The cell and molecular biology of the human pathogen, Leishmania, is investigated as a model of both intra- and extracellular parasitism. Emphasis is placed on characterizing both the biochemical functions and gene structure of their surface membrane and secreted proteins toward defining roles for these constituents in parasite survival and development. The gene encoding the unique, trypanosomatid surface membrane enzyme 3'- nucleotidase/nuclease (3'-NT/Nu) was isolated and characterized from L. donovani (Ld). The nutrient-induced over-expression of Ld 3'-NT/Nu enzyme activity was shown to be regulated at the mRNA level by transciptionally-mediated events. A construct of this gene was transfected into E. coli and the resulting expressed protein possessed 3'-NT enzymatic activity. In other studies, three different Ld secretory acid phosphatase genes (SAcP1, -2 and -3) were identified, characterized and their encoded proteins expressed in E. coli. RT-PCR analyses demonstrated that mRNAs from all three SAcP genes were constitutively transcribed in both Ld procyclic promastigotes (Pros) and amastigotes (Am). Using immuno-cytochemistry, SAcP was shown to be constitutively expressed by Ld Am's and to continuously traffick out of parasitophorus vacuoles into numerous secondary lysosomal vesicles which accumulate and fill the cytoplasm of infected macrophages. In other studies, we identified a secretory chitinase from Ld Pros and characterized its biochemical properties. Further, the gene for this enzyme was isolated, characterized and expressed in E. coli as a 51 kDa protein. Transcripts of this chitinase gene were identified via RT-PCR using mRNA from Ld Pros. We have also identified and characterized several other Ld genes which are either uniquely or differentially expressed by Am's and these are being used as probes to study parasite gene-regulated differentiation and development. Finally, arbitrary primed PCR methods were devised and used to identify both intra- and inter-specific gene polymorphisms in various Leishmania as well as to identify and characterize genetic loci which were either differentially or developmentally expressed in these parasites. The current results are of relevance toward identifying targets for the development of new diagnostic, chemotherapeutic and immunoprophylactic agents against these important human pathogens.
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