Single point mutation in Vibrio cholerae cytolysin compromises the membrane pore‐formation mechanism of the toxin

Abstract

Vibrio cholerae cytolysin (VCC) belongs to the family of b-barrel poreforming protein toxins. VCC is secreted by the bacteria as water-soluble monomers, which upon binding to target eukaryotic cells form transmembrane heptameric b-barrel channels. High-resolution 3D structures are described both for the water-soluble monomeric form and the transmembrane oligomeric pore; albeit that our understanding of the mechanistic details of the membrane pore-formation process remains incomplete. Here, we report the characterization of a nonfunctional VCC variant harboring a single point mutation of Ala425Val positioned within a potential membrane-interacting loop in the VCC structure. The mutation appears to affect interaction of the toxin with erythrocytes as well as cholesterolcontaining liposome membrane, without affecting the oligomerization ability of the membrane-bound toxin molecules. The membrane-bound oligomers formed by this VCC mutant do not appear to represent the functional pore assembly of the toxin; rather, such assembly could be considered as being trapped in an abortive, nonfunctional oligomeric state. Our results suggest that the Ala425Val mutation in VCC critically compromises its cholesterol-dependent membrane-interaction mechanism and also abrogates the process of functional membrane pore formation by the toxin.

Functional Mapping of the Lectin Activity Site on the β-Prism Domain of Vibrio cholerae Cytolysin

“…It is also important to note here that the VCC-⌬␤-Prism protein, despite having critically compromised hemolytic activity, could form SDS-stable oligomeric assembly in the membrane lipid bilayer of human erythrocytes, a characteristic feature of the archetypical ␤-PFTs including VCC (Fig. 1D, inset) (24).…”

“…Flow Cytometry-Binding of the full-length VCC and the VCC-⌬␤-Prism variant to human erythrocytes were quantitatively estimated using a flow cytometry-based assay following the methods as described in (24). Briefly, human erythrocytes were incubated with VCC variants for 30 min at 4°C in PBS.…”

“…Briefly, human erythrocytes were incubated with VCC variants for 30 min at 4°C in PBS. Cells were then treated with rabbit anti-VCC antiserum, followed by treatment with fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit antibody as described previously (24). Cells were analyzed by FACSCalibur (BD Biosciences) flow cytometer.…”

“…However, such association may not necessarily represent specific mode of interaction of VCC with the human erythrocytes, as VCC is known to display nonspecific hydrophobicity-driven interactions with the lipid components of the target cell membrane (26). Therefore, toward obtaining a more quantitative estimation of specific mode of interaction of the VCC variants with erythrocytes, binding of the VCC-⌬␤-Prism variant to human erythrocytes was monitored and compared with that of the full-length VCC protein using a quantitative flow cytometry-based assay (24,32). In this assay, we monitored the binding ability of the VCC variant with intact human erythrocytes at low temperature of 4°C.…”

Trapping of Vibrio cholerae Cytolysin in the Membrane-bound Monomeric State Blocks Membrane Insertion and Functional Pore Formation by the Toxin

“…In particular, membrane lipid components like cholesterol have been widely implicated in regulating the membrane pore formation process (25,26). In the case of VCC, the presence of cholesterol in the membrane lipid bilayer has been shown to be an obligatory requirement for efficient membrane oligomerization and functional membrane pore formation by the toxin (27)(28)(29)(30)(31)(32)(33). Cholesterol appears to regulate the mode of action of VCC by physically interacting with the toxin molecule and not by simply altering the physicochemical environment of the target membrane (32).…”