Abstract
Cholera toxin (CT), the major virulence factor of Vibrio cholerae, is an AB5 toxin secreted through the type II secretion system (T2SS). Upon secretion, the toxin initiates endocytosis through the interaction of the B pentamer with the GM1 ganglioside receptor on small intestinal cells. In addition to the release of CT in the free form, the bacteria secrete CT in association with outer membrane vesicles (OMVs). Previously, we demonstrated that strain 569B releases OMVs that encapsulate CT and which interact with host cells in a GM1-independent mechanism. Here, we have demonstrated that OMV-encapsulated CT, while biologically active, does not exist in an AB5 form; rather, the OMVs encapsulate two enzymatic A-subunit (CTA) polypeptides. We further investigated the assembly and secretion of the periplasmic CT and found that a major fraction of periplasmic CTA does not participate in the CT assembly process and instead is continuously encapsulated within the OMVs. Additionally, we found that the encapsulation of CTA fragments in OMVs is conserved among several Inaba O1 strains. We further found that under conditions in which the amount of extracellularly secreted CT increases, the concentration of OMV-encapsulated likewise CTA increases. These results point to a secondary mechanism for the secretion of biologically active CT that does not depend on the CTB-GM1 interaction for endocytosis.