GENETICALLY MODIFIED COCCIDIAN PARASITES USEFUL AS VACCINES

Abstract

A genetically modified coccidian parasites wherein expression of phosphatidylthreonine synthase (PTS) is disrupted, a polynucleotide including a nucleotide sequence encoding a phosphatidylthreonine synthase (PTS) enzyme, which catalyzes the production of a lipid, phosphatidylthreonine (PtdThr). PtdThr is an exclusive, major and physiologically important lipid in selected coccidian parasites, which is required for a normal growth and virulence of coccidian parasites. Coccidian parasites, having the expression of PTS disrupted as described herein, are useful as vaccines. The phosphatidylthreonine synthase enzyme and the nucleotide encoding sequences thereof as well as the phosphatidylthreonine phospholipid can find use in diagnostic methods and diagnostic kits or in vaccine and drug development applications.

Claims

1. A coccidian parasite selected from Toxoplasma, Neospora and Eimeria species wherein the expression of endogenous phosphatidylthreonine synthase (PTS) enzyme is disrupted thereby reducing or eliminating the synthesis of a phosphatidylthreonine (PtdThr).

2. The coccidian parasite of claim 1 which is selected from Toxoplasma gondii, Neospora caninum and Eimeria tenella, or is Toxoplasma gondii.

3. The coccidian parasite of claim 1 wherein the PtdThr is a PtdThr of formula (I): (I) wherein R1 and R2 are independently selected from satu-rated and/or unsaturated acyl groups having from 8 to 46 carbon atoms.

4. The coccidian parasite of claim 1, wherein the expression of PTS enzyme is disrupted by inactivating or deleting the corresponding PTS-encoding gene.

5. The coccidian parasite of claim 1 wherein the expression of PTS enzyme is disrupted by inactivating or deleting a nucleotide sequence encoding a protein sequence with at least 30% identity to Toxoplasma gondii PTS (SEQ ID No. 2), Neospora canium PTS (SEQ ID No. 4) or Eimeria tenella PTS (SEQ ID No. 6).

6. The coccidian parasite of claim 1, wherein the expression of PTS enzyme is disrupted by deleting or replacing the polynucleotide sequence, which comprises the nucleotides encoding for the catalytic site of PTS.

7. The coccidian parasite of claim 1 for use as a vaccine.

8. A method for preparing a genetically modified coccidian parasite of claim 1, which comprises disrupting the expression of endogenous phosphatidylthreonine synthase (PTS) enzyme in a coccidian parasite selected from Toxoplasma, Neospora and Eimeria species by inactivating or deleting the gene encoding PTS thereby reducing or eliminating the synthesis of phosphatidylthreonine (PtdThr).

9. The method of claim 8, which comprises inactivating or deleting the gene encoding PTS enzyme by single or double homologous recombination.

10. A polynucleotide comprising a nucleotide sequence encoding a phosphatidylthreonine synthase (PTS) enzyme.

11. The polynucleotide of claim 10 comprising a nucleotide sequence encoding a PTS enzyme having an amino acid sequence with at least 30% identity to Toxoplasma gondii PTS (SEQ ID No. 2), Neospora caninum PTS (SEQ ID No. 4) or Eimeria tenella PTS (SEQ ID No. 6).

12. A phosphatidylthreonine synthase (PTS) enzyme encoded by the nucleotide sequence of claim 10.

13. A phosphatidylthreonine (PtdThr) lipid of formula (I): ##STR00002## wherein R1 and R2 are independently selected from saturated and/or unsaturated acyl groups having from 8 to 46 carbon atoms.

14. The phosphatidylthreonine of claim 13 wherein R1 and R2 independently selected from saturated and/or unsaturated acyl groups having from 16 to 24 carbon atoms.

15. A method to vaccinate an animal or a human by administrating the coccidian parasite of claim 1 to said animal or human.

16. The method of claim 15 to vaccinate an animal selected from sheep, pig, poultry and cattle.

Description

[0037] Accordingly, in several aspects the instant invention also relates to a phosphatidylthreonine synthase (PTS), encoded by the nucleotide sequences described above, and to a phosphatidylthreonine (PtdThr) lipid as also described above.

[0038] The instant invention is further illustrated by the following examples without being limited thereto or thereby.

EXAMPLE 1—IDENTIFICATION AND CHARACTERIZATION OF PTDTHR

[0039] PtdThr was identified by HPLC fractionation of the lipids produced in T. gondii parasites as a major lipid peak. The acyl chain composition of PtdThr was determined by lipidomic analyses of the HPLC-derived fraction using mass spectrometry as described in detail below. The results are shown in FIG. 2. The structure of a PtdThr is shown in FIG. 2(C).

[0040] In a similar way PtdTHr was identified in E. tenella using chromatography and mass spectrometry (FIGS. 8 (A) and (B), wherein PtdThr is referred to as PT in FIG. 8(A)).

Parasite and Host Cell Cultures

[0041] A Δku80 (type I) strain of T. gondii was provided by Vern B. Carruthers (University of Michigan, USA) (Huynh M H, Carruthers V B, agging of endogenous genes in a Toxoplasma gondii strain lacking Ku80, Eukaryot Cell. 2009 Apr.; 8(4):530-9). Tachyzoites of the Δku80 strain of T. gondii were propagated in human foreskin fibroblast (HFF) cells (obtained from ATCC, American type culture collection) using Dulbecco's Modified Eagle Medium (DMEM) containing fetal bovine serum (10%), glutamine (2 mM), minimum essential medium (MEM) non-essential amino acids (100 μm glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, serine), sodium pyruvate (1 mM), penicillin (100 U/ml) and streptomycin (100 μg/ml) in a humidified incubator (37° C., 5% CO.sub.2). Parasites were routinely cultured at a multiplicity of infection (MOI) of 3 every 2-3 days unless stated otherwise. HFF were harvested by trypsinization, and grown to confluence in fresh flasks, dishes or plates for infection assays as per experimental requirements.

Lipid Extraction

[0042] Parasites were syringe-released from infected HFF (MOI, 3; 42-48 hours of infection) and passed twice through 23G and 27G needles. Host debris was removed by filtering the parasite suspension through a 5 μm filter (Merck Millipore, Germany). Cell pellets were re-suspended in 0.4 ml of PBS and lipids were extracted according to Bligh-Dyer (Bligh, E. G. & Dyer, W. J. “A rapid method of total lipid extraction and purification”, Canadian Journal Of Biochemistry And Physiology vol. 37, pp. 911-917, 1959). Briefly, 0.5 ml chloroform and 1 ml methanol were added to the samples, which were then vortexed, allowed to stand for 30 min and then centrifuged (2000 g, 5 min). The supernatant was transferred to a glass tube followed by addition of chloroform and 0.9% KCl (1 ml each). Samples were mixed, centrifuged and the lower chloroform phase containing lipids was transferred to a conical glass tube. Samples were stored at −20° C. in the airtight glass tubes flushed with nitrogen gas until further use.

Lipidomics Analyses

[0043] Total lipids were fractionated on chloroform-equilibrated silica 60 columns. Neutral lipids were eluted by acetone washing of the column. Phospholipids were purified by 5× washing with 1 column-volume of chloroform/methanol/water (1:9:1). Each lipid fraction was collected, dried under nitrogen stream at 30° C., and stored at −20° C. for downstream assays. Lipidomics was performed using automated HPLC electrospray or atmospheric-pressure-ionization tandem mass spectrometry. Internal standard PtdCho (44:2) was mixed with extracted lipids to calibrate the recovery of major lipids. A 10-20 μl aliquot of phospholipid extract in chloroform and methanol (1:1) was introduced onto a HILIC column (Kinetex, 2.6 μm) at a flow rate of 1 ml/min to separate phospholipid classes. MS data were collected using either a 4000 QTRAP (AB Sciex, Concord) or a LTQ-XL (Thermo Scientific, Hampton) mass spectrometer. Data were processed using the proprietary software of the respective instrument manufacturers.

[0044] The main lipidomic results of T. gondii tachyzoites are shown in FIG. 2. FIG. 2 (A) shows the HPLC elution profile with the retention times and relative abundance of phospholipids isolated from extracellular parasites (10.sup.7). X1, eluting between 3.5-3.8 min, represents the lipid identified as PtdThr. FIG. 2 (B) shows the MS analysis of the X1 fraction revealing PtdThr, PtdSer and phosphoethanolamine-ceramide (PEtnCer) species. Individual lipids were identified by their fragmentation patterns and m/z ratios in the negative ionization mode. FIG. 2 (C) shows the MS/MS spectrum of the X1-derived peak (m/z 850) from FIG. 2(B). Note the neutral loss of 101 Da (850.5-749.5 transition) corresponding to the loss of polar head group threonine. The acyl chains were identified by their masses. The acyl chain of position sn-1 was identified as acyl 20:1 and the acyl of position sn-2 was identified as acyl 20:4. Sn-1 and sn-2 refer to the first and second carbon of the glycerol backbone in a given phospholipid. The third carbon contains the polar head group, such as threonine in PtdThr.

EXAMPLE 2—IDENTIFICATION AND CHARACTERIZATION OF PTS

[0045] The genetic origin of PtdThr was determined by searching the parasite database Toxoplasma genomics resource database (ToxoDB) for expression of a relevant enzyme based on the structural similarity of threonine with serine. Two putative PtdSer synthases were found and their complete open reading frames were subsequently cloned (ToxoDB reference numbers TGGT1_273540 and TGGT1_261480). They were found to encode for 614 and 540 amino acid residues respectively. By sequence alignments with mammalian enzymes they were tentatively identified as close homologs of base-exchange type PtdSer synthases (PSS). The results of the alignment are presented in FIG. 1. Based on in silico analyses and the wet-lab results described herein, the two synthases were named TgPSS (Toxoplasma gondii phosphatidylserine synthase) and TgPTS (Toxoplasma gondii phosphatidylthreonine synthase). Unlike PSS occurring across the phyla, homologs of PTS could only be found in selected parasitic (Neospora, Eimeria, Phytophtora) and free-living (Perkinsus) chromalveolates organisms. Of note is the fact that distinct asparagine, histidine and cysteine residues are strictly conserved in all PSS orthologs including in TgPSS, however not in TgPTS, which contains substitutions to glutamate, tryptophan and serine at the equivalent positions (FIG. 1).

Molecular Cloning of T. gondii PTS

[0046] Parasites were syringe-released from infected HFF cells as described above for Example 1. The parasite RNA was isolated using Trizol-based method (Invitrogen) and was subsequently reverse-transcribed into first-strand cDNA.

[0047] The open reading frames of TgPSS and TgPTS were amplified from first-strand cDNA using PfuUltra II Fusion polymerase (Agilent Technologies). The primers used for amplification of the open reading frames are listed on table 1 (SEQ ID Nos. 7-10). The open reading frames were cloned into a commercial pDrive vector (Qiagen, Germany) following the manufacturer's protocol, which allowed DNA sequencing of the amplicons.

TABLE-US-00001 TABLE 1 PCR Primers for annotation of TgPTS and TgPSS open reading frames SEQ ID Primer Number Name Nucleotide Sequence  7 TgPTS-ORF-F 5′-ATGCAACTCCCTTCAAGA-3′  8 TgPTS-ORF-R 5′-TCACTGACTTCGTTCCATTTTCACG-3′  9 TgPSS-ORF-F 5′-ATGTGTCGGGGACCGCCGCT-3′ 10 TgPSS-ORF-R 5′-TCACTCGTCTTTTTGGCCTTC-3′

EXAMPLE 3—GENETIC ABLATION OF PTS IN T. GONDII

[0048] The PTS gene of T. gondii (TgPTS) was disrupted by double homologous cross over. A knockout plasmid was constructed, which contained 5′ and 3′ crossover sequence (COS) of TgPTS flanking a hypoxanthine xanthine guanine phosphoribosyltransferase (HXGPRT) marker as depicted in FIG. 3 (A). The HXGPRT marker was used to allow transgenic selection by mycophenolic acid (MPA) and xanthine (XA) (Donald, et al. “Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation”, The Journal of Biological Chemistry, vol. 271, pp. 14010-14019, 1996).

Knockout Vector Construction

[0049] The 5′ and 3′ crossover sequences (5′COS, 3′COS) of TgPTS were amplified using the genomic DNA isolated from fresh extracellular tachyzoites and using the primers of Table 2 (SEQ ID Nos. 11-14).

[0050] The 5′COS (0.9 kb) and 3′COS (0.8 kb) were cloned at NotI/EcoRI and HpaI/HpaI sites of a pTKO-HXGPRT vector, respectively. The resulting knockout vector contained 5′ and 3′COS of the TgPTS gene flanking a hypoxanthineguanine-phosphoribosyltransferase (HXGPRT) selection marker (pTKO-5′COS-HXGPRT-3′COS).

TABLE-US-00002 TABLE 2 PCR Primers for cloning of the TgPTS-5′COS and the TgPTS-3′COS in the resulting TgPTS knockout vector Primer Name SEQ ID (restriction Nucleotide Sequence Number site) (restriction site underlined) 11 TgPTS-5′COS-F 5′-CTCATCGCGGCCGCGTTCGCCTCGAGTGCTTG-3′ (NotI) 12 TgPTS-5′COS-R 5′-CTCATCGAATTCACGAGCCAGTGGAACGAC-3′ (EcoRI) 13 TgPTS-3′COS-F 5′-CTCATCGTTAACAGCATCTTTATCGATGCGCT-3′ (HpaI) 14 TgPTS-3′COS-R 5′-CTCATCGTTAACTCACTGACTTCGTTCGATTTTC-3′ (HpaI)
Genetic Modification of T. gondii

[0051] The knockout plasmid constructs (also referred to as knockout vector) were transfected into fresh tachyzoites (Δku80) suspended in Cytomix (120 mM KCl, 0.15 mM CaCl.sub.2, 10 mM K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 pH 7.6, 25 mM HEPES pH 7.6, 2 mM EGTA, 56 mM MgCl.sub.2) using a BTX instrument (BTX Harvard Apparatus, USA) with the following conditions: 50 μg DNA, about 10.sup.7 parasites, 2 kV, 50Ω, 25 ρF and 250 μs. The knockout vector (pTKO-5′COS-HXGPRT-3′COS) permitted disruption of the TgPTS gene to generate a Δtgpts mutant by mycophenolic acid (25 μg/ml) and xanthine (50 μg/ml) selection.

[0052] Single (clonal) drug-resistant mutant parasites were isolated by the limiting dilution method. The isolated PTS gene-disrupted parasites (i.e. lacking the conserved catalytic (ECWWD) site, also referred to as the Δtgpts mutant or Δtgpts strain), were screened for 5′- and 3′-crossover events at the TgPTS gene locus by PCR, using screening primers 5′Scr-F/R and 3′Scr-F/R (Table 3, SEQ ID Nos. 15-18) pDrive vector (Qiagen, Germany) for standard cloning and DNA sequencing.

TABLE-US-00003 TABLE 3 PCR primers for 5′ and 3′ recombination screening of the Δtgpts mutant SEQ ID Number Primer Name Nucleotide Sequence 15 TgPTS-KO-5′Scr-F 5′-CGATTCCTTGAGAGCAACTG-3′ 16 TgPTS-KO-5′Scr-R 5′-GACGCAGATGTGCGTGTATC-3′ 17 TgPTS-KO-3′Scr-F 5′-ACTGCCGTGTGGTAAAATGAA-3′ 18 TgPTS-KO-3′Scr-R 5′-GCCATAGAGTTCATTGCGGACTC-3′

[0053] The results of the screening are presented in FIG. 3 (B), which displays PCR images of a representative clonal Δtgpts strain showing specific amplification of 3-kb and 2.4-kb long DNA bands by 5′ and 3′ screening. The parental gDNA and knockout plasmid were included as negative controls, respectively.

[0054] The Δtgpts mutant parasites were validated by the successful insertion of the selection marker (HXGPRT) at the TgPTS locus by ORF-specific PCR using the primers TgPTS-ORF-F and TgPTSORF-R of Table 1 (SEQ ID Nos. 7-10).

[0055] The results are presented in FIG. 3 (C), which displays the resulting ORF-specific PCR confirming a successful insertion of the selection marker in the PTS gene. The Δtgpts cDNA shows amplification of an expected 4.2-kb band as opposed to the 1.8-kb band in the parental cDNA, and none in the plasmid DNA, which corroborated the targeted insertion of selection marker and deletion of the predicted catalytic site (342ECWWD346).

[0056] The identity of all PCR amplicons was confirmed by sequencing, which was performed by the genomics company LGC Limited (Germany) using standard DNA sequencing methods.

EXAMPLE 4—EFFECT OF PTS ABLATION ON PTDTHR PRODUCTION IN T. GONDII

[0057] The phospholipid composition of the Δtgpts and parental strains was investigated by thin layer chromatography (TLC), lipid phosphorus assays and lipidomics analyses. The total lipids were isolated from parasites as explained above for Example 1.

Thin Layer Chromatography and Phosphorus Quantification

[0058] Lipids were resolved by two-dimensional TLC on silica 60 plates (Merck, Germany) using chloroform/methanol/ammonium hydroxide (65:35:5) and chloroform/acetic acid/methanol/water (75:25:5:2.2) as the solvents. They were visualized by staining with iodine vapors and/or ninhydrin spray, and were identified based on their co-migration with authentic standards (Avanti Lipids). The major iodine-stained phospholipid bands were scraped off the TLC plate, and quantified by phosphorus assay as described elsewhere (Rouser et al., “Two dimensional thin layer chromatographic separation of polar lipids and determination of phospholipids by phosphorus analysis of spots”, Lipids, vol. 5, pp. 494-496, 1970).

[0059] The results are presented in FIG. 4.

[0060] FIG. 4 (A) shows the lipid profiles of the indicated strains by two-dimensional TLC. The total lipids (0.8-1×10.sup.8 parasites) were resolved and detected by iodine vapor staining. It is noted that the PdtThr band is absent in the Δtgpts mutant and that an increased production of PtdSer is also observed. FIG. 4 (B) shows the lipid phosphorus measurements of bands from the TLC plates of FIG. 4 (A) showing the lack of PtdThr and induction of PtdSer in the Δtgpts mutant. The reproducibility of the results was confirmed in four independent biological experiments (n=4).

[0061] As expected, the parental parasites harbored a notable amount of PtdThr (FIGS. 4 A & B). Synthesis of PtdThr was completely abrogated in the Δtgpts strain. Concurrently, a three-fold increase in PtdSer was observed, whereas other lipids remained largely unaffected (FIG. 4B). Genetic complementation of the mutant with a functional TgPTS recovered PtdThr, as well as reversed PtdSer content to the normal level.

[0062] Lipidomic Analyses

[0063] The lipidomic analyses using HPLC/MS as described above for Example 1.

[0064] The results are presented in FIG. 5. The lipidomics results confirmed the absence of all PtdThr-derived species in the mutant. In particular, FIG. 5 (A) shows the representative MS profiles of the HPLC-resolved lipid (retention time, 3.5-3.8 min) from the parental and Δtgpts strains (˜107 parasites each) confirming the presence of a major (m/z 850.5, 40:5) and two minor (m/z 824.5, 38:4; m/z 878.5, 42:5) PtdThr species in the parental parasites, which are completely absent in the mutant. It is noted that PtdSer peaks are more intense in the Δtgpts sample, which is in agreement with lipid analysis by TLC and chemical phosphorus assays (FIG. 4).

[0065] Taken together, these results show an autonomous synthesis of PtdThr by TgPTS enzyme, and the loss of PtdThr lipid following its functional ablation in T. gondii.

EXAMPLE 5—EFFECT OF PTS ABLATION IN T. GONDII ON GLIDING MOTILITY AND VIRULENCE OF T. GONDII

Gliding Motility Assays

[0066] Parasites (syringe-released from infected HFF as explained above) were incubated on BSA (0.01%)-coated coverslips in Hanks Balanced Salt Solution (HBSS) for 15 min at 37° C. Samples were fixed in 4% paraformaldehyde and 0.05% glutaraldehyde (10 min), and stained with anti-TgSag1 primary antibody (donated by Jean-Franois Dubremetz, University of Montpellier, France) and Alexa488-conjugated secondary antibody (Life Technologies, Germany). The motile fraction and the trail length were quantified using the ImageJ software (National Institute of Health, USA).

[0067] The results are shown in FIGS. 6 (A & B).

[0068] FIG. 6 (A) shows the motile fractions of different strains on BSA-coated glass coverslips. In total, 500-1000 parasites from 6 independent assays were scored for the presence or absence of a motility trail (immuno-stained with anti-TgSagl antibody) to calculate the motile fractions for each strain. FIG. 6 (B) shows the motility of individual parasite strains, as deduced by the length of TgSagl-stained trails. The error bars for FIGS. 6 (A & B) indicate the mean±SEM. *p<0.05, **p<0.01, and ***p<0.001.

[0069] As depicted in FIGS. 6 (A & B), the Δtgpts strain showed a distinguished reduction in the motile fraction and the trail length when compared to the parental. The strain complemented with PTS activity showed a reverted phenotype similar to the parental strain, ascertaining the specificity of the observation. Such reduced parasite motility eventually causes a poor invasion and egress of/from HFF host cells by the Δtgpts mutant. A collective defect in motility, invasion and egress impairs the lytic cycle and virulence of the Δtgpts mutant.

In Vivo Parasite Infection for Virulence Testing

[0070] C57BL/6 mice (obtained from Janvier Labs, Saint Berthevin, France) were infected with tachyzoites of the Δku80 (parental) or Δtgpts strains. Parasites for in vivo infections were propagated in HFF cells; fresh host-free tachyzoites were released 40 hrs post-infection and filtered (5 μm), as described above. They were injected via intra-peritoneal route (50 parasites of the parental strain and 5×10.sup.2 or 5×10.sup.3 parasites of the Δtgpts strain). 12 animals were monitored for the mortality and morbidity 3 times a day over a period of 4 weeks. An inoculum of 50 parental tachyzoites was used to challenge the Δtgpts-infected surviving animals, which were monitored for additional 4 weeks. A control group of naïve mice (n=4) was also infected with the same parental inoculum. The results are shown in FIG. 6 (C).

Quantification of T. gondii in the Mouse Brain

[0071] Cysts of the ME49 strain of T. gondii were harvested from the brains of female NMRI mice infected with T. gondii cysts 5 to 6 months earlier intraperitoneally as described elsewhere (Agrawal, G. G. van Dooren, W. L. Beatty, B. Striepen, Genetic evidence that an endosymbiont-derived endoplasmic reticulum-associated protein degradation (ERAD) system functions in import of apicoplast proteins, The Journal of Biological Chemistry 284, 33683-91 (2009)). The Δtgpts-vaccinated mice (500 parasites) were challenged with the ME49 T. gondii strain (3 cysts in 200 μl) parasites 4 weeks after vaccination. A control group of naïve animals was also included. Parasite burden in the mouse brain was estimated by counting cysts and semi-quantitative real-time PCR following another 4 weeks of infection with the ME49 T. gondii strain. Brain tissue was mechanically homogenized in 1 ml sterile phosphate-buffered saline and the cysts were counted using a light microscope. For quantitative PCR (qPCR), perfused brain tissue samples were snap-frozen and stored at −80° C. 30 mg tissue was used to purify nucleic acids (QIAgen, Germany). FastStart Essential DNA Green Master (Roche, Germany) was mixed with genomic DNA (90 ng) in triplicate reactions, which were developed in a LightCycler® 480 Instrument II (Roche, Germany). The parasite burden (target: TgB1 gene) was analyzed relative to mouse (reference: argininosuccinate lyase (MmASL)) by estimating target-to-reference ratio (LightCycler® 480 software v1.5.0). Primers used to amplify the TgB1 and MmASL genes are listed in Table 4 (SEQ ID Nos. 19-22).

TABLE-US-00004 TABLE 4 PCR primers for amplification of the TgB1 and MmASL genes SEQ ID Number Primer Name Nucleotide Sequence 19 TgB1-F 5′-TCCCCTCTGCTGGCGAAAAGT-3′ 20 TgB1-R 5′-AGCGTTCGTGGTCAACTATCGATTG-3′ 21 MmASL-F 5′-TCTTCGTTAGCTGGCAACTCACCT-3′ 22 MmASL-R 5′-ATGACCCAGCAGCTAAGCAGATCA-3′

[0072] All in vivo assays were in compliance with the German animal protection laws directed by Landesverwaltungsamt Sachsen-Anhalt, Germany.

[0073] The results are shown in FIG. 7.

[0074] Examination of virulence in a murine model demonstrated that nearly all animals infected with the Δtgpts mutant survived as opposed to the parental strain that was explicitly lethal (FIG. 6C). Importantly, all mice enduring the mutant infection became categorically resistant to a subsequent lethal challenge by a hypervirulent Δku80 strain of T. gondii (type I infection) causing acute toxoplasmosis (FIG. 6C). To further expand the utility of our strain as a potential vaccine against chronic infection, the Δtgpts-infected animals were challenged with the cyst-forming ME49 T. gondii strain (type II infection). Surprisingly, in contrast to naïve animals, the mutant-vaccinated mice showed no signs of chronic stage cysts in their brain tissue (FIG. 7 A-B). Consistently, unlike the infected naïve control mice, no inflammatory lesions in the cortex or meninges of the Δtgpts-immunized animals were observed. In brief, these results demonstrate the in vivo requirement of PtdThr for the parasite virulence, and illustrate the prophylactic potential of a metabolically attenuated whole-cell ‘vaccine’ against acute as well as chronic toxoplasmosis.