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A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion.

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.

SUBMITTER: Nadeem A

PROVIDER: S-EPMC8617504 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Vibrio cholerae is a Gram-negative bacterium with a monotrichous flagellum that causes the human disease cholera. Flagellum-mediated motility is an integral part of the bacterial life cycle inside the host and in the aquatic environment. The V. cholerae flagellar filament is composed of five flagellin subunits (FlaA, FlaB, FlaC, FlaD, and FlaE); however, only FlaA is necessary and sufficient for filament synthesis. flaA is transcribed from a class III flagellar promoter, whereas the other four flagellins are transcribed from class IV promoters. However, expressing flaA from a class IV promoter still facilitated motility in a strain that was otherwise lacking all five flagellins (?flaA-E). Furthermore, FlaA from V. parahaemolyticus (FlaAVP; 77% identity) supported motility of the V. cholerae ?flaA-E strain, whereas FlaA from V. vulnificus (FlaAVV; 75% identity) did not, indicating that FlaA amino acid sequence is responsible for its critical role in flagellar synthesis. Chimeric proteins composed of different domains of FlaAVC and FlaD or FlaAVV revealed that the N-terminal D1 domain (D1N) contains an important region required for FlaA function. Further analyses of chimeric FlaAVC-FlaD proteins identified a lysine residue present at position 145 of the other flagellins but absent from FlaAVC that can prevent monofilament formation. Moreover, the D1N region of amino acids 87 to 153 of FlaAVV inserted into FlaAVC allows monofilament formation but not motility, apparently due to the lack of filament curvature. These results identify residues within the D1N domain that allow FlaAVC to fold into a functional filament structure and suggest that FlaAVC assists correct folding of the other flagellins.IMPORTANCEV. cholerae causes the severe diarrheal disease cholera. Its ability to swim is mediated by rotation of a polar flagellum, and this motility is integral to its ability to cause disease and persist in the environment. The current studies illuminate how one specific flagellin (FlaA) within a multiflagellin structure mediates formation of the flagellar filament, thus allowing V. cholerae to swim. This knowledge can lead to safer vaccines and potential therapeutics to inhibit cholera.

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A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion.

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