Sphingolipids protect ergosterol in the Leishmania major membrane from sterol-specific toxins

Susceptibility of Leishmania to 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 in Leishmania is unknown, sterols in mammalian cells can be sheltered from sterol-binding agents by membrane components, including sphingolipids. Here, we tested the ability of the Leishmania major sphingolipids 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, Leishmania sphingolipids 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 protect L. major promastigotes 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. major offers 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 Importance Leishmaniasis 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 of Leishmania. 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 kill Leishmania. Unlike human cells, toxins bound to ergosterol—indicating that they had access—when sphingolipids were present. However, sphingolipids protected Leishmania from toxins and amphotericin. Thus, Leishmania organizes sterol-protective components differently from humans. Further, toxins and Leishmania serve as a system to understand fundamental rules governing sterol-protecting component membrane organization. We can use this information to help improve drugs targeting sterols.