Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1‐independent manner

The primary virulence factor of Vibrio cholerae, cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhoea characteristic of cholera. Secreted CT binds to GM1 on the surface of host cells to facilitate internalisation. Many bacterial toxins, including CT, have been shown to be additionally delivered via outer membrane vesicles (OMVs). A fraction of the closely related heat labile toxin produced by enterotoxigenic Escherichia coli has been demonstrated to reside on the surface of OMVs, where it binds GM1 to facilitate OMV internalisation by host cells. In this work, we investigated whether OMV‐associated CT is likewise trafficked to host cells in a GM1‐dependent mechanism. We demonstrated that a majority of CT is secreted in its OMV‐associated form and is located exclusively inside the vesicle. Therefore, the toxin is unable to bind GM1 on the host cell surface, and the OMVs are trafficked to the host cells in a GM1‐independent mechanism. These findings point to a secondary, noncompeting mechanism for secretion and delivery of CT, beyond its well‐studied secretion via a Type II secretion system and underscore the importance of focusing future studies on understanding this GM1‐independent delivery mechanism to fully understand Vibrio cholerae pathogenesis.