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Regulatory cascade controls virulence in Vibrio cholerae
V J DiRita1, C Parsot, G Jander
1Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115.
Abstract:
Expression of more than 17 virulence genes in Vibrio cholerae is under the coordinate control of the ToxR protein. ToxR is a transmembrane protein that binds to and activates the promoter of the operon encoding cholera toxin. As yet, the ability of ToxR to activate directly other genes in this regulon has not been demonstrated. We have cloned a gene called toxT from V. cholerae 569B; the toxT gene product, like ToxR, can activate the ctx promoter in Escherichia coli. In addition, expression of other genes identified as members of the ToxR regulon (tcpA, tcpI, aldA, and tagA) can be activated in E. coli by the toxT gene product but not by ToxR. When expressed from a constitutive promoter, the toxT gene product partially suppresses the ToxR- phenotype of a toxR deletion mutant of V. cholerae. The level of toxT mRNA is greatly reduced in a toxR mutant of V. cholerae. In addition, growth conditions under which the ToxR regulon is not expressed also repress the synthesis of toxT mRNA. These results suggest that ToxR controls transcription of toxT, whose product in turn is directly responsible for activation of several virulence genes under ToxR control.
Insights
ToxR protein controls Vibrio cholerae virulence by regulating the toxT gene. The toxT gene product then directly activates multiple virulence genes, revealing a key regulatory pathway for cholera pathogenesis.
Area of Science:
- Microbiology
- Molecular Biology
- Genetics
Background:
- The virulence of Vibrio cholerae, the causative agent of cholera, is orchestrated by a complex regulatory network.
- The ToxR protein, a transmembrane regulator, is known to control the expression of cholera toxin and other virulence factors.
- The precise mechanisms by which ToxR coordinates the expression of its regulon, particularly the direct activation of multiple genes, remained incompletely understood.
Purpose of the Study:
- To investigate the role of a newly identified gene, toxT, in the ToxR regulon of Vibrio cholerae.
- To determine if the ToxR protein directly activates genes beyond the cholera toxin operon.
- To elucidate the hierarchical relationship between ToxR and other virulence gene regulators.
Main Methods:
- Cloning of the toxT gene from Vibrio cholerae 569B.
- Functional analysis of the toxT gene product in Escherichia coli, assessing its ability to activate promoters of known ToxR-regulated genes (ctx, tcpA, tcpI, aldA, tagA).
- Complementation studies using a toxR deletion mutant of Vibrio cholerae to evaluate the phenotypic effects of toxT expression.
- Analysis of toxT mRNA levels in wild-type and toxR mutant strains of Vibrio cholerae under different growth conditions.
Main Results:
- The toxT gene product was shown to activate the ctx promoter in Escherichia coli, similar to ToxR.
- Crucially, the toxT gene product, but not ToxR, directly activated the expression of other ToxR-regulated genes (tcpA, tcpI, aldA, tagA) in Escherichia coli.
- Expression of toxT partially restored virulence gene expression in a toxR deletion mutant, and toxT mRNA levels were significantly reduced in the absence of functional ToxR.
- toxT mRNA synthesis was repressed under conditions that also repressed the ToxR regulon, indicating ToxR-dependent regulation of toxT.
Conclusions:
- ToxR controls the transcription of the toxT gene in Vibrio cholerae.
- The ToxR-inducible ToxT protein acts as a direct activator of multiple virulence genes within the ToxR regulon.
- This study reveals a critical two-step regulatory cascade (ToxR controlling toxT, and ToxT activating downstream genes) essential for coordinating Vibrio cholerae virulence.