A Genomic Island of Vibrio cholerae Encodes a Three- Component Cytotoxin with Monomer and Protomer Forms Structurally Similar to Alpha-Pore-Forming Toxins

Alpha-pore-forming toxins (a-PFTs) are secreted by many species of bacteria, including Escherichia coli, Aeromonas hydrophila, and Bacillus thuringiensis, as part of their arsenal of virulence factors, and are often cytotoxic. In particular, for a-PFTs, the membrane-spanning channel they form is composed of hydrophobic a-helices. These toxins oligomerize at the surface of target cells and transition from a soluble to a protomer state in which they expose their hydrophobic regions and insert into the membrane to form a pore. The pores may be composed of homooligomers of one component or heterooligomers with two or three components, resulting in bi- or tripartite toxins. The multicomponent a-PFTs are often expressed from a single operon. Recently, motility-associated killing factor A (MakA), an a-PFT, was discovered in Vibrio cholerae. We report that makA is found on the V. cholerae GI-10 genomic island within an operon containing genes for two other potential a-PFTs, MakB and MakE. We determined the X-ray crystal structures for MakA, MakB, and MakE and demonstrated that all three are structurally related to the a-PFT family in the soluble state, and we modeled their protomer state based on the a-PFT AhlB from A. hydrophila. We found that MakA alone is cytotoxic at micromolar concentrations. However, combining MakA with MakB and MakE is cytotoxic at nanomolar concentrations, with specificity for J774 macrophage cells. Our data suggest that MakA, -B, and -E are a-PFTs that potentially act as a tripartite pore-forming toxin with specificity for phagocytic cells.