BIOCHEMICAL AND MOLECULAR CHARACTERIZATION OF ENZYMES SECRETED BY LEISHMANIA

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Ongoing studies in this project concern the basic cell biology, biochemistry and molecular biology of Leishmania, a group of protozoan pathogens of humans. All Leishmania parasites undergo a cyclic developmental cycle: 1) in mammals, they transform into and multiply as obligate intracellular [amastigote] forms within the phago-lysosomal system of host macrophages and 2) in their hematophagous sandfly-vector hosts, they differentiate into, and multiply as, extracellular [promastigote] forms within the insect?s alimentary tract. Various species of this organism cause over 12 million cases of human disease worldwide. In infected humans, these parasites destroy macrophages within the skin or internal organs (spleen, liver and bone marrow) causing either disfiguring ulcerative cutaneous lesions (e.g., caused by L. mexicana) or degenerative and fatal visceral disease (e.g., caused by L. donovani). Studies from our laboratory have established that Leishmania parasites are apically polarized secretory cells and that they constitutively secrete over 40 different soluble protein, glycoprotein and carbohydrate constituents into their growth media in vitro. Such soluble, extracellularly secreted products can readily diffuse away from these parasites and permeate their environments. In nature, these organisms reside and multiply within various aqueous micro-environments in their several hosts. Since these organisms actively secrete a variety of different enzymes which could alter such host microenvironments, an understanding of the nature of these parasite enzymes seems essential. To that end, several parasite secretory enzymes are investigated toward defining their functional roles in the survival, maintenance, growth and transmission of these organisms. Further, genes encoding these proteins are being 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 L. donovani. We also showed that all pathogenic Leishmania possess a highly conserved locus for this gene and express antigenically related secretory chitinase activities. Similarly, the genes for several different members of the unique leishmanial secretory acid phosphatase family were characterized from L. donovani and shown to be functionally conserved in other related leishmanial species. Fusion constructs of these genes in tandem with green fluorescent protein (-GFP) were made and used to delineate the targeting signals encoded in these enzymes. Experiments involving gene-deletion, -mutation, -over expression and - reconstitution are in progress toward determining whether their encoded enzymes are, in fact, essential to the survival of these human pathogens.