| Abstract: | The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae. Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans. Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum. We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.Vibrio cholerae is known as the cause of cholera, a disease that can lead to fatal dehydration (1). The disease is caused by a few serogroups, and the main factor behind the symptoms is the cholera toxin (CT) encoded by genes located on a prophage mobile genetic element (CTX-φ) that induce severe disruption of intestinal cell function, leading to watery, secretory diarrhea (2). Most serogroups do not cause cholera, as they do not possess the genes for CT, but they cause other diseases e.g., skin, wound, and gastrointestinal infections as well as bacteremia (3)]. The natural reservoirs of V. cholerae are aquatic sources such as rivers, brackish waters, and estuaries and are often associated with copepods, aquatic plants, and shellfish (4). The factors and mechanisms allowing V. cholerae and other Vibrionaceae to survive and thrive in harsh natural environments are of major interest to researchers (5).V. cholerae is motile by virtue of a single polar flagellum. The flagellum export machinery and the virulence-associated type-III secretion system (fT3SS and vT3SS, respectively) are suggested to share a common ancestor (6), explaining their similar structure and molecular organization. The vT3SS allows the delivery of effector proteins through a hollow channel directly to the eukaryotic host cell (7), and flagellar proteins are delivered via the fT3SS channel during flagellum assembly. In the bacterial cytoplasm, effectors secreted by the vT3SS are stabilized by chaperones to prevent aggregation. These chaperones are often encoded by genes adjacent to those encoding the effectors (8). Flagellar proteins are similarly protected by chaperones before they are transported to the growing distal end of the flagellum (9).We use Caenorhabditis elegans as a predatory organism model for identifying and assessing V. cholerae factors, other than CT, that may contribute to bacterial survival and persistence (10). With this model, we discovered a cytotoxin, MakA (motility-associated killing factor A), which we demonstrated to be an essential factor for the cytotoxic activity of V. cholerae in both C. elegans and Danio rerio (zebrafish) (11). We also demonstrated that secretion of MakA occurs via the flagellum in a manner that is undocumented in V. cholerae. Our crystal structure of MakA revealed similarities to ClyA (11), the pore-forming toxin first identified in nonpathogenic Escherichia coli (12, 13) and, subsequently, also in Salmonella enterica (14). ClyA from E. coli is expressed from a monocistronic operon and oligomerizes into a dodecameric pore upon release via membrane vesicles (13, 15, 16). MakA is also structurally related to two proteins from Bacillus cereus, the hemolysin BL binding component B (HBL-B) and the NheA component of the Nhe nonhemolytic enterotoxin. Both of these are considered components of tripartite toxins (17). Recently, a tripartite toxin, AhlABC, was identified and structurally characterized as a pore-forming toxin in Aeromonas hydrophila, and the structure of soluble AhlB shares the general structure described for MakA (18). A similar toxin complex of three proteins, SmhABC from Serratia marcescens, was also reported (19). However, if and how the Ahl and Smh proteins are released during normal growth, or if there is a dedicated secretion system, remain unclear.Here, we identify the proteins from the five V. cholerae genes, vca0880 through vca0884, that are coexpressed from the operon makDCBAE and analyze the crystal structures of MakA, MakB, and MakE. Our in vitro studies revealed that an equimolar combination of the MakA/B/E proteins acted as a tripartite cytotoxin causing lysis of red blood cells and cytotoxicity to epithelial cells. Examination of a large number of bacterial genomes revealed that the mak operon is present in many V. cholerae and other Vibrionaceae strains. These include Vibrio (Listonella) anguillarum, an inhabitant of estuarine and marine coastal ecosystems worldwide and the etiological agent of vibriosis in warm- and cold-water fish (20). The identification and structural characterization of the Mak proteins in V. cholerae presented here reveals a hitherto-unrecognized potential of many pathogenic Vibrionaceae strains to produce the tripartite Mak cytolytic toxin. |
