Research Project
10300829
PROJECT ABSTRACT The anti-Leishmaniasis drug amphotericin B has a low therapeutic index, which may be due to protection of ergosterol in the Leishmania plasma membrane. The extent to which Leishmania hides its ergosterol and how it reseals its membrane have never been examined. Thus, there is a critical need to determine the mechanisms by which Leishmania protect their sterols to prevent membrane damage, and by which they reseal damaged membranes. Without this information, the full potential of anti-ergosterol drugs like amphotericin B will not be realized, and improved membrane disrupting strategies will not be identified. The long-term goal is to identify membrane mechanisms and lipids in Leishmania that will provide high selectivity for controlling the parasite. The overall goal is to identify the mechanisms Leishmania use to prevent and reseal membrane damage. The central hypothesis is that Leishmania prevent membrane damage by protecting ergosterol from access to sterol-binding toxins and drugs via the primary Leishmania sphingolipid, inositol phosphorylceramide (IPC), and potently reseals membrane damage via Ca2+-independent repair. The rationale for the project is that Leishmania are genetically tractable protozoa evolutionarily distinct from mammals that are likely to provide key insights into resisting and resealing plasma membrane damage. Determining the differences in membrane defense and repair between Leishmania and mammals will provide a strong scientific framework in which existing anti-Leishmania therapies can be improved, and new therapies developed. To attain the objectives, these specific aims will be pursued: 1) Determine the mechanisms that reduce membrane damage in Leishmania, and 2) Determine the mechanisms that promote membrane repair in Leishmania. In Aim 1, the working hypothesis that Leishmania prevent membrane damage by sheltering vulnerable sterols under IPC will be tested by challenging L. major promastigotes genetically or enzymatically lacking sphingolipids, phospholipids or virulence factors with sterol- binding toxins or detergents and measuring sterol accessibility, toxin binding, and lethality by flow cytometry. 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 membrane shedding, patch repair and ESCRT trafficking in toxin-challenged L. major expressing GFP-tagged ESCRT proteins by flow cytometry, ultracentrifugation, and live cell imaging. The expected outcomes of completing this work are to have defined the mechanisms by which Leishmania protect their sterols from attack, and membrane repair pathways that restore homeostasis in human pathogens evolutionarily distant from mammals. The proposed research is innovative because it departs from the status quo by revealing new paradigms of sterol accessibility and membrane repair. These results will have a positive impact because a better understanding of how Leishmania protect their membrane will provide new drug targets. Targeting protective mechanisms in Leishmania may potentiate drugs like amphotericin B, and provide a selective target for Leishmania and other protozoa.
