Functional dissection of Leishmania major membrane components in resistance to cholesterol-dependent cytolysins

ABSTRACT Bacteria use cholesterol-dependent cytolysins (CDCs) to damage eukaryotes. While well-studied in mammals, the mechanisms by which CDCs bind to and kill protozoans remains unclear. CDCs bind to the human pathogen Leishmania major, but only kill in the absence of sphingolipids. The contribution of other leishmanial membrane components to CDC binding and cytotoxicity remains unknown. Here, we used genetic knockouts and inhibitors to determine the contribution of key membrane components to CDC binding and killing in L. major. We analyzed toxin binding and killing using flow cytometry and western blotting. Loss of the virulence factor GP63 enhanced toxicity of perfringolysin O, but not streptolysin O. Plasmenylethanolamine and lipophosphoglycan had minimal contributions to CDC binding and cytotoxicity. Removal of sterols protected cells from CDCs, yet failed to reduce binding. We used CDCs defective in engaging glycans or cholesterol to confirm that CDCs deficient in sterol binding, but not glycan binding, could bound to L. major. Thus, in non-mammalian systems, CDCs may rely on glycans for binding, while using sterols for pore-formation. This suggests that CDCs may not be sterol-specific probes in some non-mammalian systems. We conclude that early-branching eukaryotes use distinct mechanisms from mammals to limit CDC pore-formation and killing. Competing Interest Statement PAK is a co-founder of Ardiyon Bio. The funders had no role in the study's design, data collection, analysis, or interpretation, manuscript writing, or decision to publish the results. Footnotes Key Contribution: Cholesterol-dependent cytolysins streptolysin and perfringolysin rely more on glycans for binding to Leishmania major, while requiring sterol engagement for cytotoxicity.