Document Type



Doctor of Philosophy


Chemical Engineering

First Adviser

Angela C. Brown


Toxigenic Vibrio cholerae is the causative organism responsible for cholera, an infectious disease characterized by acute watery diarrhea. Cholera toxin (CT), the primary virulence factor of V. cholerae, is a prototypical AB5 toxin secreted through the type II secretion pathway. Upon secretion, the toxin initiates endocytosis through the interaction of the CTB subunit with the GM1 ganglioside receptor on the small intestinal cells. In addition to the secretion of the toxin in the free, water-soluble form, V. cholerae secrete biologically active CT in association with outer membrane vesicles (OMVs). OMVs are naturally released spherical buds of the outer membranes of Gram-negative bacteria with an enveloped periplasmic content. Pathogen-derived OMVs are known to mediate the delivery of active toxins and other virulence factors to distant host cells.

In this work, we sought to characterize the association of CT with V. cholerae OMVs and the role of CT-GM1 interaction in cellular uptake of vesicles. 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 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.