Vibrio cholerae cytolysin (VCC) is a water-soluble, membrane-damaging, pore-forming toxin (PFT) secreted by pathogenic V. cholerae, which causes eukaryotic cell death by altering the plasma membrane permeability. VCC self-assembles on the cell surface and undergoes a dramatic conformational change from prepore to heptameric pore structure. Over the past few years, several high-resolution structures of detergent-solubilized PFTs have been characterized. However, high-resolution structural characterization of small β-PFTs in a lipid environment is still rare. Therefore, we used single-particle cryo-EM to characterize the structure of the VCC oligomer in large unilamellar vesicles, which is the first atomic-resolution cryo-EM structure of VCC. From our study, we were able to provide the first documented visualization of the rim domain amino acid residues of VCC interacting with lipid membrane. Furthermore, cryo-EM characterization of lipid bilayer–embedded VCC suggests interesting conformational variabilities, especially in the transmembrane channel, which could have a potential impact on the pore architecture and assist us in understanding the pore formation mechanism.

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Pore-forming toxins (PFTs) are the unique class of membranedamaging protein toxins that generally act to kill their target cells by forming oligomeric pores in the cell membranes. Pore formation-mediated cytotoxicity exerted by the PFTs is considered to be an ancient and evolutionarily conserved mechanism of cell killing documented in the diverse kingdoms of life, starting from bacteria to higher vertebrates (Iacovache et al., 2010). Particularly, many pathogenic bacteria employ PFTs as their potent virulence factors.PFTs are structurally classified into two distinct categories: (i) α-PFTs that employ α-helices to generate the transmembrane pores, and (ii) β-PFTs that utilize β-strands for the pore-formation (Mondal & Chattopadhyay, 2020;Mondal et al., 2018).Vibrio cholerae cytolysin (VCC) is a prominent member in the bacterial β-PFT family (De & Olson, 2011;Olson & Gouaux, 2005).VCC exhibits potent membrane-damaging hemolytic activity, cytotoxicity, as well as enterotoxicity (Ichinose et al., 1987;Yamamoto et al., 1984). Based on such properties, VCC is considered as a prominent virulence factor of the cholera pathogen, V. cholerae. VCC is secreted by the bacteria in the form of an inactive precursor, termed

cytolysin (VCC) is a β-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

A wide variety of bacterial pathogens secrete a unique class of proteins that attack target cell membranes and form transmembrane oligomeric pores with distinct β-barrel structural scaffolds. Owing to their specific mode of action and characteristic structural assembly, these proteins are termed as β-barrel pore-forming toxins (β-PFTs). The most obvious consequence of such pore-forming activity of bacterial β-PFTs is the permeabilization of cell membranes, which eventually leads to cell death. Bacterial β-PFTs have been studied extensively for nearly past four decades, and their mechanisms of actions have revealed some of the most enigmatic aspects of the protein structure–function paradigm. In most of the cases, β-PFTs are released by the bacteria as water-soluble monomeric precursors, which upon encountering target cell membranes assemble into membrane-inserted oligomeric pores. Structural descriptions are now documented for the water-soluble precursor forms, as well as for the membrane-anchored oligomeric pores of many β-PFTs. These studies have revealed that β-PFTs undergo a series of well-orchestrated structural rearrangements during membrane pore formation. Nevertheless, mechanisms that trigger and regulate distinct steps of the pore-formation processes still remain obscure. Here, we discuss our current understanding regarding structure–function mechanisms in the β-PFT family, with particular emphasis on some of the unsolved issues associated with the β-barrel pore-formation mechanism.