Leishmania-macrophage cellular interactions in vitro

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DESCRIPTION: (provided by the applicant): Our long-term objectives are to elucidate multiple determinants of Leishmania virulence. Specifically addressed in this proposal are the invasive/evasive determinants of these parasites to achieve intracellular entry, survival and replication for a successful infection of their host cells or the macrophages. Leishmaniaamazonensis molecules of relevance proposed for study are nucleoside diphosphate kinases (NdK) and possibly ATPase. Presumably, Leishmania secret these enzymes to deplete extracellular ATP (eATP) accumulated at the site of infection, thereby preventing eATP-induced cytolysis of macrophages and their activation - events detrimental to the parasite survival. NdK and ATPase activities were detected in the spent medium of cultured parasites. The enyzmes were partially purified and found to reverse ATPdependent cytolysis of the J774 macrophages in vitro. The NdK genes are organized in the genome as two different tandem-repeated clusters. Transfection of wild type cells with this gene increases the release of NdK, facilitating their infection of macrophages in vivo. It is proposed to continue the studies as follows: (1) Further attempts to purify NdK isoforms from Leishmania wild type cells, ndk transfected Leishmania and E. coli transformants for biochemical and biological characterization of the secreted enzyme; (2) Analysis of the genes and intergenic regions in the two genomic clusters for sequence heterogeneity and assessment of their differential functions by transfection with particular emphasis on secretory NdK: (3) Further evaluating the biological functions of Leishmania secretory NdK as a scavenger of eATP for: (A) reducing the eATP-induced production of the microbicidal radicals by macrophages: and (B) preserving the integrity of these host cells by preventing their premature eATP-induced cytolysis; and (4) Further evaluation of Leishmania NdK functions by molecular genetic approaches. Understanding the cellular and molecular mechanisms of these and other invasive/evasive determinants of Leishmania will help us develop better therapeutic strategy to deal with leishmaniasis more effectively.