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 sandflies), 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. Such secretory products can readily permeate throughout and presumably alter the host micro-environments in which Leishmania reside. Thus, an understanding of the nature of these parasite products seems essential. To that end, several parasite secretory enzymes and regulatory proteins 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 toward defining their expression and regulation during parasite growth, development and differentiation. For example, our studies have elucidated the enzymatic activity, gene structure and chromosomal locus of the L. mexicana chitinase. Expression constructs of this gene are being used to examine the role of this enzyme in parasite growth, development and differentiation within their sandfly vector and mammalian hosts. In addition, we identified and characterized a new gene encoding a unique parasite invertase [sucrase] enzyme. Results of both biochemical and molecular studies showed that this enzyme was highly developmentally upexpressed in promastigotes suggesting that it presumably affords a unique nutritional advantage to the parasite within its insect vector host. Recently, we also demonstrated that promastigotes of all pathogenic Leishmania species released very high levels of protease enzyme activity into their culture supernatants during their growth in vitro. Results of biochemical and immunochemical labeling studies showed that this protease activity was a soluble, secretory isoform of GP-63 , the major surface membrane zinc-matallo-protease of these organisms. Our results suggest that this secretory protease isoform aids in facilitating parasite growth in its various host environments. In other studies involving gene-deletion, -mutation, and -over expression of the L. donovani calreticulin, we demonstrated that this resident ER chaperone-folding protein is in fact, essential to the survival of these human pathogens. In parallel, 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, at least in part, to regulate protein over-expression of these organisms.