A Leishmania major protein with extensive homology to silent information regulator 2 of Saccharomyces cerevisiae.

We have isolated a cDNA from the protozoan parasite Leishmania major (Lm) that encodes a protein homologous to the Saccharomyces cerevisiae and Kluyveromyces marxianus silent information regulator 2 (SIR2) proteins. The deduced Lm SIR2-related protein (termed LmSIR2rp) consists of 381 amino acids that share 40.5% identity with yeast SIR2, increasing to 60% when substitutions are included. Moreover, the LmSIR2rp aa sequence contains a single potential zinc-binding domain with a CysXaa2CysXaa20CysXaa2Cys motif, and its C-terminal part is rich in Ser (16 Ser residues over 40 aa) which constitute potential sites for phosphorylation. The characterization of a novel Lm gene product which shows considerable similarity to a yeast mating-type regulatory protein provides a new tool to investigate the parasite differentiation control mechanisms and gene expression regulation.

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.

Characterization of a Leishmania antigen associated with cytoplasmic vesicles resembling endosomal-like structure.

In the present study we have used antibodies to Leishmania major promastigote antigens which were eluted from a glutathione-agarose column (LmGbp) and could identify several parasite components among different Leishmania species by using immunoprecipitation and Western blot techniques. The results also showed that some of LmGbp are present among the molecules released into the culture medium. Moreover, immunofluorescence assays clearly demonstrated that LmGbp are expressed by intracellular amastigotes. The electron micrographs of thawed cryosections of L. major-infected cells revealed that the antigens were associated with the membrane of the phagocytic vacuole. Moreover, the Western blot technique allowed us to identify, using other Leishmania species extracts and anti-LmGbp antibodies, a major polypeptide of an apparent molecular mass of 66 kDa. Immunofluorescence studies suggested that the 66 kDa polypeptide is associated with intracytoplasmic vesicles. Cryosections of Leishmania promastigotes improved the fine structure preservation of the organelles and enabled a number of features to be seen, particularly the structures considered as vesicles, which appeared as a complex tubulo-vesicular structure resembling mammalian cell endosomes and Leishmania organelles previously named 'megasomes'. Further studies using antibodies against the native 66 kDa protein will be needed to investigate the localization of the protein at the ultrastructural level and to follow its intracellular vesicular traffic.

Improved specificity of Trypanosoma cruzi identification by polymerase chain reaction using an oligonucleotide derived from the amino-terminal sequence of a Tc24 protein.

In the present study, the diagnostic value of Trypanosoma cruzi recombinant protein (Tc24) was examined. Although antibodies against Tc24 were detected during natural and experimental T. cruzi infections, specificity studies revealed that sera from T. rangeli-infected mice also recognized to some extent Tc24 protein. In addition, sera from Tc24-immunized mice reacted against a 21 kDa polypeptide in T. rangeli extracts. Detailed analysis of the antibody response against 20-40 peptide localized in the Tc24 amino-terminal domain suggests that this sequence is not expressed by T. rangeli 21 kDa antigen. Therefore, the PCR reaction using oligonucleotides corresponding to a 20-26 peptide clearly demonstrated the specificity of the oligoprobes for T. cruzi identification. Positive signals were also found when using blood samples from T. cruzi-infected mice. Taken together, these results suggest that the PCR-based 20-26 assay may be useful in the specific diagnosis of Chagas' disease.

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.