Analysis of ToxR‐dependent transcription activation of <i>ompU</i>, the gene encoding a major envelope protein in <i>Vibrio cholerae</i>

Crawford, J. Adam; Kaper, James B.; DiRita, Victor J.

doi:10.1046/j.1365-2958.1998.00925.x

Cited by 117 publications

(124 citation statements)

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“…ToxT then directly activates transcription of the genes encoding CT and TCP and autoregulates its own gene (Yu and DiRita, 1999). In the other branch, independent of TcpP and ToxT, ToxR differentially regulates the expression of two outer membrane porins, OmpU and OmpT (Chakrabarti et al, 1996), resulting in almost exclusive expression of OmpU in a toxR + strain and exclusive expression of OmpT in a toxR -strain Crawford et al, 1998;Li et al, 2000). The genes encoding CT are part of the genome of a lysogenic phage (Waldor and Mekalanos, 1996), and the genes encoding TCP are part of the Vibrio pathogenicity island, which is also apparently of phage origin (Karaolis et al, 1998;.…”

Section: Introductionmentioning

confidence: 99%

“…At the ompU promoter, DNase I footprinting revealed the protection of three distinct sites by ToxR. It was proposed that the binding of ToxR to a region extending from -238 to -149 of the promoter is followed by co-operative binding to the downstream regions extending from -116 to -58 and from -53 to -24, where ToxR interacts directly with RNA polymerase (RNAP) and activates transcription (Crawford et al, 1998). For toxT, ToxR binding to the -108 to -65 region of the promoter recruits TcpP to bind downstream at -58 to -44, where TcpP can activate transcription directly (Krukonis et al, 2000;Li et al, 2000).…”

Section: Introductionmentioning

confidence: 99%

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ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae

Merrell

Camilli

et al. 2002

Molecular Microbiology

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SummaryIn pathogenic Vibrio cholerae, the transmembrane DNA-binding protein ToxR co-ordinates the expression of over 20 genes, including those encoding important virulence factors such as cholera toxin and the toxin-co-regulated pilus. The outer membrane protein OmpT is the only member of the ToxR regulon known to be repressed by ToxR. In this study, we examined the environmental conditions that regulate OmpT expression and demonstrated that ompT transcription is upregulated 14-fold when the bacteria enter late log phase from early log phase. Deletion of the crp gene completely abolishes OmpT expression. Comparison of ompT transcription levels in the isogenic crp -, toxR -and crp -toxR -mutants revealed that (i) in the absence of ToxR, constitutive high-level ompT transcription is dependent on cAMP receptor protein (CRP); (ii) ToxR not only interferes with CRPdependent ompT activation, but also abolishes the CRP-independent, basal level ompT transcription; thus, the mechanism by which ToxR represses ompT transcription involves both antiactivation and direct repression; (iii) both CRP and ToxR are required for the regulation of OmpT expression by growth phase. To provide further insights into the molecular mechanism of CRP-dependent activation of ompT transcription, we demonstrated that CRP-dependent activation requires a CRP binding site centred at -310 of the ompT promoter, without which the interaction of CRP with other CRP binding site(s) more proximal to the promoter results in repression. Mutations in two regions on CRP (AR1 and AR2) that directly

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae

Merrell

Camilli

et al. 2002

Molecular Microbiology

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“…In addition to its role as a porin, OmpU has been speculated to play a crucial role in the pathogenesis of V. cholerae. This speculation is mainly based on the fact that expression of OmpU is under the control of the ToxR regulon, which controls major virulence factors required for the pathogenesis of V. cholerae (32)(33)(34). Moreover, OmpU has been reported to facilitate intestinal colonization of the bacterium by conferring resistance against bile and anti-microbial peptides.…”

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Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria

Gupta

Prasad

Mukhopadhaya

2015

Journal of Biological Chemistry

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Porins, a major class of outer membrane proteins in Gramnegative bacteria, primarily act as transport channels. OmpU is one of the major porins of human pathogen, Vibrio cholerae. In the present study, we show that V. cholerae OmpU has the ability to induce target cell death. Although OmpU-mediated cell death shows some characteristics of apoptosis, such as flipping of phosphatidylserine in the membrane as well as cell size shrinkage and increased cell granularity, it does not show the caspase-3 activation and DNA laddering pattern typical of apoptotic cells. Increased release of lactate dehydrogenase in OmpU-treated cells indicates that the OmpU-mediated cell death also has characteristics of necrosis. Further, we show that the mechanism of OmpU-mediated cell death involves major mitochondrial changes in the target cells. We observe that OmpU treatment leads to the disruption of mitochondrial membrane potential, resulting in the release of cytochrome c and apoptosis-inducing factor (AIF). AIF translocates to the host cell nucleus, implying that it has a crucial role in OmpU-mediated cell death. Finally, we observe that OmpU translocates to the target cell mitochondria, where it directly initiates mitochondrial changes leading to mitochondrial membrane permeability transition and AIF release. Partial blocking of AIF release by cyclosporine A in OmpU-treated cells further suggests that OmpU may be inducing the opening of the mitochondrial permeability transition pore. All of these results lead us to the conclusion that OmpU induces cell death in target cells in a programmed manner in which mitochondria play a central role.

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“…It is responsible for in vitro induction of CT in a biotype-specific manner, for a significant albeit incomplete response to pH and temperature (19), and for regulation of virulence gene expression in response to a quorum-sensing system (20). These effects are mediated by AphA and AphB, transcriptional regulators that directly activate tcpPH transcription and ultimately regulate ToxT and CT (21).ToxRS regulates multiple genes other than ToxT (22), including the ompU and ompT genes (3,7,23). ToxR activity, independent of ToxT, results in the reciprocal expression of OmpU and repression of OmpT, two outer-membrane proteins that may act as adhesins (24) or porins (25), and that may affect virulence factor expression, intestinal colonization, and resistance to bile acids (26).…”

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confidence: 99%

“…ToxRS regulates multiple genes other than ToxT (22), including the ompU and ompT genes (3,7,23). ToxR activity, independent of ToxT, results in the reciprocal expression of OmpU and repression of OmpT, two outer-membrane proteins that may act as adhesins (24) or porins (25), and that may affect virulence factor expression, intestinal colonization, and resistance to bile acids (26).…”

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Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner

Hung

Mekalanos²

2005

Proc. Natl. Acad. Sci. U.S.A.

146

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The production of cholera toxin (CT) during Vibrio cholerae infection results in the hallmark diarrhea that characterizes the disease cholera. The transmembrane protein ToxR was originally identified as a functional transcriptional activator of ctxAB in a heterologous Escherichia coli system. However, direct ToxR activation of the ctxAB promoter in V. cholerae has not been previously demonstrated. Instead, a regulatory cascade has been defined in which the activators ToxRS and TcpPH modulate ctxAB expression by acting in concert to transcriptionally activate another regulator, ToxT. ToxT, in turn, directly activates ctxAB expression as well as expression of the tcp genes and other virulence-associated genes. In this study, we show that ToxRS directly activates ctxAB in a ToxT-independent manner in a classical biotype V. cholerae, and that this activation requires the presence of bile acids. Although the levels of CT induced by this mechanism are lower than levels induced under other in vitro conditions, the bile-dependent conditions described here are more physiologic, being independent of pH and temperature. We further show that the inability of bile acids to stimulate ToxRS-dependent expression of CT in El Tor biotype strains is related to the differences between classical and El Tor ctxAB promoters, which differ in the number of heptad TTTTGAT repeats in their respective upstream regions. The ability of bile acids to stimulate direct activation of ctxAB by ToxRS depends upon the transmembrane domain of ToxR, which may interact with bile acids in the inner membrane of V. cholerae. The Gram-negative bacterium Vibrio cholerae is the infectious agent that causes the severe human diarrheal disease cholera (1). Two major virulence factors, cholera toxin (CT) and the toxin-coregulated pilus (TCP), play major roles in the pathogenesis of this infection. Secretion of CT, an ADP-ribosylating toxin encoded in the genome of the filamentous, lysogenic CTX⌽ phage, results in elevated cAMP levels in intestinal epithelial cells and subsequent secretory diarrhea (2). The regulation of CT and TCP expression has been studied intensely and has been reported to respond to temperature, pH, osmolarity, bile salts, and certain amino acids in vitro (3, 4); nevertheless, a clear understanding of the in vivo environmental stimuli that trigger CT and TCP production remains elusive.The first transcriptional regulator of CT production identified was ToxR, a 32-kDa inner-membrane protein that activates the ctx promoter in Escherichia coli and whose deletion in V. cholerae results in the inability to produce CT (5). Deletion analysis of the ctxAB promoter localized the binding site of ToxR to multiple heptad repeats, TTTTGAT, upstream of ctxAB (6). However, ToxR activation of ctxAB transcription in V. cholerae has not been demonstrated (7). Instead, ToxR in V. cholerae binds with its downstream enhancer ToxS to the upstream region of the toxT gene, which encodes another transcriptional activator (8). ToxT belongs to the AraC family of trans...

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Analysis of ToxR‐dependent transcription activation of ompU, the gene encoding a major envelope protein in Vibrio cholerae

Cited by 117 publications

References 48 publications

ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae

ToxR interferes with CRP‐dependent transcriptional activation of ompT in Vibrio cholerae

Vibrio cholerae Porin OmpU Induces Caspase-independent Programmed Cell Death upon Translocation to the Host Cell Mitochondria

Bile acids induce cholera toxin expression in Vibrio cholerae in a ToxT-independent manner

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