Pore Assembly of Bacterial Alpha Pore-Forming Toxin (αPFT), Cytolysin a on Lipid Membranes

Abstract

Due to its significance as the primary barrier to cellular entry, cell membrane integrity is key to its sustenance. To overcome this barrier, pathogens have devised diverse cell membrane disruption strategies. Once such mechanism is the formation of nanopores by pore forming toxins (PFTs) released by several bacteria. PFTs, a major class of bacterial toxins are released as water soluble monomers. Upon binding the target bilayer membrane, they undergo structural rearrangements and oligomerize to form a stable transmembrane pore. The molecular mechanisms that result in efficient pore formation on eukaryotic membranes have been unclear. We dissect the assembly pathway of Cytolysin A (ClyA), a representative αPFT by devising single-molecule fluorescence assays that decouple pore formation stages and report on membrane binding, membrane insertion and oligomerization on artificial membrane bilayers. By evaluating the kinetics of intermediates along assembly pathway in membranes of different compositions, testing putative membrane binding domains with mutations, and combining them with mathematical modeling of erythrocyte lysis kinetics, we present a comprehensive mechanistic framework for ClyA pore formation. Our results show that cellular receptors are not essential for ClyA activity but cholesterol, a key component of mammalian membranes, is critical for cell lysis. Interestingly, initial binding of ClyA to membranes is not cholesterol dependent. However, cholesterol enhances the pore formation dynamics by driving the transmembrane domain insertion and oligomerization. Newly discovered cholesterol binding motifs within the N-terminus and β-tongue regions of the protein impart this selective enhancement of ClyA activity on cholesterol membranes. Finally, a cell lysis kinetics model which incorporates the cholesterol dependence of various pathway intermediates is developed to capture ClyA mediated erythrocyte lysis across different mutants.