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Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae.

ABSTRACT: The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to construct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes of V. cholerae is ToxR, a transmembrane DNA binding protein. The V. cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot analysis and toxR transcriptional fusions, we demonstrated a reduction in expression of the toxR gene in the dnaK mutant strain together with a concomitant increase in expression of a htpG-like heat shock gene that is located immediately upstream and is divergently transcribed from toxR. This may be due to increased heat shock induction in the dnaK mutant. In vivo, however, although expression from heat shock promoters in the dnaK mutant was similar to that observed in vitro, expression of both toxR and htpG was comparable to that by the parental strain. In both strains, in vivo expression of toxR was significantly higher than that observed in vitro, but no reciprocal decrease in htpG expression was observed. These results suggest that the modulation of toxR expression in vivo may be different from that observed in vitro.

SUBMITTER: Chakrabarti S

PROVIDER: S-EPMC96425 | biostudies-literature | 1999 Mar

REPOSITORIES: biostudies-literature

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Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae.

Chakrabarti S S Sengupta N N Chowdhury R R

Infection and immunity 19990301 3

The dnaK gene of Vibrio cholerae was cloned, sequenced, and used to construct a dnaK insertion mutant which was then used to examine the role of DnaK in expression of the major virulence factors of this important human pathogen. The central regulator of several virulence genes of V. cholerae is ToxR, a transmembrane DNA binding protein. The V. cholerae dnaK mutant grown in standard laboratory medium exhibited phenotypes characteristic of cells deficient in ToxR activity. Using Northern blot anal ...[more]

PMID: 10024539

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Project description:The relA gene product determines the level of (p)ppGpp, the effector nucleotides of the bacterial stringent response that are also involved in the regulation of other functions, like antibiotic production and quorum sensing. In order to explore the possible involvement of relA in the regulation of virulence of Vibrio cholerae, a relA homolog from the organism (relA(VCH)) was cloned and sequenced. The relA(VCH) gene encodes a 738-amino-acid protein having functions similar to those of other gram-negative bacteria, including Escherichia coli. A deltarelA::kan allele was generated by replacing approximately 31% of the open reading frame of wild-type relA of V. cholerae El Tor strain C6709 with a kanamycin resistance gene. The V. cholerae relA mutant strain thus generated, SHK17, failed to accumulate (p)ppGpp upon amino acid deprivation. Interestingly, compared to the wild type, C6709, the mutant strain SHK17 exhibited significantly reduced in vitro production of two principal virulence factors, cholera toxin (CT) and toxin-coregulated pilus (TCP), under virulence gene-inducing conditions. In vivo experiments carried out in rabbit ileal loop and suckling mouse models also confirmed our in vitro results. The data suggest that (p)ppGpp is essential for maximal expression of CT and TCP during in vitro growth, as well as during intestinal infection by virulent V. cholerae. Northern blot and reverse transcriptase PCR analyses indicated significant reduction in the transcript levels of both virulence factors in the relA mutant strain SHK17. Such marked alteration of virulence phenotypes in SHK17 appears most likely to be due to down regulation of transcript levels of toxR and toxT, the two most important virulence regulatory genes of V. cholerae. In SHK17, the altered expression of the two outer membrane porin proteins, OmpU and OmpT, indicated that the relA mutation most likely affects the ToxR-dependent virulence regulatory pathway, because it had been shown earlier that ToxR directly regulates their expression independently of ToxT.

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Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae.

Publications

Role of DnaK in in vitro and in vivo expression of virulence factors of Vibrio cholerae.

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