Fig 1.
TtrRS increases the host colonization of V. parahaemolyticus and the ttr gene cluster is widely distributed in Proteobacteria.
(A) Deletion of 09830 decreased the colonization of V. parahaemolyticus. Colonization (CFU) of V. parahaemolyticus was measured from feces in the streptomycin-treated adult mouse model at 48 h post-infection. Groups containing six mice each were intragastrically administered with the indicated strains at a dose of 2.0 × 109 CFU/mouse. The Mann-Whitney test was used for statistical analysis, and asterisks indicate significant differences (**, P < 0.01). (B) Homology analyses of amino acid sequences encoded by 09830–09850 genes using BLAST of NCBI. The identities of the amino acid sequences of each protein are shown between V. parahaemolyticus strain HZ and S. typhimurium strain LT2. (C) Phylogenetic analysis of Ttr homologs in prokaryotes. The phylogenetic tree was constructed with the MEGA7 software using the neighbor-joining method. The ttrRS-ttrBCA-tsdBA (09830–09860) gene cluster in V. parahaemolyticus and other bacteria strains were used for homology analyses based on amino acid sequences using BLAST. The ttrR box (GTGG-N4-CCAC) is denoted by filled circle for presence and empty circle for absence in the putative promoter region of ttrB and tsdBA.
Fig 2.
Identification of TtrSR-regulated genes.
(A) The expression level of ttrBCA was assessed by measuring luminescence in PttrB-lux transcriptional fusion strains. V. parahaemolyticus containing promoter-lux transcriptional fusion plasmids were grown in MLB at 37°C. (B) The expression level of ttrBCA was assessed by measuring luminescence in E. coli harboring PttrB-lux reporter. The E. coli strains were grown in LB at 37°C, which also contain a pBAD24 vector control or pBAD24-ttrS-ttrR. Luminescence expression was calculated as the luminescence per unit of OD600. The unpaired two-tailed Student’s t-test was used for statistical analysis (****, P < 0.0001). (C) EMSA showing that TtrR binds to the promoter region of ttrB. Each reaction mixture contains DNA probe (30 nM) and TtrR protein (0 to 500 nM), and 16S rRNA served as negative control. (D) Volcano plot of the differentially expressed genes was analyzed between the ΔttrR and WT strains by RNA-seq. The x-axis displays the value of log2 (Fold change), and the y-axis represents the value of -log10(P value). Red dots represent up-regulated genes, while green dots represent down-regulated genes. (E) Validation of gene regulation by qRT-PCR. Ten genes, including 5 upregulated genes and 5 downregulated genes identified by RNA-seq analysis, were randomly selected to perform qRT-PCR. Each sample was run in triplicate, and the housekeeping gene 16S rRNA was detected as a control.
Fig 3.
TtrRS regulates the transcription of ttrRS-ttrBCA-09855-09860 gene cluster.
(A) The expression level of ttrRS and 09855–09860 was assessed by measuring luminescence in PttrS-lux and P09855-lux transcriptional fusion strains, respectively. V. parahaemolyticus containing promoter-lux transcriptional fusion plasmids were grown in MLB at 37°C. (B) The expression level of ttrSR, 09855–09860, and 05905–05910 was assessed by measuring luminescence in E. coli harboring PttrS-lux, P09855-lux, and P05905-lux reporter, respectively. The E. coli strains were grown in LB at 37°C, which also contain a pBAD24 vector control or pBAD24-ttrS-ttrR. (C-D) EMSA showing that TtrR binds to the promoter region of ttrS and 09855, respectively. Each reaction mixture contains DNA probe (30 nM) and TtrR protein (0 to 500 nM), and 16S rRNA served as negative control. (E) EMSA showing that TtrR binds to the promoter region of 05905. Each reaction mixture contains DNA probe (30 nM) and TtrR protein (0, 200, 400 nM). The 16S rRNA served as negative control, and P09855 served as positive control. (F) Homology analyses of amino acid sequences encoded by 05905–05910 genes using BLAST. The identities of the amino acid sequences of each protein are shown between V. parahaemolyticus strain HZ and S. typhimurium strain LT2. (G) The 05910 influences the transcription of TtrRS target genes. V. parahaemolyticus containing promoter-lux transcriptional fusion plasmids were grown in MLB at 37°C. Luminescence expression was calculated as the luminescence per unit of OD600. The unpaired two-tailed Student’s t-test was used for statistical analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 4.
Verification of the TtrR binding sites.
(A) WebLogo generated from the alignment of binding sequences to show the TtrR binding box. N stands for any nucleotide. (B-C) Validation of TtrR binding to the box in the promoter regions by EMSA. The triangle indicates the protein concentration gradient, which was 0 nM, 200 nM, 300 nM, and 400 nM. (D-E) Validation of TtrR binding to the box in the promoter regions by bioluminescence reporter assay. V. parahaemolyticus containing promoter-lux transcriptional fusion plasmids were grown in MLB at 37°C. Luminescence expression was calculated as the luminescence per unit of OD600. (F) mRNA level of ttrC and 09860 in WT and box deleted strain was determined by using qRT-PCR. The results are expressed as means ± SD from three independent experiments. The unpaired two-tailed Student’s t-test was used for statistical analysis (**, P < 0.01; ***, P < 0.001).
Fig 5.
Tetrathionate triggers TtrRS phosphorylation and increases the expression of target genes.
(A) Tetrathionate supports the growth of V. parahaemolyticus. (B) Deletion of ttrA reduces bacterial growth in the presence of tetrathionate. The strains were grown in M9 in the absence or in the presence of tetrathionate at 37°C under micro-aerobic conditions. Bacteria cell density was measured and reported as the value of OD600. (C) The effect of tetrathionate on transcription of TtrRS target genes. V. parahaemolyticus containing promoter-lux transcriptional fusion plasmids were grown in M9 in the absence or the presence of tetrathionate at 37°C. Luminescence expression was calculated as the luminescence per unit of OD600. (D) Tetrathionate induces the transcription of TtrRS target genes. mRNA level of ttrA in WT and ΔttrR strains was determined by using qRT-PCR. The results are expressed as means ± SD from three independent experiments. (E-F) Tetrathionate and phosphorylation promote TtrR dimerization. The recombinant pKNT25 and pUT18 plasmids were co-transformed into E. coli BTH101. The strains were grown in M9 in the absence or the presence of tetrathionate at 30°C for 8 h. The β-galactosidase activity was measured and reported as Miller Units. (G) Mutation of TtrR Asp58 or TtrS His397 abolished the transcription of TtrRS target genes. The Asp58 of TtrR or His397 of TtrS was substituted with alanine on the genome of V. parahaemolyticus. The strains containing PttrB-lux plasmid were grown in M9 in the absence or the presence of tetrathionate at 37°C. (H) Mutation of phosphorylation sites influences the TtrRS activity. The E. coli strain contains both a P09855-lux and a recombinant pBAD24 plasmid. Luminescence expression was calculated as the luminescence per unit of OD600. The unpaired two-tailed Student’s t-test was used for statistical analysis (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 6.
TtrSR and their target genes participate in sulfur metabolism in V. parahaemolyticus.
(A-B) Comparison of tetrathionate reduction and thiosulfate generation in the cultures of WT and the relevant mutants. The strains were grown in a modified M9 minimal medium containing sodium tetrathionate under micro-aerobic conditions. The complemented strains harbor a recombinant pBAD24 carrying the relevant deleted gene, while other strains have an empty pBAD24 plasmid. Experiments were repeated at least three times. (C-D) Comparison of thiosulfate oxidation and tetrathionate generation in the cultures of WT and the relevant mutants. The strains were grown in a modified M9 minimal medium containing sodium thiosulfate under micro-aerobic conditions. (E-F) H2S generation by WT and the relevant mutants. The strains were grown in a modified M9 minimal medium containing sodium thiosulfate or sodium tetrathionate. H2S generation was monitored by lead acetate strips. (G-H) Growth curves of WT and the relevant mutants. The strains were grown in M9 in the presence of tetrathionate or thiosulfate at 37°C under micro-aerobic conditions. Bacteria cell density was measured and reported as the value of OD600.
Fig 7.
Target genes influence TtrRS regulation and V. parahaemolyticus colonization.
(A-B) Deletion of ttrA or tsdBA affects the transcription of TtrRS target genes. V. parahaemolyticus containing promoter-lux transcriptional fusion plasmids were grown in a modified M9 minimal medium, which contained sodium thiosulfate or sodium tetrathionate at 37°C. Luminescence expression was calculated as the luminescence per unit of OD600. (C-E) TtrSR and their target genes contribute to intestinal colonization of V. parahaemolyticus. Colonization (CFU) of V. parahaemolyticus was measured from feces in the streptomycin-treated adult mouse model. Groups containing six mice each were intragastrically administered with the indicated strains at a dose of 2.0 × 109 CFU/mouse. The unpaired two-tailed Student’s t-test (A-B) or Mann-Whitney test (C-E) was used for statistical analysis (ns, P > 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).
Fig 8.
Proposed model of TtrRS-mediated tetrathionate-responsive genetic circuit that contributes to intestinal colonization of V. parahaemolyticus.
Gut microbiota produces large quantities of H2S, while the intestinal mucosa could convert it to thiosulfate (S2O32-) [17, 18]. Once inside the host gut, V. parahaemolyticus reacts with thiosulfate to form tetrathionate (S4O62-) by TsdBA. V. parahaemolyticus then senses the presence of tetrathionate through TtrS, which, in turn, transphosphorylates TtrR; phosphorylated TtrR is an active dimer form and therefore binds to the ttrR box of the promoter region of ttrRS-ttrBCA-tsdBA gene cluster and upregulates their transcription, which enhances TsdBA to oxidize thiosulfate to tetrathionate that further activates TtrRS. Meanwhile, the increased TtrBCA functions to conduce the reduction of both tetrathionate and thiosulfate, which promotes bacterial growth and colonization in the host gut. Therefore, TtrRS and their target genes constituted a tetrathionate-responsive genetic circuit to sufficiently exploit the host available sulfur compounds, which further contributes to the intestinal colonization of V. parahaemolyticus.