(464i) Recognition of Target Cells By Vibrio cholerae outer Membrane Vesicles

Vibrio cholerae is the etiologic agent of cholera, a disease characterized by severe, often fatal diarrhea. Cholera toxin (CT), the major virulence determinant of disease, binds a specific ganglioside, GM1, on intestinal epithelial cells, allowing internalization of the toxin. CT is secreted by the bacteria both in a water-soluble “free” form, as well as associated with outer membrane vesicles (OMVs). OMVs are spherical, lipid-encapsulated vesicles, on the order of 100 nm in diameter, that are secreted by Gram negative bacteria. These vesicles contain bacterial virulence factors, implicated in disease, as well as DNA and other molecules. OMV-associated CT has been demonstrated to be physiologically active; however, the mechanism by which delivery of OMV-associated CT occurs has not yet been demonstrated. In the present study, we sought to characterize the delivery of CT-containing OMVs with the goal of identifying novel therapeutic targets to prevent disease. We hypothesized that CT on the OMV surface might act as a targeting agent, allowing the OMV to bind to the CT receptor, GM1. We have demonstrated that V. cholerae OMVs contain CT both inside and on the surface. At neutral pH, the toxin has no affinity for the anionic lipid components of the bacterial membrane, with the exception of lipid A. This affinity suggests that the CT is located on the OMV membrane surface, where it is obscured by the polysaccharide chains of lipopolysaccharide (LPS) component of the OMV membrane so that the OMV-associated CT is unable to bind GM1 on the cell surface. As a result, V. cholerae OMVs do not bind GM1, and soluble GM1 does not inhibit binding or toxicity of V. cholerae OMVs. Additionally, OMVs purified from a strain of Vibrio cholerae that does not produce CT are trafficked in an identical method as CT-containing OMVs. These results indicate that V. cholerae OMVs recognize target cells in a CT-independent mechanism.