Abstract
Nephrotoxicity is one common side effect of the first line anti-leishmanial treatment, liposomal amphotericin B. Amphotericin B targets ergosterol, so one approach to reducing dose and side effects could be improving the access of the drug to its target. 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-binding 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. These findings suggest that 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.
Author Summary Leishmaniasis is a neglected tropical disease with ~1.5-2 million new cases and ~70,000 deaths per year. One first-line treatment for leishmaniasis is liposomal amphotericin B, which is expensive and can damage the kidneys. One way to reduce cost and side effects is to reduce the needed dose by improving efficacy. In order to improve its efficacy, we need to learn how its target—ergosterol—is protected by other components of Leishmania. The human equivalent of ergosterol is protected by components called sphingolipids. We tested the ability of sphingolipids to protect ergosterol using pore-forming toxins. Pore-forming toxins need ergosterol to first bind and then kill Leishmania. Unlike human cells, toxins bound to ergosterol—indicating that they had access—even when sphingolipids were present. However, sphingolipids prevented the toxins from killing the cells, and provided protection from amphotericin B. These data reveal that Leishmania organizes its sterol-protective components differently from humans. Further, we can use toxins and Leishmania as a system to understand the fundamental rules governing how sterol-protecting components are organized in the membrane. We can then use this information to help improve liposomal amphotericin B efficacy.
Competing Interest Statement
The authors have declared no competing interest.