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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. [ABSTRACT FROM AUTHOR]

ToxR is a transmembrane regulatory protein that controls virulence gene expression in Vibrio cholerae. Previous experiments using λ repressor–ToxR chimeric proteins and a λ repressor-controlled reporter system (OR1 PR–lacZY ) established that ToxR sequences can effectively dimerize the amino-terminal domain of λ repressor in Escherichia coli. However, in E. coliToxR does not respond to environmental signals that control virulence gene expression in V. cholerae. Here, we report the results of experiments designed to test whether environmental signals that modulate virulence gene expression in V. cholerae also modulate a monomer to dimerization transition of λ–ToxR chimeras. When the OR1 PR–lacZY reporter fusion and chimeric proteins were transferred to V. cholerae, we unexpectedly found that λ–ToxR chimeras did not dimerize significantly. Interestingly, experiments evaluating the ability of λ–ToxR proteins to form tetramers in E. coli suggested that λ–ToxR dimers could act co-operatively. Using a redesigned reporter system containing multiple λ operator sites (OR1 OR2 OR3 PR–lacZY ), we found that λ–ToxR could dimerize quite efficiently in V. cholerae. These data imply that multiple DNA binding sites might enhance the ability of ToxR to dimerize in V. cholerae and suggest that ToxR dimers might be capable of co-operative interactions. However, we failed to correlate a monomer–dimer transition of the λ–ToxR chimeras with changes in virulence gene expression in response to environmental signals in V. cholerae. Finally, because of conflicting results in the literature, the importance of membrane localization of ToxR and dimerization of the ToxR periplasmic domain was re-evaluated. This was accomplished by measuring the ability of various chimeric proteins to activate toxin gene expression in both E. coli and V. cholerae. These assays suggest that, in V. choleraedeletion of the transmembrane domain has a profound effect on ToxR activity, although it is not an absolute requirement when ToxR is dimerized by a heterologous domain. In addition, we noted differences in chimeric protein activity when expressed in E. coli and V. cholerae. A construct substituting the monomeric MalE domain for the periplasmic domain of ToxR was unable to activate a ctx::lacZ reporter fusion in E. coli. Although the addition of leucine zipper sequences to this construct resulted in enhanced activity of the chimera in E. coli, both chimeras were able to produce wild-type levels of toxin in V. cholerae. These data support the notion that dimerization of ToxR stimulates its activity as a transcriptional activator in E. coli. In V. cholerae, however, we present data that do not demonstrate a correlation between dimerization of the periplasmic domain and ToxR activity. [ABSTRACT FROM AUTHOR]

The ToxR protein of Vibrio cholerae is an integral membrane protein that coordinately regulates virulence determinant expression. ToxR directly activates the cholera toxin operon, but maximal activation is achieved in the presence of ToxS, an integral membrane protein thought to interact with ToxR periplasmic sequences. Studies that substitute alkaline phosphatase sequences for the periplasmic domain of ToxR have led to a model for ToxR activation based on dimerization and ToxS interaction. We constructed λ-ToxR chlmeric proteins using the DNA-binding domain of the phage λ repressor, which cannot effectively dimerize by itself, to assess the ability of ToxR to form dimers in Escherichia coli. The results suggest that ToxR sequences can propagate dimerization, and that ToxS can influence the ability to dimerize. [ABSTRACT FROM AUTHOR]