Hfq regulates the expression of the thermostable direct hemolysin gene in Vibrio parahaemolyticus

Abstract

Background: The hfq gene is conserved in a wide variety of bacteria and Hfq is involved in many cellular functions such as stress responses and the regulation of gene expression. It has also been reported that Hfq is involved in bacterial pathogenicity. However, it is not clear whether Hfq regulates virulence in Vibrio parahaemolyticus. To evaluate this, we investigated the effect of Hfq on the expression of virulence-associated genes including thermostable direct hemolysin (TDH), which is considered to be an important virulence factor in V. parahaemolyticus, using an hfq deletion mutant.

Results: The production of TDH in the hfq deletion mutant was much higher than in the parental strain. Quantification of tdh promoter activity and mRNA demonstrated that transcription of the tdh gene was up-regulated in the mutant strain. The hfq-complemented strain had a normal (parental) amount of tdh expression. The transcriptional activity of tdhA was particularly increased in the mutant strain. These results indicate that Hfq is closely associated with the expression level of the tdh gene. Interestingly, other genes involved in the pathogenicity of V. parahaemolyticus, such as VP1680, vopC, and vopT, were also up-regulated in the mutant strain.

Conclusion: Hfq regulates the expression of virulence-associated factors such as TDH and may be involved in the pathogenicity of V. parahaemolyticus.

Figure 1

Genetic structure and sequence alignment of Hfq. (A) Genetic structure of hfq and neighboring regions. Vp, V. parahaemolyticus strain RIMD2210633 (accession no. BA000032); Vc, V. cholerae (accession no. NP_230001); Ec, E. coli serotype O157:H7 (accession no. NP_313175). Arrows indicate the direction of transcription. The numbers of amino acids (aa) that comprise the proteins are indicated. (B) Sequence alignment of Hfq from various pathogens. Dashes indicate amino acid residues that are identical to those in V. parahaemolyticus. All Hfq amino acid sequences were obtained from GenBank. Vp, V. parahaemolyticus strain RIMD2210633 (accession no. BA000032); Vc, V. cholerae (accession no. NP_230001); Vv, V. vulnificus (accession no. NP_760222); Pa, P. aeruginosa (accession no. NP_233631); O157, E. coli serotype O157:H7 (accession no. NP_313175); St, S. typhi (accession no. NP_458797).

Figure 2

Growth of the Δhfq strain in LB medium. Bacteria were cultured in LB medium supplemented with 3% NaCl at 37°C and the cell density was measured with a spectrophotometer. The assay was performed independently 3 times. Closed circle, parental strain; closed square, hfq-complemented deletion strain (pHfqΔhfq); open circle, hfq deletion strain (Δhfq); open square, hfq deletion strain with empty vector (pSN1Δhfq).

Figure 3

Hemolytic activity in culture supernatants of parental and derivative strains. Bacteria were cultured in 10 ml of LB medium supplemented with 3% NaCl at 37°C. One hemolytic unit (HU) was defined as in a previous report [45,46]. The results are expressed as means ± SDs of 5 independent experiments. *, p < 0.05 versus LB; #, p < 0.01 versus WT. LB, negative control; WT, V. parahaemolyticus strain RIMD2210633 (parent strain); Δhfq, hfq deletion strain; pHfq/Δhfq, hfq-complemented deletion strain; pSN1/Δhfq, deletion strain with empty vector.

Figure 4

Production of TDH by the Δhfq strain. (A) Detection of TDH in cell lysates from mid-log phase (OD₆₀₀ = 0.6) and stationary phase (OD₆₀₀ = 1.5) cultures. (B) Detection of TDH in mid-log phase cell lysates from the parent strain and derivatives. (C) Quantification of tdh mRNA. Fold change in the gene transcription level from mid-log phase in the Δhfq strain versus the parent strain, as measured by quantitative real-time RT-PCR. Data analysis was as previously described [42,43]. Data are expressed as means ± SDs of 5 independent experiments. *, p < 0.05.

Figure 5

Activities of the tdhA and tdhS gene promoters in the WT and Δhfq strains. β-galactosidase activity measured in the parental WT strain (black bars) and the Δhfq strain (open bars). pHN1/WT, pHN1 harboring tdhA gene promoter in V. parahaemolyticus strain RIMD2210633; pHN2/WT, pHN2 harboring tdhS gene promoter in V. parahaemolyticus strain RIMD2210633; pHRP309/WT, pHRP309 in V. parahaemolyticus strain RIMD2210633; pHN1/Δhfq, pHN1 harboring tdhA gene promoter in Δhfq strain; pHN2/Δhfq, pHN2 harboring tdhS gene promoter in Δhfq strain; pHRP309/Δhfq, pHRP309 in Δhfq strain. Data in Miller units are the means ± SDs of 5 independent experiments. *, P <0.01.

Figure 6

Expression of virulence-associated genes in the Δhfq strain. (A) Western blot detection of proteins in total cell lysates. (B) Quantification of mRNAs encoding virulence-associated proteins. The fold changes in gene transcription levels in the Δhfq and parental WT strains grown to mid-log phase were determined by quantitative real-time RT-PCR. Data analysis was as previously described [42,43]. Data are the means ± SDs of 5 independent experiments. *, p < 0.05.