Sphingomyelin-cholesterol synergy facilitates conformational maturation of the cytolysin A pore.

Abstract

Bacterial pore-forming toxins breach host barriers by converting water-soluble monomers into transmembrane pores extracellularly, among the most radical transitions in structural biology. We show that synergy between host sphingomyelin (SM) and cholesterol (CHOL) facilitates the conformational maturation of Cytolysin A (ClyA), lowering the energetic barrier to membrane insertion. Combining vesicle and suspended-lipid-bilayer assays, molecular dynamics simulations, cell permeabilization and biophysical measurements, we demonstrate that SM-CHOL synergy selectively lowers the barrier for unfolding the membrane-bound β-tongue motif-the critical step in ClyA maturation-generating a reactive molten-globule intermediate, and directs its refolding into the α-helical conformation required for pore formation. Conserved lysines K175 and K206 are the cationic anchors for this interaction; anchor mutants uncouple membrane binding from conformational maturation, distinguishing this specific lipid-mediated folding event from bulk membrane order. As assembly proceeds, the toxin reciprocally remodels the membrane and dismantles liquid-ordered domains, coupling folding to disruption of host lipid organization. These findings establish a paradigm of lipid-mediated conformational catalysis, revealing how pathogens might have co-evolved to exploit host lipid complexity to overcome membrane-insertion barriers.

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