Attenuation of Leishmania infantum chagasi Metacyclic Promastigotes by Sterol Depletion

Abstract

ABSTRACTThe infectious metacyclic promastigotes ofLeishmaniaprotozoa establish infection in a mammalian host after they are deposited into the dermis by a sand fly vector. SeveralLeishmaniavirulence factors promote infection, including the glycosylphosphatidylinositol membrane-anchored major surface protease (MSP). MetacyclicLeishmania infantum chagasipromastigotes were treated with methyl-beta-cyclodextrin (MβCD), a sterol-chelating reagent, causing a 3-fold reduction in total cellular sterols as well as enhancing MSP release without affecting parasite viabilityin vitro. MβCD-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, MβCD-treated promastigotes caused a higher initialin vitroinfection 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 MβCD-treated promastigotes exposed to healthy human serumin vitro, as well as loss of MSP, a protease that enhances C3b cleavage to iC3b. Mass spectrometry showed that MβCD 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 ofLeishmaniaprotozoa at least in part through retention of membrane virulence proteins.