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.

FIG. 1

Cloning and physical map of the dnaK locus of V. cholerae. (A) Construction of plasmid pSC830 by ligation at the NcoI site of two overlapping clones, pSC350 and pSC510. All plasmids are derivatives of pACYC177. Only insert sequences are shown. Complementation of temperature-sensitive and phage λ-resistant phenotypes of E. coli CG800 and CG2682 by plasmids pSC350, pSC510, and pSC830 is indicated. (B) Physical map of the dnaK locus. The region of plasmid pSC830 within the hatched lines, which contains the complete dnaK gene and terminal portions of the dnaJ and grpE genes, was sequenced. Organization of the genes is indicated. The restriction sites shown are BamHI (B), EcoRI (E), EcoRV (EV), HincII (Hi), HindIII (H), HpaI (Hp), NcoI (N), and PstI (P).

FIG. 2

Identification of V. cholerae DnaK by Western blot analysis. E. coli GW4813 (ΔdnaK) cells containing plasmid pACYC177 (lane a), pKP31 (lane b), or pSC830 (lane c) and V. cholerae O395 cells (lane d) were heat shocked; then total cellular proteins were separated by SDS-PAGE (12.5% acrylamide), transferred to nitrocellulose membranes, and probed with anti-E. coli DnaK serum. Sizes are indicated in kilodaltons.

FIG. 3

Sequences of two putative promoters, P1 and P2, of the V. cholerae dnaK gene. Positions of the −35 and −10 regions were assigned by alignment with the consensus ς³² recognition sequence.

FIG. 4

CT production in V. cholerae O395 ▨ and O395K1 ▥. Cells were grown in LB (pH 6.6) at 30°C, in LB (pH 8.6) at 37°C, or in rabbit ileal loops, and CFU per milliliter was assayed. CT was measured in culture supernatants or intestinal fluids corresponding to 10⁹ CFU and expressed as a percentage of the amount obtained in culture supernatants of strain O395 grown at pH 6.6, 30°C.

FIG. 5

Northern blot analysis. V. cholerae O395 (lane a) and O395K1 (lane b) were grown in LB (pH 6.6) at 30°C, total RNA was isolated, and Northern blots were prepared and probed with [³²P]dCTP-labelled fragments of ctxAB (A), tcpA (B), and toxR (C) genes; 15 μg of RNA was loaded per well.

FIG. 6

Outer membrane proteins of V. cholerae O395 (lane a) and O395K1 (lane b) analyzed by SDS-PAGE and Coomassie blue staining. The arrowhead denotes the position of the 23-kDa outer membrane-located heat shock protein. The mobilities of protein molecular mass standards (in kilodaltons) are shown to the left.

FIG. 7

Swarming behavior of V. cholerae O395 and O395K1 on motility agar plates at 30°C (A) and 37°C (B).

FIG. 8

Transcriptional fusion plasmids for analysis of htpG and toxR expression. (A) The top diagram shows the organization of the htpG and toxR genes in the V. cholerae O395 chromosome. A 220-bp BamHI (B)-EcoRV (EV) fragment containing the intergenic region was inserted at the BamHI-Stu1 (S) sites between the phoA and lacZ genes in the promoter probe vector pTAC3734, to give plasmid pSCGR. The arrows denote directions of transcription. (B) β-Galactosidase and alkaline phosphatase activities in V. cholerae O395 containing plasmid pTAC3734 or pSCGR grown in vitro (LB, pH 7.2, 37°C) or in vivo. Results (means ± standard deviation of five independent experiments) are expressed as enzyme activities in Miller units, corresponding to 10⁹ CFU.