Mucosal penetration primes Vibrio cholerae for host colonization by repressing quorum sensing

Abstract

To successfully infect a host and cause the diarrheal disease cholera, Vibrio cholerae must penetrate the intestinal mucosal layer and express virulence genes. Previous studies have demonstrated that the transcriptional regulator HapR, which is part of the quorum sensing network in V. cholerae, represses the expression of virulence genes. Here, we show that hapR expression is also modulated by the regulatory network that governs flagellar assembly. Specifically, FliA, which is the alternative sigma-factor (sigma(28)) that activates late-class flagellin genes in V. cholerae, represses hapR expression. In addition, we show that mucin penetration by V. cholerae is sufficient to break flagella and so cause the secretion of FlgM, the anti-sigma factor that inhibits FliA activity. During initial colonization of host intestinal tissue, hapR expression is repressed because of low cell density. However, full repression of hapR expression does not occur in fliA mutants, which results in attenuated colonization. Our results suggest that V. cholerae uses flagellar machinery to sense particular intestinal signals before colonization and enhance the expression of virulence genes by modulating the output of quorum sensing signaling.

Fig. 1.

FliA represses quorum sensing in flgD mutants. (A) Proposed model for repression of the hapR promoter by secretion of FlgM. (B) FlgM secretion is increased in the flgD mutant. Strains containing a plasmid expressing a functional flgM-his₆ were grown to midlog. Culture pellets (P) and TCA-precipitated supernatants (S) were isolated and subjected to Western blot analysis using anti-His-6 antiserum. All samples were normalized to contain 10⁹ bacterial cells. (C) Reduced production of HapR is due to hapR repression by FliA. Strains harboring hapR-lacZ transcriptional fusions were grown to midlog in LB and harvested to measure β-galactosidase activity (Upper). Results, reported in Miller units, are means of three experiments ± standard deviations. Whole-cell extracts were subjected to Western blot analysis using anti-HapR antiserum (Lower). All samples were normalized to contain 10⁹ bacterial cells.

Fig. 2.

The expression of hapR is repressed by FliA during the colonization of infant mice. (A) In vivo competition assays. Infant CD-1 mice were orally inoculated with ≈10⁶ wild-type and mutant bacteria. For each mutant, the competitive index is defined as the number of colony-forming units (CFU) for the mutant compared with the corresponding CFU number for a wild-type strain recovered from the intestines 18 h after inoculation. (B) Expression of hapR during colonization. Infant CD-1 mice were orally inoculated with ≈10⁶ bacteria that contain a hapR-K_m^r reporter. At each time point, mouse intestines were homogenized, treated with or without kanamycin (500 μg/ml) for 10 min, and plated on LB plates. The number of colonized bacteria per mouse (above each bar) was calculated based on the number of CFU recovered from samples not treated with kanamycin. Expression of hapR is defined as the percentage of K_m-resistant CFU of the total CFU. Results are means from experiments with three to five mice, and the bars represent the corresponding standard deviations.

Fig. 3.

V. cholerae cells lose flagella during mucin penetration. (A) Flagellar mutants swim more slowly through mucin than do wild-type cells. Midlog cultures (100 μl) of wild-type V. cholerae, the flgD mutant (white bars), or cultures premixed with 100 μl of 1% mucin were loaded into a column containing 1 ml of 1% mucin. After incubation for 30 min at 37°C, 500 μl were collected from the bottom of the column and plated on LB medium. (B) Images of V. cholerae during mucin column penetration. Flagellar staining (Upper) and transmission electron microscopy (Lower) of V. cholerae cells in the presence or absence of mucin. [Scale bars : 2 μm (Upper) and 500 nm (Lower).]

Fig. 4.

Quorum sensing is repressed when V. cholerae penetrate mucin. (A) Secretion of FlgM increases during mucin penetration. Midlog cultures of wild-type bacteria expressing flgM-his₆ were incubated for 30 min in LB medium with or without 1% mucin. Cytoplasmic and cell-free supernatant fractions were subjected to Western blot analysis. All samples were normalized to contain 10⁸ bacterial cells. (B) Real-time RT-PCR analysis of hapR expression. Mucin penetration assays were performed as described above. RNA was extracted, and real-time RT-PCR was performed for hapR and flaD transcripts. Transcript levels were normalized by 16S RNA. Results for each promoter are reported as the ratio of transcript level in the presence of mucin to the transcript level in the absence of mucin. (C) Expression of hapR in V. cholerae cells on the surface of HEp-2 cells. Confluent HEp-2 cell monolayers were submerged in 1 ml of 1% mucin or LB. Midlog cultures of wild-type (wt) and fliA-overexpressing (fliA^C) strains of V. cholerae that contain the hapR-K_m^r reporter were inoculated onto HEp-2 cell cultures. After 2-hr incubation, the bacteria bound to HEp-2 cells were collected, treated with or without kanamycin (500 μg/ml) for 10 min, and plated onto LB agar. The hapR-K_m^r expression was defined as the percentage of CFU that survive treatment with kanamycin of the total CFU. All results are means of three experiments ± standard deviations.