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 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. ? ? In light of the foregoing, for example, in FY 2008, we identified, characterized and expressed the full length gene that encodes a unique L. donovani LdK39B-kinesin protein. Kinesins are a superfamily of motor proteins that are important components of the microtubule cytoskeletons of most eukaryotic cells. Given the importance and extensive nature of the microtubule cytoskeleton in Leishmania parasites, kinesins are also likely to play central roles in the growth and differentiation of these organisms. In support of that concept, during the course of human visceral leishmaniasis, patients produce a very significant antibody response against the LdK39-kinesins. Despite its clinical relevance, the molecular functions of the LdK39 kinesin proteins have remained unknown. However, in the current studies, a variety of cell and molecular biology approaches were used to identify the subcellular localization of both the endogenous LdK39B kinesin protein and to assess the behavior of several separate LdK39B expressed chimeric gene constucts in transfected parasites. Results of these studies demonstrated that this large (>261 kDa) motor protein was constitutively expressed in both the insect vector and mammalian developmental forms of this organism. Moreover, results of confocal fluorescence, immunoelectron microscopy and subcellular fractionation studies demonstrated that the LdK39-kinesin protein was localized and tightly/integrally bound to the apical poles of the parasite kinetoplast, a unique mitochondrial-DNA containing organelle which is found only in these organisms. It is significance to note that this is the first kinesin to be found associated with this unique parasite organelle. Based on its motor functions and its unique subcellular structural association with the parasite kinetoplast, the LdK39 kinesin might represent a logical target for chemotherapeutic intervention against these pathogens. ? Leishmania and other related trypanosomatid parasites are all obligate purine auxotrophs i.e. they are incapable of synthesizing purines and therefore must salvage these compounds from their hosts in order to survive and multiply. Thus, these organisms have evolved a number of enzymes to salvage essential purines from their hosts. Therefore, to address this issue, in other parallel studies in FY 2008, we used variety of anti-sense RNA methods, to completely abrogate the expression and functional activity of a unique, bi-functional trypanosomatid parasite purine salvage enzyme, i.e. the 3-nucleotidase/nuclease. Results of these studies demonstrated for the first time that this unique parasite purine-salvage enzyme was essential for the survival, growth and development of these organisms. Thus, these results indicated that this critical enzyme might represent a logical target for potential therapeutic intervention against these auxotropic parasitic organisms.? In higher eukaryotes, it has been shown that protein translocation across the endoplasmic reticulum (ER) is often mediated by the Signal Recognition Particle (SRP). Thus, in FY 2008 this SRP hypothesis was investigated in collaborative studies, using several different trypanosomatids. In these studies, we down-regulated the SRP using two approaches, i.e. RNAi silencing of genes encoding SRP proteins and the over-expression of dominant-negative mutants of 7SL RNA. These studies showed that down-expression of the SRP was not detrimental to the expression of proteins containing endogenous signal peptides. However, the overall results of these studies demonstrated that, as in bacteria, but in contrast to mammalian cells, the trypanosomatid SRP are generally essential/necessary for biogenesis of surface membrane targeted proteins.? 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.
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