Determining the mechanisms of membrane repair in the human pathogen Leishmania major

About This Grant

PROJECT ABSTRACT Membrane integrity is key to the survival of pathogenic protozoa, but their mechanisms of maintaining membrane integrity are poorly understood. For example, the causative agent of cutaneous leishmaniasis, Leishmania major, maintains membrane integrity using distinct lipids compared to mammalian cells. Targeting key functional differences between mammalian cells and pathogenic eukaryotes in maintaining membrane integrity represents one strategy to developing new and improved therapeutics. There is a critical need to determine the mechanisms by which Leishmania sense damage and reseal damaged membranes. Without this information, the full potential of anti-ergosterol drugs (which compromise membrane integrity) may not be realized, and new membrane- disrupting strategies will not be identified. The long-term goal is to exploit the unique membrane architecture and repair mechanisms in Leishmania to develop highly selective therapies that limit infection. The overall objective for this project is to determine the mechanisms Leishmania uses to prevent and reseal membrane damage. The central hypothesis is that Leishmania senses the influx of oxidizing agents, which triggers Endosomal Sorting Complexes Required for Transport (ESCRT)-mediated membrane resealing and shedding via a Ca2+- independent mitochondrial pathway. The rationale for the project is that Leishmania are evolutionarily distinct from mammals, yet genetically tractable organisms that have unique systems of membrane repair. Determining the differences in membrane resealing between Leishmania and mammals will provide a strong scientific framework in which existing anti-Leishmania therapies can be improved, and new therapies can be developed. To attain the objectives, these specific aims will be pursued: 1) Determine the mechanisms by which Leishmania senses membrane damage, and 2) Determine the contribution of ESCRT to membrane repair in Leishmania. In Aim 1, the working hypothesis that Leishmania sense damage via the influx of reactive oxygen species that depolarize the mitochondrion and alter lipid trafficking will be tested in lipid-deficient L. major by flow cytometry and live cell imaging after challenge with multiple toxins. In Aim 2, the working hypothesis that Leishmania reseal their membrane using Ca2+-independent, ESCRT-dependent shedding of damaged membranes will be tested by measuring repair responses to L. major expressing tagged ESCRT proteins using flow cytometry and high resolution imaging and transiently deleting ESCRT proteins. The expected outcomes are to have determined the mechanisms by which Leishmania sense membrane damage, and to have determined the contribution of Leishmanial ESCRT to membrane repair. The proposed research is innovative because it departs from the status quo by revealing the mechanism of damage-sensing in Leishmania and providing a new paradigm of membrane repair. These results are expected to have a positive impact because a better understanding of how Leishmania sense damage and repair their membrane will provide new drug targets and new paradigms of membrane repair.