Our current understanding of the biogenesis of extracellular vesicle (EVs) and their functions in Leishmania infections is still rudimentary. All eukaryotic cells including macrophages that harbor Leishmania parasites, release EVs into the culture medium and presumably into the lesion environment as well. Leishmania infection may alter either the quantity or the composition of EVs that are released from infected cells (iEVs). Parasite- derived molecules that are released in iEVs could become biomarkers in the blood of an infected host, which could inform on the progress of the infection. Alternatively, parasite-derived molecules in iEVs, working in concert with host cell-derived molecules, could condition cells in the vicinity of infected cells to promote lesion development. It is presently not known how Leishmania parasites contribute to the development of the parasite lesion. That is an important gap in our understanding of Leishmania pathogenesis. Knowledge of the identity of host and parasite-derived molecules in iEVs and the underlying mechanisms involved in their release could reveal new targets for interventions to control Leishmania infections. There are less than a handful of primary reports that have described the composition of iEVs from Leishmania-infected cells and their functions. Those studies have shown that at least one parasite derived molecule, gp63 is included in iEVs released from Leishmania mexicana-infected cells. In our preliminary studies, we have identified more than 25 parasite-derived proteins in iEVs from L. donovani-infected macrophages. One of those parasite molecules is a putative parasite homolog of a family of mammalian molecules that function in angiogenesis. We have also identified over 100 host cell molecules that are preferentially released in iEVs. Using the Ingenuity Pathway Analysis program, it was revealed that approximately seventeen of those molecules had been shown to contribute to angiogenesis in other systems. From those and other preliminary findings, our working hypothesis is that Leishmania infection induces the release of host and parasite-derived molecules in iEVs that promote neovascularization of Leishmania lesions. We propose to fully elucidate the molecular composition of iEVs from Leishmania infected cells (Aim 1). To obtain a more detailed understanding of iEV biogenesis, we will characterize the expression of a putative parasite homolog of the Vasohibin family of molecules that we have identified in the proteome of iEVs (Aim 2). In mammals, Vasohibins have been implicated in angiogenesis. Finally, we will commence to evaluate the contributions of iEVs to lesion neovascularization (Aim 3). Together, we propose studies that will lead to a greater understanding of iEV biogenesis and the contributions of iEVs in the promotion of lesion development. These studies will reveal new targets for approaches to control Leishmania infections.
Studies are proposed to elucidate the molecular composition of extracellular vesicles released by Leishmania- infected cells and to then evaluate their contributions to the neovascularization in Leishmania lesions.