Advances in the use of genetically engineered parasites to study immunity to Toxoplasma gondii.

Studying in vivo biology and the host immune response to Toxoplasma gondii has yielded many insights into the pathogenesis of this parasitic organism. It is recognized that this infection in immune competent hosts elicits a strong Th1-type response, which is characterized by the generation of parasite-specific CD4(+) and CD8(+) T cells that produce IFN-gamma and provide protective immunity. One of the problems associated with studying resistance to Toxoplasma has been the lack of reagents to track parasite-specific T cell responses with a high degree of specificity. To overcome this difficulty, it is possible to use a combination of transgenic parasites that are engineered to express well-characterized heterologous reporters or antigens, and T cell hybridomas or naïve T cells that express a T cell receptor specific for the processed peptide. These approaches have provided new insights into parasite dissemination, antigen presentation, as well as immune regulation.

Peptides that mimic Candida albicans-derived beta-1,2-linked mannosides.

Beta-1,2-linked mannosides from Candida albicans phosphopeptidomannan (PPM) bind to macrophages through a receptor independent from the macrophage alpha-linked mannose receptor and stimulate these cells to secrete immune mediators. Anti-beta-1,2-linked mannoside but not anti-alpha-linked mannoside antibodies produced after immunization with neoglycoproteins protect animals from disseminated candidiasis. In this study, peptides that mimic beta-1,2-linked mannosides were isolated using phage display methodology. A phage library expressing random peptides was panned with an anti-beta-1,2-linked mannoside monoclonal antibody (mAb). After three rounds of biopanning, the isolated phages were able to inhibit recognition of C. albicans by the mAb. Sixty percent of the phages had an identical DNA insert corresponding to the peptide sequence FHENWPS that was recognized specifically by the mAb. Injection of KLH-coupled peptide into mice generated high titers of polyclonal antibodies against C. albicans yeast cell walls. The anti-FHENWPS antibodies bound to C. albicans PPM and were inhibited by soluble beta-1,2-mannotetraose. Together, these data provide evidence for mimotopic activity of the peptide selected by biopanning with the anti-beta-1,2-oligomannoside mAb.

Differential expression of two plant-like enolases with distinct enzymatic and antigenic properties during stage conversion of the protozoan parasite Toxoplasma gondii.

The precise molecular mechanisms underlying the switch between the two developmental stages of Toxoplasma gondii, and the metabolic adaptations occurring during this stage conversion are poorly understood. Because inhibitors of mitochondrial respiration are known to trigger differentiation from tachyzoite into bradyzoite stages, we believe that some of the switch components may be sought in the regulation of central carbohydrate metabolism. We have previously described a cDNA encoding a bradyzoite-specific enolase, ENO1. We now report the isolation and characterization of another enolase-encoding cDNA (ENO2) that is expressed preferentially in the tachyzoite stage. The deduced amino acid sequences of ENO1 and ENO2 share 73.65 % identity. They both display significant homologies to plant enolases with the presence of two plant-like peptide insertions, a pentapeptide EWGW(Y)C(S) and a dipeptide EK (or DK). We demonstrate that deletions of the ENO1 pentapeptide motif on its own or together with the dipeptide reduce drastically the affinity for the 2PGA substrate, suggesting that the evolutionary acquisition of these peptides in enolases of land plants and apicomplexan parasites contribute a specific function to their enzymatic activities. T. gondii ENO1 and ENO2 were also expressed as active recombinant enzymes in Escherichia coli. While ENO1 and ENO2 display similar K(m) values, the pure tachyzoite-specific enzyme (ENO2) has a threefold specific activity at V(max) compared with that of the bradyzoite-specific enolase (ENO1). Moreover, immunoblot analyses performed using polyclonal antibodies raised against the recombinant enzymes revealed that the native enolase in tachyzoite and bradyzoite are also antigenically distinct. Taken together, our results indicate that the differences witnessed between the two activities may be instrumental in maintaining glycolysis in pace with the distinct stage-specific requirements of carbohydrate metabolism.

Molecular cloning, functional complementation in Saccharomyces cerevisiae and enzymatic properties of phosphatidylinositol synthase from the protozoan parasite Toxoplasma gondii.

The obligate intracellular parasite Toxoplasma gondii, the causative agent of toxoplasmosis, switches between the rapidly dividing tachyzoite and the slowly replicating bradyzoite in intermediate hosts such as humans and domestic animals. We have recently identified a bradyzoite cDNA encoding a putative phosphatidylinositol (PtdIns) synthase using a subtractive library [Yahiaoui, B., Dzierszinski, F., Bernigaud, A., Slomianny, C., Camus, D., and Tomavo, S. (1999) Mol. Biochem. Parasitol. 99, 223-235]. Here, we report the cloning of another cDNA encoding PtdIns synthase that is exclusively expressed in the tachyzoite stage. The two transcripts are encoded by two different genes, which are stage-specifically regulated. The deduced amino-acid sequence (258 amino acids with a calculated total molecular mass of 27.8 kDa) of the tachyzoite-specific cDNA shares a significant degree of identity (between 26.5 and 30.1%) to the PtdIns synthases from human, rat, Arabidopsis thaliana and yeast. Interestingly, the putative protein encompasses an N-terminal extension that is approximately 40 amino-acids longer than that of PtdIns synthases from other organisms. Functional complementation realized by tetrad analysis of segregants of a Saccharomyces cerevisiae PtdIns synthase-deficient mutant (PIS1/pis1:kanMX4) showed that only the T. gondii putative PtdIns synthase truncated at its N-terminal extension is able to restore the viability of the cells. We demonstrate that this protein expressed in yeast transformants is functionally active in the membrane preparation and requires manganese and magnesium ions for activity. To our knowledge, this is the first report on the molecular cloning and functional analysis of a gene encoding a PtdIns synthase in protozoan parasites.

Targeted disruption of the glycosylphosphatidylinositol-anchored surface antigen SAG3 gene in Toxoplasma gondii decreases host cell adhesion and drastically reduces virulence in mice.

The protozoan parasite Toxoplasma gondii is able to invade a broad range of cells within its mammalian hosts through mechanisms that are not yet fully understood. Several glycosylphosphatidylinositol-anchored antigens found in the parasite membrane are considered as major determinants in the critical interactions with the host cell. We have discovered that two of these surface antigens, SAG1 and SAG3, share significant identity, with considerable similarities in structure, suggesting an overall conserved topology. To investigate their physiological roles further, we have generated T. gondii mutants deficient in SAG3 through gene disruption. The disrupted strains display at least a twofold reduction in host cell invasion when compared with wild-type parasites. This correlated with a similar decrease in host cell adhesion in the SAG3 null mutants. Importantly, the null SAG3 mutants show attenuated infectivity, with a markedly reduced capacity to cause mortality in mice, whereas both wild-type and complemented mutants that re-expressed SAG3 were lethal at the same doses. Taken together, our results indicate that SAG3 is one member of the redundant system of T. gondii receptors that act as ligands mediating host cell recognition and attachment.

Molecular cloning, organellar targeting and developmental expression of mitochondrial chaperone HSP60 in Toxoplasma gondii.

The obligate intracellular protozoan parasite Toxoplasma gondii has a single tubular mitochondrion. During infection, it recruits the host cell's mitochondria abutting to the intracellular vacuole, that contains the parasites. The respective contribution of host and parasitic mitochondria in the intracellular growth of T. gondii remains unknown. Heat shock protein, HSP60 has been reported in all eukaryotes examined, as an essential chaperone required for the folding and multimeric complex assembly of mitochondrial proteins. Here, we report the isolation and molecular characterization of two cDNAs corresponding to a single T. gondii gene coding for HSP60. Using a model fusion protein, preHSP60-chloramphenicol acetyl transferase (CAT), we demonstrate that the classical 22 amino acid mitochondrial presequence and the adjacent 32 amino acids of the mature protein are both required for the in vivo import into T. gondii mitochondria. The T. gondii HSP60 gene composed of five introns and six exons is transcribed into two related but differently spliced transcripts. Whereas the two transcripts can be detected in both developmental stages within the intermediate host, their levels are significantly increased in bradyzoites when compared to tachyzoites. By immunoblot analysis, the predicted 60-kDa protien corresponding to HSP60 was detected in both tachyzoite and bradyzoite forms. Using immunofluorescence assays. the polyclonal antibodies specific to T. gondii HSP60 recognized the mitochondrion in tachyzoites, as expected. In contrast, these antibodies reacted against two unknown vesicular bodies which are distinct from the classical mitochondrial pattern in bradyzoites. Taken together. these expression patterns of mitochondrial chaperone HSP60 suggests stage-specific induction of the respiratory pathway in the protozoan parasite T. gondii.

Effects of water activity, NaCl and smoke concentrations on the growth of Lactobacillus plantarum ATCC 12315.

The effect of water activity (a(w)), NaCl and liquid smoke concentrations on the growth of Lactobacillus plantarum, was studied in MRS broth under anaerobic conditions. A complete 2(3) factorial design was used to determine influential factors and interactions among these factors. NaCl concentration and a(w) had a major effect on the maximum biomass obtained but no interaction influenced this response. Smoke did not affect either biomass nor acidifying capacities.

The protozoan parasite Toxoplasma gondii expresses two functional plant-like glycolytic enzymes. Implications for evolutionary origin of apicomplexans.

The recent discovery of a vestigial, nonphotosynthetic plastid ("apicoplast") in the Apicomplexa has considerably modified our perception of the evolutionary origin of these parasites. Phylogenetic analysis and the presence of four surrounding membranes of the apicoplast provide important support for the hypothesis that apicomplexans have acquired their apicoplast by secondary endosymbiosis, probably from a green alga. This suggests that genes encoding predicted homologs of proteins of green algae or related photosynthetic lineages could have entered the nucleus of apicomplexan parasites by transfer from the ancestor harboring the apicoplast. We describe here complementary DNAs encoding two Toxoplasma gondii glycolytic enzymes, glucose-6-phosphate isomerase (G6-PI) and enolase, which have considerable identities with land plant counterparts. Both cDNAs of T. gondii complement Escherichia coli mutants lacking G6-PI and enolase genes and lead to the expression of active enzymes. In the drug untreatable encysted bradyzoites of T. gondii, G6-PI and enolase genes are overexpressed or exclusively expressed at both transcriptional and protein levels. Moreover, three-dimensional models and protein phylogeny confirmed that G6-PIs and enolases of T. gondii, Plasmodium falciparum, and land plants are closely related. Because these glycolytic enzymes are plant homologs, which differ from those of animals, they will be useful to trace the evolutionary origin of Apicomplexa and might offer novel chemotherapeutic targets in diseases caused by apicomplexan parasites.

Isolation and characterization of a subtractive library enriched for developmentally regulated transcripts expressed during encystation of Toxoplasma gondii.

To survive within infected hosts, Toxoplasma gondii undergoes profound metabolic and morphological changes by differentiating into a cyst characterized by its resistance to the immune system and chemotherapy. The stimulus that triggers Toxoplasma encystation and the molecular mechanisms regulating the bradyzoite phenotype are still unknown. Here, we developed a differentiation method in conjunction with a selective and subtracted cDNA strategy devised to identify developmentally regulated transcripts. We isolated and analyzed 65 cDNA clones. In addition to bradyzoite specific cDNAs previously reported, we demonstrate that twelve genes are exclusively or preferentially transcribed in the encysted bradyzoite forms of T. gondii using semi-quantitative RT-PCR. Among cDNAs identified, are those encoding predicted homologues of chaperones (mitochondrial heat shock protein 60, T-complex protein 1), DNA-damage repair protein, phosphatidylinositol synthase, glucose-6-phosphate isomerase and enolase. The identification of these genes opens the way for further study of molecular mechanisms controlling gene expression during T. gondii encystation.