Membrane localization of the ToxR winged‐helix domain is required for TcpP‐mediated virulence gene activation in <i>Vibrio cholerae</i>

Crawford, J. Adam; Krukonis, Eric S.; DiRita, Victor J.

doi:10.1046/j.1365-2958.2003.03398.x

Cited by 72 publications

(85 citation statements)

References 63 publications

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“…After 3 h of growth at 26°C in the presence of 0.2% arabinose to induce RovA expression, there was no significant difference in the transcription of hreP (82.1 Ϯ 5. 4 Miller units after RovA overproduction compared to 69.7 Ϯ 8. 9 Miller units for the strain carrying the control plasmid pBAD18Kan).…”

Section: Resultsmentioning

confidence: 91%

“…In the ToxR/ToxS system of Vibrio cholerae, ToxR is the transmembrane transcriptional regulator, which interacts with the transmembrane effector protein, ToxS, to activate the transcription of ompU and ompT (5,30). In conjunction with a second transmembrane transcriptional regulator, TcpH, and its effector, TcpP, ToxR activates the transcription of toxT, which results in the activation of virulence genes, including those coding for the cholera toxin and the toxin-coregulated pilus (TCP) (4,12,22).…”

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confidence: 99%

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A Regulatory Network Controls Expression of the In Vivo-Expressed HreP Protease of Yersinia enterocolitica

et al. 2009

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The human enteropathogen Yersinia enterocolitica survives and replicates in the lymphoid tissues of its host. Previous in vivo analyses of gene expression revealed that various chromosomal genes are expressed at this stage of infection, but not in vitro. One of these, termed hreP, encodes a protease that is necessary for full virulence of Y. enterocolitica. Using transposon mutagenesis, we identified three genes, pypA, pypB, and pypC, as positive regulators of hreP transcription. PypA is an inner membrane protein with no significant similarity to any known proteins; PypB is a ToxR-like transmembrane transcriptional regulator; and PypC is a cytoplasmic transcriptional regulator with an OmpR-like winged helix-turn-helix DNA binding motif. We show that all Pyp proteins are able to activate hreP independently of each other and that PypB and PypC interact directly with the hreP promoter region. Furthermore, pypB and pypC are autoregulated and regulate each other. Additional data indicate that transcription of hreP is repressed by the histone-like nucleoid-structuring protein H-NS in a temperature-dependent manner. Our data reveal a new regulatory network that might have implications for the controlled expression of further virulence-associated functions in Yersinia.

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Section: Resultsmentioning

confidence: 91%

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confidence: 99%

A Regulatory Network Controls Expression of the In Vivo-Expressed HreP Protease of Yersinia enterocolitica

et al. 2009

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“…The ToxR effector protein, ToxS, is dispensable for the stability of ToxR in V. cholerae (13), suggesting that neither YaeL nor the first protease that degrades TcpP acts on ToxR under the conditions of our experiments. Furthermore, periplasmically truncated forms of TcpP are generally unstable in V. cholerae (13), whereas periplasmically truncated forms of ToxR are much more stable in the cell (36). When a TcpP periplasmic truncation was tested in a tcpPH͞yaeL mutant, a stable degradation product similar in size to TcpP* accumulated (Fig.…”

Section: Discussionmentioning

confidence: 96%

Degradation of the membrane-localized virulence activator TcpP by the YaeL protease in Vibrio cholerae

Matson

DiRita

2005

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

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132

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A common mechanism inhibiting the activity of transcription factors is their sequestration to the membrane until they are needed, at which point they are released from the membrane by proteolysis. Acting in contrast to this inhibition mechanism are virulence regulators of Vibrio cholerae, the ToxR and TcpP proteins, which are localized to the inner membrane of the cell, where they bind promoter DNA and activate gene expression. TcpP is rapidly degraded in the absence of another protein, TcpH. We used a genetic screen to identify regulators of TcpP stability and identified the YaeL membrane-localized zinc metalloprotease as responsible for degrading TcpP in the absence of TcpH. In Escherichia coli, DegS and YaeL cooperate to degrade RseA, an antisigma factor that sequesters E to the inner membrane, thereby inhibiting the activity of E . When yaeL was disrupted in a V. cholerae tcpH mutant, we observed accumulation of a lower molecular weight species of TcpP. This observation is consistent with TcpP being partially degraded in the absence of YaeL. A mutant lacking both DegS and YaeL continued to accumulate the TcpP degradation product, indicating that protease other than DegS is acting before YaeL in degrading TcpP. The YaeL-dependent degradation pathway is active in TcpH ؉ cells under conditions that are not favorable for virulence gene activation. This work expands the knowledge of YaeL-dependent processing in the bacterial cell and reveals an unexpected layer of virulence gene regulation in V. cholerae.gene regulation ͉ pathogenesis ͉ membrane regulators

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“…Twenty to 100 l of culture was used to measure ␤-galactosidase activity as described previously (36). For quantification of ToxR activation of ompU-lacZ and toxT-lacZ, the chromosomal reporter strains EK383 and EK733 were used (4,22). The optical density at 600 nm (OD 600 ) was determined by spectrophotometry and used to normalize cultures for subsequent Western blot analysis.…”

Section: Methodsmentioning

confidence: 99%

“…This model is further supported by the fact that overexpressed TcpP can activate toxT in the absence of ToxR, but ToxR cannot activate toxT expression in the absence of TcpP (3)(4)(5)18). Membrane localization of ToxR is required for toxT activation in conjunction with TcpP but not activation of the TcpP-independent ompU promoter (21)(22)(23). Thus, ToxR is believed to serve as a coactivator, enhancing transcriptional activation of toxT by promoting TcpP recruitment and/or binding to the toxT promoter (5,17,18).…”

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confidence: 90%

Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

et al. 2016

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TcpP and ToxR coordinately regulate transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP and ToxR are membrane-localized transcription factors, each with a periplasmic domain containing two cysteines. In ToxR, these cysteines form an intramolecular disulfide bond and a cysteine-to-serine substitution affects activity. We determined that the two periplasmic cysteines of TcpP also form an intramolecular disulfide bond. Disruption of this intramolecular disulfide bond by mutation of either cysteine resulted in formation of intermolecular disulfide bonds. Furthermore, disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP. While the decreased stability of TcpP-C207S resulted in a nearly complete loss of toxT activation and cholera toxin (CT) production, the second cysteine mutant, TcpP-C218S, was partially resistant to proteolytic degradation and maintained ϳ50% toxT activation capacity. TcpP-C218S was also TcpH independent, since deletion of tcpH did not affect the stability of TcpP-C218S, whereas wild-type TcpP was degraded in the absence of TcpH. Finally, TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, suggesting that the single periplasmic cysteine in TcpH may assist with disulfide bond formation in TcpP by interacting with the periplasmic cysteines of TcpP. Consistent with this finding, a TcpH-C114S mutant was unable to stabilize TcpP and was itself unstable. Our findings demonstrate a periplasmic disulfide bond in TcpP is critical for TcpP stability and virulence gene expression. IMPORTANCEThe Vibrio cholerae transcription factor TcpP, in conjunction with ToxR, regulates transcription of toxT, the master regulator of numerous virulence factors in Vibrio cholerae. TcpP is a membrane-localized transcription factor with a periplasmic domain containing two cysteines. We determined that the two periplasmic cysteines of TcpP form an intramolecular disulfide bond and disruption of the intramolecular disulfide bond in TcpP decreased the stability of TcpP and reduced virulence gene expression. Normally TcpH, another membrane-localized periplasmic protein, protects TcpP from degradation. However, we found that TcpH was also unstable when intramolecular disulfides could not be formed in TcpP, indicating that the periplasmic cysteines of TcpP are required for functional interaction with TcpH and that this interaction is required for both TcpP and TcpH stability. C holera is caused by ingestion of the aquatic Gram-negative bacterium Vibrio cholerae. The profuse watery diarrhea characteristic of cholera is induced by cholera toxin (CT), an ADPribosylating toxin that induces cyclic AMP production in intestinal epithelial cells, resulting in massive secretion of water and electrolytes. Microcolony formation and colonization of the intestine require expression of the toxin-coregulated pilus (TCP) (1). Transcription of the genes encoding CT and TCP, as well as several other virulence factors, is regulated by...

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Membrane localization of the ToxR winged‐helix domain is required for TcpP‐mediated virulence gene activation in Vibrio cholerae

Cited by 72 publications

References 63 publications

A Regulatory Network Controls Expression of the In Vivo-Expressed HreP Protease of Yersinia enterocolitica

A Regulatory Network Controls Expression of the In Vivo-Expressed HreP Protease of Yersinia enterocolitica

Degradation of the membrane-localized virulence activator TcpP by the YaeL protease in Vibrio cholerae

Formation of an Intramolecular Periplasmic Disulfide Bond in TcpP Protects TcpP and TcpH from Degradation in Vibrio cholerae

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