Biochemical And Molecular Characterization Of Enzymes

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Ongoing studies in this project concern the cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a dimorphic life cycle: 1) in mammals (humans), they multiply as obligate intracellular forms [amastigotes] within the lysosomal system of macrophages, eventually destroying these cells and 2) within their insect vectors (blood-sucking sandfies), they differentiate and multiply as, extracellular forms [promastigotes] within the alimentary tract and eventually migrate to the mouth parts for transmission. By World Health Organization estimates, Leishmania parasites annually cause well-over 12 million cases of human disease (leishmaniasis) worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (i.e. spleen, liver and bone marrow) causing either large and disfiguring, malignant skin ulcers (e.g. caused by L. mexicana) or degenerative and most often fatal visceral disease (e.g. caused by L. donovani). Previous studies from our laboratory have established that Leishmania parasites constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents. Since such secretory products can readily permeate and presumably alter the host micro-environments in which Leishmania reside, an understanding of the nature of these parasite products seems essential. To that end, several parasite secretory enzymes are being investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins have been identified and characterized for the first time toward defining their expression and regulation during parasite growth, development and differentiation. During the past year, our studies have elucidated the enzymatic activity, gene structure and chromosomal locus of the leishmanial chitinase in both L. donovani and L. mexicana. Further, we showed that all pathogenic Leishmania possess a highly conserved locus for this gene and all of these organisms express structurally related secretory chitinase activities. Similarly, the genes for several new members of the unique leishmanial secretory acid phosphatase (SAcP) family were identified and characterized from L. donovani. Our biochemical and molecular studies showed that this family of enzymes was functionally conserved among all pathogenic leishmanial species including L. major as well as, other more distantly related trypanosomatid parasites of humans. Further, we demonstrated that these conserved SAcPs are synthesized by amastigote forms of all pathogenic Leishmania species during the course of both human cutaneous and visceral disease. Moreover, we made chimeric constructs of these genes in tandem with the green fluorescent protein (-GFP) to determine the unique molecular signals responsible for targeting these enzymes into the parasite secretory pathway. Experiments involving gene-deletion, -mutation, and -over expression are in progress toward demonstrating that these secretory enzymes are, in fact, essential to the survival of these human pathogens. In addition, using GFP constructs of several different truncated surface membrane proteins , we defined a unique endosomal trafficking pathway in Leishmania which interfaces with the secretory pathway and functions to regulate protein over-expression of these organisms.