Characterization of V. cholerae T3SS-dependent cytotoxicity in cultured intestinal epithelial cells.
AM-19226 is a pathogenic, non-O1/non-O139 serogroup strain of Vibrio cholerae that uses a Type 3 Secretion System (T3SS) mediated mechanism to colonize host tissues and disrupt homeostasis, causing cholera. Co-culturing the Caco2-BBE human intestinal epithelial cell line with AM-19226 in the presence of bile results in rapid mammalian cell death that requires a functional T3SS. We examined the role of bile, sought to identify the mechanism, and evaluated the contributions of T3SS translocated effectors in in vitro cell death. Our results suggest that Caco2-BBE cytotoxicity does not proceed by apoptotic or necrotic mechanisms, but rather displays characteristics consistent with osmotic lysis. Cell death was preceded by disassembly of epithelial junctions and reorganization of the cortical membrane skeleton, although neither cell death nor cell-cell disruption required VopM or VopF, two effectors known to alter actin dynamics. Using deletion strains, we identified a subset of AM-19226 Vops that are required for host cell death, which were previously assigned roles in protein translocation and colonization, suggesting that they function other than to promote cytotoxicity. The collective results therefore suggest that cooperative Vop activities are required to achieve cytotoxicity in vitro, or alternatively, that translocon pores destabilize the membrane in a bile dependent manner.
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Kelly A
KA
Miller
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Mudit
M
Chaand
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Stacy
S
Gregoire
Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Takeshi
T
Yoshida
Department of Dermatology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Lisa A
LA
Beck
Gastroenterology and Hepatology Division, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Andrei I
AI
Ivanov
Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Michelle
M
Dziejman
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001646:Bile / Q000737:chemistry; D001647:Bile Acids and Salts / Q000633:toxicity*; D018938:Caco-2 Cells; D016923:Cell Death / Q000187:drug effects; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D054884:Host-Pathogen Interactions; D006801:Humans; D009997:Osmotic Pressure; D015398:Signal Transduction; D000069296:Type III Secretion Systems / Q000235:genetics* / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D014774:Virulence
D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D000069296:Type III Secretion Systems