plants is inhibited by the herbicide glufosinate (2 amιno-4-fhydroxymethyiphosphinyl) butanoic acid Glutamine synthase also is present in animals
ATP-phosphofructokinasc: (ATP-PFK) May exert control ov er glycolytic pathway because a step when hexoses phosphate cannot also be used to form sucrose or starch Nearly all animals lack PPi-PFK with plant-like substrate specificity (i c PPi, not ATP) Auxins Growth regulators in plants, which are tryptophan deπvatives Herbicides modeled on auxins ai e structural mimics of fl.esc compounds rather than inhibitor s of auxin function Biochcmical pathw ays: Biochemical pathways include metabolic pathwa\ s A y chemical reaction in life Herein "biochemical pathways" and metabolic pathways" are used interchangeably
Bradyzoite: The slowly replicating life cycle stage of the Apicomplexan parasite Toxoplasma gondii This stage is responsible for latent and recrudescent infection due to this parasite The morphologic features which characterize this parasite stage are electron dense rhoptπes and amylopectin granules Bradyzoites contain a plastid organeUe as do other life cycle stages of this parasite This parasite stage also has specific antigens w hich other life cycle stages do not have, including bradyzoite surface antigen 4 and bradv zoite antigen 5 (lactate dehydrogenase), w hich is an intracellular and cyst matrix antigen Bradyzoites exist together in a structure called a cyst which has a cyst wall and matrix Cysts contain a few to thousands of bradyzoites The cyst containing bradyzoites is a major means of transmission of the organism Toxoplasma gondii hen it is ingested in meat which is not cooked to w ell done It is also a form of the organism responsible for recrudescent eye and brain Jisease in infants and children who are congenitally infected with the parasite and also in patients whose immune system is not normal
Branched chain am o acid sy nthesis (valine leucine and isoleucine) inv olv ing acetohv droxv acid synthase is the first of the series of reactions is another metabolic pathw v present plants but not in animals
Bi .niched ch mi .uniiio acids Ammo acids (v aline leucine ? a isoleucine) the s\ nihe-is ol \ Inch can be inhibited bv sullonv lurca and imid? 7 Jnone herbicide,. There are examples in plants (Kuriki et al., 1996. Morell et al. 1997, Kortostee et al.,
1996, Grula -v al, 1995; Khoshnoodi ei al, 1996).
Branching or Q enzyme: Forms branches in amylopectins between C6 of the main chain and C I of the branch chain. 5 Catabolism: Degradation or breakdown of large molecules to small molecules, often releasing energy.
Calmodulin: is a calcium binding protein (Robson et al, 1993)
Catechol 1,2-deoxygenase (phenol hydroxylase): An enzyme which functions in phenylalanine synthesis. 10 Chloroplast: A DNA-containing multilamellar organeUe of plants and algae associated with metabolic pathways important for photosynthesis and other energy production. Chloroplasts utilize proteins encoded in their ow n DNA and also proteins encoded by nuclear DNA
Chorismate: The product of the action of the enzyme EPSP synthase on shikimate 15 Cliorisniate lvase: An enzyme responsible for the conversion of chorismate to
3 ,4-dihydroxybenzoate
Cliorisniate mutase (7-phosplιo-2-dclιydro-3-dcoxy-arabino-hcplulate-aldolasc):
An enzyme which functions in chorismate synthesis
Cliorisniate s nthase: An enzyme responsible for the conversion of 3-phospho 5- — '-' enolpyruvy! shikimate to chorismate
Chorismate: The product of the action of the enzvme EPSP sv nthase on shikimate Competitive inhibitors: Structures sufficiently similar to the substrate that they compete for the active site of the enzyme. Addition of more natural substrate overcomes effect of the inhibitor.
Components: includes nucleic acids, proteins, peptides, enzymes, peptide targeting sequences, transit peptides, carbohydrates, starch, lipids, hormones, for example those listed in Table 1 and other constituents of metabolic pathways or products derived from these components.
Conventional energy generation: Usual pathways of generation of energy in mitochondria utilizing cytochromes for the transfer of electrons. Conversion of Fats to Sugars in Plants: Occurs by oxidation and the glyoxylate cycle.
Cryptosporidiosis: The disease due to the Apicomplexan parasite Cryptosporidium parvum. It causes self-limited diarrhea or no symptoms in immunologically normal individuals. In individuals who have immunocompromising illnesses, such as the acquired immune deficiency syndrome, Cryptosporidiosis causes life-threatening, persistent, copious, watery diarrhea.
Cryptosporidium parvum: Cryptosporidium parvum is an Apicomplexan parasite which causes cryptosporidiosis
Cyanidc-inscnsilivc, non-liciuc "alternative" oxidase is a metabolic activity that is found in most eukaryotic plants and algae and is absent from multicellular animals The alternative oxidase is a single polypeptide enzvme that lacks heme and can serve as the lei minal election acceptor tυ support respiratory growth of /-.', coli in the absence of heme The coupling efficiency of this oxidase is lower than that of the cyanide-sensiti e cytochrome oxidase That is, not as many protons are pumped across the mitochondrial inner membrane in parallel with electron transfer through the alternative oxidase as they are through the cytochrome oxidase The alternative oxidase appears to be used by plants and algae only under certain conditions The alternative oxidase also is used during different life-cycle stages or under different environmental conditions. Thus, inhibitors of the alternative oxidase may act cooperatively or synergistically with GSAT inhibitors Cyclohexadienyl dehydratase: An enzyme which functions in phenylalanine synthesis
Cyclohexadienyl dehydrogenase: An enzyme which functions in tyrosine synthesis
Cytochrome oxidase: An enzyme utilized in the conventional pathway of energy generation
Dehydroquinate dehydratase: An enzyme which functions in chorismate synthesis Dcoxyribonuclcascs: Enzymes which are hydrolases which hydrolyze DNA (phosphate esters)
Ei eria bovis: Causes bovine eimeπosts
Eimeria maxima and Eimeria tenella: Cause eimeriosis in chickens Eimeria: A group of Apicomplexan parasites which cause gastrointestinal disease in agnculturallv important animals including poultry and cattle These economically important parasites mclude l.imei ia tenella /." maxima and L hovis Endosymbiont: An organism which is taken up by another organism and then lives within it
Enzyme: A protein which catalyzes (makes more rapid) the conversion of a substrate into a product Enzymes are catalysts which speed reaction rates generally by factors between 10s and I O20 They may require ion or protein cofactors Control is by products and environmental changes There are more than 5000 enzymes in living systems. Enzymes are named with common or trivial names, and the sufFix-ase which characterizes the substrate acted upon (e g , cytochrome oxidase removes an electron from a cytochrome) Sequential series of steps in a metabolic pathway Enzymes that govern the steps in a metabolic pathway are sometimes arranged so that a kind of assembly-line production process occurs
EPSP synthase: An enzyme important in the conversion of shikimate to chorismate EST: Expressed sequence tag, a short, single pass cDNA sequence generated from randomly selected library clones Eukaryote: Microorganism or phylogenetically higher organism, the cells of which have a nucleus with a limiting membrane
Fatty Acid Synthesis in Plants: Occurs in chloroplasts of leaves and proplastids of seeds and roots Mainly palmitic acid and oleic acid AcetylCo A carboxylases differ in plants and animals Linoleic acid synthase and linoleneic acid synthase are lipid synthases present in plants and not animals Glv colysis → pyruv ate — >acetv l CoA Example
8 acetyl CoA +7 ATP' + 14 NADPH + 1+H'~* palmityl CoA + 7 CoA + 7 ADP2" + 7H2P04 + 14 NADP* + 7H20
Fragment: Refers to a sequence of nucleic acids or aminoacids, where a fragment is sufficient to function as a component of or product derived from an Apicomplexan as defined herein
Gabaculine: An inhibitor of the enzyme GSAT in the heme synthesis pathway Gene: Nucleotide sequence which encodes an amino acid sequence or another nucleotide sequence Gibcrcllin Metabolism in Plants: Plant hormones which promote plant growth, overcome dormancy, stimulate GI to S transition and shorten S phase of cell cycle, increase hydrolysis of starch and sucrose into glucose and fructose They are derivatives of ent-gibberellane skeleton synthesized from a 2acetyl CoA to mevalonic acid to isopenternyl pyrophosphate to 4 isopentenyl pyrophosphate to geranylgeranyl pyrophosphate to copalylpyrophosphate to kaurene to kaurenol to keaurenal to kaurenoic acid to GA|2 aldehyde to other giberelhns These functions are not clearly established but it is hypothesized that hydrolysis of starch to sugar occurs by inducing formation of amylase enzymes lsoprenoid compounds, diterpenes synthesized from acetate units of acetyl coenzyme A bv mevalonic acid pathway stimulate growth Inhibitors of giberellin synthesis include phosphon D Λmo I 61 S (blocks conversion of gei anyl p iopnosphatc to CO palylpv ropho .phate) phosphon D w hich aUo inhibits conv ei sion ol l oxidalion) loi nulion oi kaurene (J (_ oi cyco el ancv mi ol and pactobutrazol (blocks oxidation of karene and kaurcnoic acid). Young leaves are major sites for giberellin synthesis. These plant hormones which induce hydrolysis of polysaccharide into hexoses are used in glycolysis. When hexoses are abundant, glycolysis is more rapid. GIutamyl-tRNA reductase: An enzyme which functions in heme synthesis. GIutamyl-tRNA synthetase: An enzyme which functions in heme synthesis. Glycolysis in Plants: Several reactions of glycolysis also occur in plastids. Glycolysis = lysis of sugar; degradation of hexosis to pyruvic acid in plants. In animals, degradation of glycogen (animal starch) to pyruvate. Plants form no glycogen. Glyoxylate pathway: The pathway important for lipid degradation which takes acetyl CoA and converts it to CoA-SH through the conversion of isocitrate to C4 acids including succinate. This pathway utilizes isocitrate lyase and also converts glyoxylate to malate, a reaction catalyzed by the enzyme malate synthase. The glyoxysome or Glyoxylate pathway which is cytoplasmic in certain algae involves isocitrate lyase and malate synthase to metabolize lipids and provide C4 acids. A metabolic distinction between autotrophic eukaryotes and heterotrophs is the presence of a glyoxylate cycle This cycle employs two enzymes, isocitrate lyase and malate synthase, to bypass the two decarboxvlation steps of the TCA cycle and enables the utilization of carbon stored in fatty acids for growth. In plants, the enzymes of the glyoxylate cycle are compartmentalized within a unique single-membrane-bound organeUe, the glyoxysome In certain algae, the cycle is entirely cytoplasmic In plants, these enzvmes are most abundant during germination and senescence In animals, the glyoxylate cycle enzymes have been described as being present onlv during starvation
Glyoxysome: An organeUe which in some instances contains enzymes important in the glyoxylate cvcle GSAT: Glutamate-I semialdehyde aminotransferase is the enzyme important in heme synthesis for the conversion of glutamate semialdehyde to ALA (δ-aminolevuhnic acid) Heme synthesis pathway: A metabolic pathway important for generation of heme, porphyrins and other iron sulfated proteins used in mitochondria in the conventional pathway of energy generation This pathway occurs in plant chloroplasts and uses the nuclear encoded enzyme GSAT A metabolic distinction between plants and animals occurs in the heme biosynthesis pathway Non-photosynthetic eukaryotes, including animals, yeast, fungi and protists, produce δ-aminolevulinic acid (ALA), the common precursor of heme biosynthesis, by condensation of glycine and succinate In contrast, photosynthetic organisms, including plants, algae and cyanobacteπa, E coli and some other bacteria synthesize ALA from glutamate (a 5-carbon pathway) Euglena utilize both condensation of glycine and succinate and the 5 carbon pathway to produce δ- aminolevuhnic acid T gondii also has the ALA synthase which results in formation of heme by condensation of glycine and succinate, as does /' falcipai um (Suroha and Padmanaban 1992) Expression of this enzv me is developmentally regulated For example, in plants GS ΛT is most abundant in the leaves There are examples in plants (Matters A. Beale 1 ^ ElicluV al 19S8) I Ici IIICKIC \ compound V hn.li kills plant-, OI algae Hydrolascs: Enzymes which break chemical bonds (e.g., amides, esters, glycosides) by adding the elements of water.
Imidazolinones: Inhibitor of acetohydroxy acid synthase (an enzyme involved in the synthesis of branched chain amino acids, a pathway not in or rarely present in animals, Indolc-3-glyceroI phosphate synthase (antliranilatcisomerasc), (indolcglyccrol phosphate synthase): An enzyme which functions in tryptophan synthesis.
Inhibitor: A compound which abrogates the effect of another compound.
A compound which inhibits the replication or survival of a microorganism or the function of an enzyme or key component of a metabolic pathway or otherwise abrogates the function of another key molecule in a microorganism or other organisms or plant.
Isocitrate lyase: An enzyme which functions in glyoxylate cycle.
Isomcrascs: Enzymes which rearrange atoms of a molecule to form a structural isomer. Isoprenoid Metabolism in Plants: Terpenes are isoprenoids that lack oxygen and are pure hydrocarbons; 5 carbon units with some of the general properties of lipids.
Giberellins and abscidic acid are others of this vast complex of compounds not found in animals
Isoprene units (head) are CH2 - CH3C = CH - CH; ( tail) and are synthesized entirely from acetate of acetyl CoA and restricted to plants Synthesized by mevalonic acid pathway because mevalonatc is an important intermediate Kinases: A subclass of transferases which transfer phosphate groups, especially from
ATP.
Latency: The dormant form of the parasitic infection. One example is with
Toxoplasma gondii in which the infection is not active and the parasite is primarily within cysts in the bradyzoite phase of the life cycle. Another example is the hypnozoite phase of 'Plasntodium falciparum.
Ligases or Synthetascs: Enzymes which join two molecules coupled with hydrolysis of ATP or other nucleoside triphosphate.
Lipascs: Enzymes which are hydrolases which hydrolyze fats (esters) Lipid and terpene synthesis associated with plant plastids. Also see fatty acid synthesis and terpenes.
Lysnscs: Enzymes which form double bonds by elimination of a chemical group.
Malaria: Disease due to pathogenic Plasmodia. Examples are Plasntodium falciparum, Plasmodium virax, Plasmodium ovale, Plasmodium malaria, in humans and Plasmodium knowlesii in monkeys
Malate synthase: An enzyme which functions in glyoxylate cycle
Metabolic pathways: Both anabolism and catabolism consist of metabolic pathways in which an initial Compound A is conv erted to another B, then B is converted to C, C to D and so on until a final product is formed In respiration, glucose is the initial compound, and CO; and H;0 are the final products There are approximately 50 distinct reactions in respiration but other metabolic pathways have fevvci reactions Herein the phrases "metabolic pathways" and "biochemical pathways" are used interchangeably.
Metabolism: Chemical reactions that make life possible. Thousands of such reactions occur constantly in each cell. Microbes: Organisms which are visible only with use of a microscope Some cause disease (are pathogenic).
Microbicidal: An agent (e.g., an antibiotic or antimicrobial compound) which kills microbes.
Mitochondria: An organeUe responsible for the generation of energy. Multilamellar: An adjective which refers to the multiple membranes within an organeUe.
Noncompctitivc inhibitors: Combine with enzymes at sites other than active site
"Not involve": Are not a starting point, a component, or a product of the metabolic pathways described in relation to this invention NPMG: An inhibitor of EPSP synthase in the shikimate pathway
Nucleic Acid: Deoxyribonucleic acid and ribonucleic acid molecules are constructed of a sugar phosphate backbone and nitrogen bases, important in the encoding, transcription and synthesis of proteins
Oocyst: A life cycle stage of a parasite, e.g . Toxoplasma gondii that contains sporozoites 7 gondii sporozoites and oocvsts form only in the cat intestine This foim of the par asite is able to persist in nature in warm, moist soil for up to a year and is highh infectious Spoiulation occui s sev eral d \ s after excretion of oocvsts by members of the cat family (e.g., domestic cats or wild cats such as lions or tigers)
Sporulation must occur before the oocyst becomes infectious
OrganeUe: A structure within a cell. Examples are plastids, mitochondria, rhoptries, dense granules and micronemes Oxidorcductases (oxidases, reductases, dehydrogenases): Remove and add electrons or electrons and hydrogen. Oxidases transfer electrons or hydrogen to 02 only.
Paraminobenzoic acid (PABA): A product of the shikimate pathway in plants.
Parasite: .An organism which lives in or on a host for a period of time during at least one life-cycle stage.
Phagcmid: Plasmid packaged within a filamentous phage particle.
Phosphoribosyl anthranilate isomerase: An enzyme which functions in tryptophan synthesis
Plant-like: Present in algae and higher plants, but not or only rarely, or in unusual circ*mstances in animals
Plasmodium falciparum: One species of Plasmodium which causes substantial human disease
Plasmodium knowlesii: A species of Plasmodium which causes malaria in monkeys
Plastid: A uliilamellar organeUe of plants, algae and Apicomplexan parasites which contains its ow n DNA separate from nuclear DNA Plastids have been described in studies of Apicomplexan parasites which used electron micrographs (Siddall, 1992, Williamson e. <//., 1994, Wilson et ai, 1991 , Wilson et al., 1994, Wilson et al , 1996,
Hackstein . α/., 1995, McFadden et ai, 1996)
Polymerascs: Enzymes which link subunits (monomers) into a polymer such as RNA or DNA PPi phosphofructokinase Type I : An enzyme present in plants that functions in glycolysis and in a number of organisms regulates glycolysis In plants and protozoans
PPi, not ATP (as in animals) is utilized to synthesize Fru-1-6P2 from Fru 6P. Activity is not stimulated in protozoa by Fru-2-6-P2 (Peng & Mansour, 1992, Denton et al.,
1996a,b) Prephenate dehydratase (phenol 2-moιιoxygcnasc): An enzyme which functions in phenylalanine synthesis.
Prephenate dehydrogenase: An enzyme which functions in tyrosine synthesis
Product: The end result of the action of an enzyme on a substrate
Prosthetic group: Smaller organic nonprotein portion of an enzyme essential for catalytic activ ity Flavin is an example
Proteinascs: Enzymes which are hydrolases which hydrolyze proteins (peptide bonds)
PS II: Important alternative means for producing energy within chloroplasts and apparently also described as being present in Apicomplexans
Pyrimethamine: An inhibitor of the conversion of folate to folinic acid and thus an inhibitor of nucleic acids production effective against Toxoplasma gondii
Recrudescence: Reactivation of the parasite Toxoplasma gondii from its latent phase Respiration: Major catabo c process that releases energy in all cells It involves breakdown of sugars to C02 and H20
Ribonuclcases: Enzymes which are hydrolases which hydrolyze RNA (phosphate esters) Salicylic Acid Metabolism in Plants: Salicylic acid is a plant hormone which promotes activity of cyanide resistant respiration SHAM: An inhibitor of the alternative oxidase
Shikimate dehydrogenase: An enzyme which functions in chorismate synthesis Shikimate kinase: (shikimate 3-phosphotransferase) An enzyme which functions in chorismate synthesis
Shikimate pathway A pathway that involves the conversion of shikimate to chorismate and subsequently the production of folate, aromatic amino acids, and ubiquinone This pathway contains enzymes which lead to production of folic acid, ubiquinone, and aromatic amino acids Folate, ubiquinone, and aromatic amino acids are products derived from this pathway in plants There is sequential use of products of these pathways as reactants in subsequent enzymatically catalyzed reactions For example, ubiquinone is an essential coenzyme for both conventional and alternative respiration There are examples in plants, bacteria and fungi (Bornemann et al , 1995 Marzabadi et al , 1996, Ozenberger el al . 1989, Shah el al 1997, Gilchπst S Kosuge I 9S0, Watsh c. _./ , 1990, Weische & Leisterner, 19S5, Green e/ al , 1992. Young -'/ _./ , 1971 ) Sliilviiuatc: I ne Mibsliate lor LI'SP sv nthase Sporozoitc: Another phase of the life cycle of Toxoplasma gondii which forms within the oocyst which is produced only within the cat's intestine. A highly infectious form of the parasite.
Stage specific: A characteristic of the parasite which is expressed or present only in a single life cycle stage or in some but not all life cycle stages.
Starch Degradation in Plants: 3 enzymes: α amylase (attack 1, 4 bonds of amylopectin (to maltose) and amylase (to dextrin). Many activated by Ca++. Located in chloroplasts. β amylase hydrolyzes starch to maltose; starch phosphorylase degrades starch beginning at nonreducing end. (Starch + H2P04 *•* glucose + - Phosphate) Only partially degrades amy lopectin debranching enzymes hydroxy 1.6 branch linkage in amylopectin. Hexoses cannot move out of chloroplasts or amyloplasts thus must be converted to triose phosphate (3-PG aldehyde and dehydroxyacetone P). sucrose + UDP *«■* fructose + UDP-glucose, *= sucrose synthase Starch Formation in Plants: Animals store starch as glycogen and plants store starch as amylose and amylopectin. Starch synthesis is dependent on starch synthase'and branching Q enzymes Mutations in genes encoding these enzymes lead to diminished production of starch In addition, amylopectin synthesis predominates in plant mutants without UDP-glucose-starch glycosyl transferase whereas wild type plants with this enzyme make predominantly amylose and a smaller amount of amylopecti In the mutant UDP-glucose-starch glycosyl transferase appears to be transcriptionally regulated Amino acid motifs that target proteins to plant plastid organeUes have been identified in UDP-glucose starch glycosyl transferase, as have other motifs that determine transit into plastids and mitochondria and these have been used to target the transported proteins in plants. Reactions include' ADPG + small amylose (in glucose) *-*larger amylose (N+l glucose units)+ADP,*= starch synthase K+. Branching or Q enzymes form branches in amylopectins between C6 of the main chain and CI of the branch chain. There are examples in plants (Abel et al., 1996; Van der Leif et al., 1991 ; Van der Steege et α/., 1992).
Starch synthase: catalyzes reaction. ADPG + small amylose (n-glucose units) → larger amylose n+l glucose units + ADP and is activated by K+. Thus, sugars not starch accumulate in plants deficient in K+.
Starch: Major storage carbohydrate of plants, used for energy regeneration Two types composed of D glucose connected by 1. 4 bonds which cause starch chains to coil into helices The two types are amylose and amylopectin Amylopectin is highly branched with the branches occurring between C-6 of a glucose in the main chain and C- 1 of the first glucose in the branch chain (- 1 ,6 bonds) Amyloses are smaller and have fewer branches Amylopectin becomes purple or blue when stained with iodine- potassium-iodine solution. Amylopectin exhibits a purple red color. Control of starch formation is by K- and a light activated sucrose phosphate synthase enzyme, invertase enzymes and the allosteric effect of fructose 2. 6 phiphosphate adenosine diphosphoglucose (ADPG) donates glucoses to form starch Starch in amyloplasts is a principal respiratory substrate for storage organs Substrate reactant: Enzyme substrates have virtually identical functional groups that are capable of reacting. Specificity results from enzyme substrate combinations similar to a lock and key arrangement.
Substrate: The protein on which an enzyme acts that leads to the generation of a product.
Sucrose Formation Reactions in Plants: UTP+glucose 1 phosphate *"»UDPG+PPi
PPi+H20 +2 Pi
UDPG+fructose 6 phosphate-^sucrose-6-phosphate+UDP
Sucrose-6-PHOSPHATE+H20-sucrose+Pi UDP+ATP^UTP+ADP
.-. glucose- 1 -phosphate+fructose 6 phosphate+2 H20+ATP— sucrose 3Pi+ADP Sulfadiazine: An antimicrobial agent effective against Toxoplasma gondii which competes with para-aminobenzoic acid important in folate synthesis Sulfonylureas: Inhibitors of acetohydroxy acid synthase (an enzyme involved in the synthesis of branched chain amino acids, a pathway not or rarely present in animals), Synergy: The effect of a plurality of inhibitors or antimicrobial agents which is greater than the additive effect would be combining effects of either used alone Synergy occurs particularly when the action of an enzyme (which is inhibited) on a substrate leads to a product which is then the substrate for another enzyme which also is inhibited, that is. when the enzymes are in series or follow one another in a pathway This effect occurs because the production of the first enzymatic reaction prov ides less substrate for the second reaction and thus amplifies the effect of the second mlnl.iioi oi antimicrobial agent In contrast, an additive effect is when the effect of the compounds used together is simply the sum of the effects of each inhibitory compound used alone
This most often occurs when the pathways are in parallel, for example, when the effect on the first enzyme does not modify the effect of the second enzyme. Tachyzoite: The rapidly replicating form of the parasite Toxoplasma gondii
Thcileria: An Apicomplexan parasite infecting cattle.
Toxoplasma gondii: A 3-5 micron, obligate, intracellular, protozoan parasite which is an Apicomplexan.
Toxoplasmosis: Disease due to Toxoplasma gondii. Transit (translocation) peptide sequence: Amino acid sequence which results in transit into or out of an organeUe. These have been described in plants (Volkner &
Schatz, 1997; Theg & Scott, 1993). Herein we also call it a "metabolic pathway," although it is part of a component of a metabolic pathway or may function independently of a metabolic pathway Triazinc: An inhibitor of PS II complex.
Tryptophan svnthasc alpha subunit: An enzyme which functions in tryptophan synthesis.
Tryptophan synthase beta subunit: An enzy me which functions in tryptophan synthesis T\ pc 1 PPi phosiihofi'uctokiiiasc is another enzyme present in plants and there is different substrate utilization In phosphofructokmases of animals UDP glucose starch glycosyl transferase: An enzyme involved in production of amylose in plants. The absence of this enzyme leads to starch formation as amylopectin rather than amylose.
USPA: Gene which encodes a universal stress protein. This has been described in E. coli (Nystrom & Neidhardt, 1992).
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