Effects of cytotoxic necrotizing factor 1 and lethal toxin on actin cytoskeleton and VE-cadherin localization in human endothelial cell monolayers

Integrity of the vascular endothelium is largely dependent on endothelial c ell shape and establishment of intercellular junctions. Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells b y modulating the GTPase activity of p21 Rho family proteins. In the present study we have analyzed the effect of Rho-directed toxins on the actin cyto skeleton and monolayer integrity of endothelial cells. We report here that Escherichia coil cytotoxic necrotizing factor 1 (CNF1) activates Rho in hum an umbilical vein endothelial cells (HUVEC). In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly mo difying peripheral localization of VE-cadherin, a specific marker of vascul ar endothelial cell adherens junctions, Further, Rho activation with CNF1 b locks thrombin-induced redistribution of VE-cadherin staining and gap forma tion in HUVEC monolayers. Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers w ithout disrupting the continuity of VE-cadherin staining, indicating that R ho-dependent stress fibers are not required for maintaining this adhesion r eceptor at sites of intercellular contact. Lethal toxin (Clostridium sordel lii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the act in microfilament system and monolayer integrity in HUVEC cultures.