Cell And Developmental Biology Of Trypanosomatid Parasites

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Abstract

Leishmania are obligate intracellular protozoan pathogens of humans. Within infected patients, various species of this organism inhabit and destroy macrophages within the skin or internal organs (i.e., spleen, liver and bone marrow). Thus, they cause either ulcerative, non-healing, disfiguring malignant skin lesions (e.g. L. mexicana) or degenerative and most often fatal visceral disease (e.g. L. donovani). According to World Health Organization estimates, these diseases afflict over 12 million patients annually in the Tropics and Neo-tropics worldwide. Our studies are aimed at defining the mechanisms involved in the pathophysiology of these organisms. In that regard, the basic cell, molecular and developmental biology of Leishmania and related trypanosomatid protozoa are investigated toward identifying and characterizing parasite molecules which are essential for the survival of these human pathogens. How these parasites are able to survive, access nutrients, multiply and differentiate within their insect vector and mammalian hosts are questions central to understanding the basic parasitic nature and evolutionary adaptations of these organisms. Since these parasites interact directly with their hosts, knowledge of the composition and functions of their surface membrane and secretory enzymes and other functional proteins seems essential. To that end, unique parasite surface membrane, secreted enzymes and regulatory proteins are identified and biochemically characterized to determine their functional roles in the survival of these organisms. Further, the genes encoding such proteins are being isolated and characterized for the first time, toward defining their expression and regulation during the course of parasite growth, differentiation and development. For example, recently we identified and characterized the genes that encode the unique Leishmania Rab5b-protein. In these biochemical and molecular studies, we demonstrated that this unique L. donovani Rab5b-protein functions in identifying and maintaining the structural identity and integrity of early endosomal compartments in this parasite. We found that this protein also helps to facilitate the proper endosomal trafficking and transit of both secretory and surface membrane proteins into and through the unique endocytic compartments of these parasites. Further, using site-specific GFP tagged-constructs in conjunction with confocal fluorescence microscopy localization studies, we showed that the Ld Rab5b was essential for the normal endocytic trafficking in these organisms. Kinesins are a superfamily of motor proteins that are important components of the microtubule cytoskeletons of most eukaryotic cells. Given the importance of the microtubule cytoskeleton in Leishmania parasites, kinesins are also likely to play central roles in the growth and differentiation of these organisms. For example, a Leishmania K39-kinesin was previously shown to be produced during the course of human visceral leishmaniasis and that infected patients mounted a strong humoral response to a K39 epitope. Thus, the K39 kinesin has been used as a serodiagnostic assays for visceral leishmaniasis but despite its clinical relevance, the molecular function of the K39 kinesin protein remained unknown. In that regard, in ongoing cell and molecular biology studies, we recently identified, cloned, characterized and analyzed the full length K39 kinesin open reading frame from Leishmania donovani. Further, we investigated the localization of the endogenous LdK39 protein in the parasite and assessed the behavior of several separate expressed LdK39 protein chimeric domains. Using a combination of molecular and cell biological approaches, we showed that the endogenous LdK39 kinesin and a truncated GFP-K39 chimeric fusion protein bind to and move along the parasite cytoskeleton in an ATP-dependant manner and that they accumulate at the posterior cell pole of these human parasites. Results of these studies demonstrated that the LdK39-kinesin was a unique motor protein involved in intracellular transport and trafficking along cytoskeletal microtubules and that it was also involved spindle pole division in these parasites. Based on its functions, the LdK39 kinesin might represent a logical target for therapeutic intervention against these parasites. It is significance to note that this is the first kinesin gene to be described from these important human pathogens. In other parallel studies, we used various anti-sense RNA methods, to completely abrogate the expression and functional activity of a unique, bi-functional trypanosomatid surface membrane enzyme, i.e. the 3-nucleotidase/nuclease. Further, results of these studies demonstrated for the first time that this unique parasite enzyme was essential for the survival, growth and development of these parasites. Thus, these results indicated that this critical enzyme might represent a logical target for potential therapeutic intervention against these auxotropic organisms. Previously, it has been shown that protein translocation across the endoplasmic reticulum (ER) is often mediated by the Signal Recognition Particle (SRP). Thus,in collaborative studies, we investigated the SRP in several different trypanosomatids. We down-regulated the SRP using two approaches, i.e. RNAi silencing of genes encoding SRP proteins and over-expression of dominant-negative mutants of 7SL RNA. The overall results of these studies demonstrated that, as in bacteria, but unlike mammalian cells, the trypanosomatid SRP are generally essential for biogenesis of surface membrane proteins.[unreadable] Cumulatively, the results of our recent and ongoing studies continue to provide pertinent and significant information toward understanding the unique pathophysiology of these parasites. In addition, these studies are of practical relevance toward demonstrating whether specific /unique parasite enzymes and regulatory proteins are logical targets for 1) the design of new chemotherapeutic drugs, 2) the development of new diagnostic tools and/or 3) useful as potential vaccines against these human pathogens.