Herbert A. Avila

Schizodeme analysis of Trypanosoma cruzi stocks from South and Central America by analysis of PCR-amplified minicircle variable region sequences

Kinetoplast DNA (kDNA) was isolated from 56 stocks of Trypanosoma cruzi isolated from human patients, animals and insects from Brazil, Venezuela, Colombia and Costa Rica. Comparison of the patterns of digested kDNA on acrylamide gels led to the grouping of several stocks into two schizodemes. Schizodeme analysis was also performed using a set of 330-bp fragments representing all the variable regions of the minicircle DNA molecules, which were obtained by PCR amplification of the kDNA using conserved region primers. The results of this analysis were consistent with the analysis using total kDNA, but the more informative restriction profiles allowed the construction of additional schizodemes. In addition, two oligomers were generated from variable region sequences of cloned minicircles from a Y and a Cl strain, and these were used as schizodeme-specific probes to detect homologous sequences in the amplified minicircle DNAs. The results indicate that a combination of restriction enzyme fingerprinting and hybridization of amplified variable region minicircle DNA with schizodeme-specific probes can be used for both sensitive detection and classification of T. cruzi.

Leishmania LPG3 encodes a GRP94 homolog required for phosphoglycan synthesis implicated in parasite virulence but not viability

Leishmania promastigotes express an abundant cell surface glycoconjugate, lipophosphoglycan (LPG). LPG contains a polymer of the disaccharide‐phosphate repeat unit Galβ1,4Manα1‐PO4, shared by other developmentally regulated molecules implicated in parasite virulence. Functional complementation of a Leishmania donovani LPG‐defective mutant (OB1) accumulating a truncated LPG containing only the Manα1‐PO4 residue of the first repeat unit identified LPG3, the Leishmania homolog of the mammalian endoplasmic reticulum (ER) chaperone GRP94. LPG3 resembles GRP94, as it localizes to the parasite ER, and lpg3− mutants show defects including down‐regulation of surface GPI‐anchored proteins and mild effects on other glycoconjugates. LPG3 binds cellular proteins and its Leishmania infantum GRP94 ortholog is highly immunogenic, suggesting a potential role in directing the immune response. However, null lpg3− mutants grow normally, are completely defective in the synthesis of phosphoglycans, and the LPG3 mRNA is regulated developmentally but not by stress or heat. Thus the role of LPG3/GRP94 in Leishmania metabolism differs significantly from other eukaryotes. Like the other glycoconjugate synthetic pathways in this parasite, its activity is focused on molecules implicated in virulence rather than viability.