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 amastigote forms (AM) 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 promastigote forms (PRO) 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, 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 toward defining their expression and regulation during parasite growth, development and differentiation. Lipases are ubiquitous enzymes that hydrolyze the ester linkages of fats to form glycerol and free-fatty acids. We hypothesized that lipase activities released by Leishmania might play important roles in the biology of these parasites. In that regard, in FY 2010-11, we continued studies concerning the characterization of the lipase activities present in the in vitro culture supernatants of Leishmania. In addition, we used a PCR-based approach to identify and clone a candidate lipase gene, designated LdLip3, from the Leishmania donovani genome. Sequence analysis revealed that LdLip3 consisted of an ORF of 927-bp and a deduced protein of 308 aa with a predicted molecular mass of 33.0 kDa. Further analysis showed it contained a putative 24aa signal peptide and the absence of an anchor motif, both consistent with a secretory molecule. Moreover, a conserved serine-lipase active site was identified. RT-PCR revealed that this gene is expressed in both PRO and AM. To further characterize this lipase, an epitope-tagged construct was episomally expressed. Western analysis revealed that transfected Leishmania secreted the expressed protein. Cumulative results of coupled immunoprecipitation-enzyme activity assay analyses demonstrated the LdLip3 expressed protein possessed high specific lipase activity. Taken together, our data represent the first report to describe a secretory lipase enzyme from this group of parasites. In that regard, LdLip3 could hold promise as a potential target for therapeutic intervention or diagnostic purposes. As indicated above, all Leishmania parasites are transmitted via the bite of phlebotomine sandflies. Within the alimentary tract of their vectors, Leishmania sp. reside and multiply as extracellular, flagellated PRO. In order to be transmitted to their mammalian hosts, PRO must move anteriorly within the vector digestive tract. During this migration, these parasites must obtain nutrients and energy sources to survive and multiply. Typically, sandflies imbibe plant juices (e.g. sucrose and other polysaccharides) and store them in the crop for subsequent regurgitation into the anterior mid-gut. Recently, we have found that Leishmania mexicana PRO secrete/release an invertase/sucrase activity into their culture medium during their growth in vitro. In contrast L. mexicana axenic AM do not appear to release any detectable invertase activity. To investigate this further, in FY 2010-11, we adopted a molecular approach to identify and characterize the gene encoding this unique parasite enzyme. Results of these studies showed that we had identified a gene that encodes a putative secretory invertase from L. mexicana (LmxM04.0310;LmINV). The LmINV encodes a 71.5 kDa protein with conserved &#946;-fructofuranosidase (&#946;FF) domains and a secretion signal peptide. We found that LmINV was developmentally expressed only by PROs during parasite growth.Further,LmINV-HA episomal constructs were generated and are being used to characterize the biochemical properties of this expressed chimeric enzyme.Interestingly, LmINV-HA transfected PROs expressed more than 100 fold higher levels of secreted LmexINV activity than vector-match controls but, such transfectants were incapable of transformating into axenic amastigotes in vitro. These results suggest that LmxINV has additional roles in the developmental biology of this human pathogen. Previously, our lab has demonstrated that virtually all Leishmania sp., like other trypanosomatid parasites are purine auxotrophs and therefore are, totally dependent upon salvaging these essential compounds from their insect vector and mammalian hosts. Thus in FY 2010-11, we continued studies concerning the biochemical characterization and functional properties of a unique new, 35 kDa, secretory nuclease from L. mexicana. Our studies showed that this enzyme was constitutively released/secreted by both AM and PRO developmental forms of this parasite. Using a molecular approach, we identified, characterized the gene, LmexNucS that encodes this new Class I nuclease family member from these organisms. Sequence analysis revealed that LmexNucS possesses a signal peptide and five structural motifs characteristic of the P1/S1 fungal/plant secretory nuclease family. Northern blot and protein analyses confirmed that LmexNucS was transcribed and differentially translated through the parasites life cycle (AMs>>PROs). Western blot and enzyme activity analyses verified that LmexNucS was constitutively secreted/released by both L.mex PRO and AM developmental forms. In order to delineate the functional properties of the LmexNucS, the gene was episomally over-expressed in LmexNucS-HA transfectants. Results of combined anti-HA immunoprecipitation/ enzyme activity assays showed that LmexNucS was N-linked glycosylated and that it could readily degrade RNA, single stranded DNA, double stranded DNA as well as various synthetic polynucleotide substrates (i.e. poly-A, -I, and -U). Further we demonstrated that LmexNucS was irreversibly inactivated by sulfhydryl reducing agents e.g. DTT. Most importantly, we demonstrated that LmexNucS was constitutively secreted by AMs within infected macrophages both in vivo and in vitro. Cumulatively, our results indicate that the L.mexNucS must play important roles in facilitating the growth, development and survival of this important human pathogen. In that regard, this leishmanial secretory nuclease might be exploited for potential diagnostic or for therapeutic purposes. Taken together, 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
NIH ExPorter
NIH ExPorter