(722j) Studies of Mechanism of Vibrio Cholerae Outer Membrane Vesicles Delivery to Target Cells for the Identification of Therapeutic Targets

Vibrio cholerae is a gram negative pathogenic bacterium which causes acute diarrheal disease in regions with poor sanitation. The enterotoxin called cholera (CT) and the endotoxin (LPS) have been identified as the major toxins produced by V. cholerae. Like other gram negative bacteria, V. cholerae naturally produces outer membrane vesicles (OMVs), which mediate the delivery of virulence factors to the host cells. In the case of CT, recent studies have demonstrated that the vesicle-associated CT is biologically active. In the present study, we sought to identify the primary components of the OMVs and host cells that facilitate OMV delivery for the identification of therapeutic targets. Isothermal titration calorimetry (ITC) was used to measure the binding of CT to the OMV-like lipid bilayers composed of varying combinations of the most common phospholipids in the bacterial outer membrane, including phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipin (CL), and LPS. The relative binding affinities of CT with the lipid components of the OMVs were also assessed using lipid dot-blot analysis. Among the lipid species examined, CT was found to bind strongly to the negatively charged CL and Lipid A, less strongly to the anionic PG and unnoticeably to PE, suggesting that the toxin binding to the OMVs is regulated by electrostatic interactions. Consequently, the effect of the ionic strength and pH on the inhibition of the toxin association with the OMVs was further investigated by ITC. Based on the higher affinity of the toxin for the charged lipid components, which comprise the inner leaflet of the OMVs inner leaflet, a trypsin digestion experiment was performed to confirm the location of the toxin relative to the OMVs. Furthermore, the role of the OMV components on the vesiclesâ?? toxicity and binding to the host cells was studied utilizing purified OMVs from V. cholerae and three sets of liposomes (model OMVs) composed of varying combinations of PE, PG, CL, LPS and CT. To address whether the OMVs require CT for cytotoxicity, the binding to and internalization with intestinal epithelial cells by OMVs secreted from both a toxic and a non-toxic strain of the bacteria were evaluated by confocal microscopy. These results shed light on the mechanism by which V. cholerae OMVs are delivered to host cells, allowing us to develop targeted anti-virulence approaches to V. cholera infection.