Sphingolipids protect ergosterol in theLeishmania majormembrane from sterol-specific toxins

Abstract

AbstractSusceptibility ofLeishmaniato the first line treatment amphotericin B remains poorly understood. Amphotericin B targets ergosterol, so one approach to improving drug efficacy and reducing side effects could be improving access to ergosterol. While the surface exposure of ergosterol inLeishmaniais unknown, sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids. Here, we tested the ability of theLeishmania majorsphingolipids inositol phosphorylceramide (IPC), and ceramide to shelter ergosterol by preventing binding and cytotoxicity of the sterol-specific toxins streptolysin O and perfringolysin O using flow cytometry. In contrast to mammalian systems,Leishmaniasphingolipids did not preclude toxin binding to sterols in the membrane. However, IPC interfered with cytotoxicity. Ceramide reduced perfringolysin O, but not streptolysin O, cytotoxicity in cells. Ceramide sensing was controlled by the toxin L3 loop. Ceramide was sufficient to protectL. majorpromastigotes from amphotericin B. We propose a mechanism whereby pore-forming toxins engage additional lipids like ceramide to determine the optimal environment to sustain pore formation. Thus,L.majoroffers a genetically tractable model organism for understanding toxin-membrane interactions. Furthermore, our findings suggest targeting ceramide may enhance the efficacy of ergosterol-targeting anti-leishmanial drugs.Abstract ImportanceLeishmaniasis is a neglected tropical disease with ∼1.5-2 million new cases and ∼70,000 deaths annually. One first-line treatment for leishmaniasis is liposomal amphotericin B, which is expensive and damages the kidneys. Cost and side effects can be minimized by improving efficacy. To improve efficacy, we must learn how amphotericin’s target—ergosterol—is protected by other components ofLeishmania. The human ergosterol equivalent is protected by components called sphingolipids. We tested the ability of sphingolipids to protect ergosterol using pore-forming toxins. Pore-forming toxins use ergosterol to bind and killLeishmania. Unlike human cells, toxins bound to ergosterol—indicating that they had access—when sphingolipids were present. However, sphingolipids protectedLeishmaniafrom toxins and amphotericin. Thus,Leishmaniaorganizes sterol-protective components differently from humans. Further, toxins andLeishmaniaserve as a system to understand fundamental rules governing sterol-protecting component membrane organization. We can use this information to help improve drugs targeting sterols.