Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

The infectious metacyclic promastigotes of Leishmania protozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. Several Leishmania virulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). Metacyclic Leishmania infantum chagasi promastigotes were treated with methyl-beta-cyclodextrin (M beta CD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viability in vitro. M beta CD-treated promastigotes were more susceptible to complement-mediated lysis than untreated controls and reduced the parasite load 3-fold when inoculated into BALB/c mice. Paradoxically, M beta CD-treated promastigotes caused a higher initial in vitro infection rate in human or murine macrophages than untreated controls, although their intracellular multiplication was hindered upon infection establishment. There was a corresponding larger amount of covalently bound C3b than iC3b on the parasite surfaces of M beta CD-treated promastigotes exposed to healthy human serum in vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that M beta CD promotes the release of proteins into the extracellular medium, including both MSP and MSP-like protein (MLP), from virulent metacyclic promastigotes. These data support the hypothesis that plasma membrane sterols are important for the virulence of Leishmania protozoa at least in part through retention of membrane virulence proteins.