The TviA auxiliary protein renders the Salmonella enterica serotype Typhi RcsB regulon responsive to changes in osmolarity - PubMed (original) (raw)

The TviA auxiliary protein renders the Salmonella enterica serotype Typhi RcsB regulon responsive to changes in osmolarity

Sebastian E Winter et al. Mol Microbiol. 2009 Oct.

Abstract

In response to osmolarity, Salmonella enterica serotype Typhi (S. Typhi) regulates genes required for Vi capsular antigen expression oppositely to those required for motility and invasion. Previous studies suggest that osmoregulation of motility, invasion and capsule expression is mediated through the RcsC/RcsD/RcsB phosphorelay system. Here we performed gene expression profiling and functional studies to determine the role of TviA, an auxiliary protein of the RcsB response regulator, in controlling virulence gene expression in S. Typhi. TviA repressed expression of genes encoding flagella and the invasion-associated type III secretion system (T3SS-1) through repression of the flagellar regulators flhDC and fliZ, resulting in reduced invasion, reduced motility and reduced expression of FliC. Both RcsB and TviA repressed expression of flhDC, but only TviA altered flhDC expression in response to osmolarity. Introduction of tviA into S. enterica serotype Typhimurium rendered flhDC transcription sensitive to changes in osmolarity. These data suggest that the auxiliary TviA protein integrates a new regulatory input into the RcsB regulon of S. Typhi, thereby altering expression of genes encoding flagella, the Vi antigen and T3SS-1 in response to osmolarity.

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Figures

Figure 1. Cluster analysis of RcsB and TviA regulated gene expression levels in S. Typhi

Gene expression profiling was performed on bacterial strains (wild-type strain, rcsB mutant, Δ_viaB_ mutant or Δ_tviB-vexE_ mutant) grown under low osmolarity condition (SOB broth). The CAST algorithm was used to identify similarities in gene expression between samples. Bars above the heat maps represent geometric means of fold change ± standard deviation for cluster of genes repressed (clusters 1–4) or activated (clusters 5–7) by RcsB and/or TviA or genes with no change in gene expression (cluster 8). The number of genes within each cluster is indicated below each heat map. ND: none detected.

Figure 2. Influence of RcsB and TviA on transcription of tviA and rcsB in S. Typhi

(A) The S. Typhi wild-type strain (Ty2) and the isogenic Δ_rcsB_ mutant (SW513), were grown in low osmolarity medium (tryptone yeast extract broth) under aerobic conditions. RNA was extracted and transcription of tviA was determined by real time qRT-PCR. Bars represent the geometric mean of mRNA levels (in percent of wild-type strain levels) ± standard error. (B) The S. Typhi wild-type strain (Ty2), an isogenic Δ_viaB_ mutant (STY2), a rcsB::lacZYA mutant (SW237), and a Δ_viaB rcsB_::lacZYA (SW239) were grown in low osmolarity medium (SOB broth) under aerobic conditions and β-galactosidase activity was measured. Bars represent the geometric mean of three independent experiments ± standard error. ND: none detected. ns: not statistically significant.

Figure 3. RcsB represses flhC and hilA transcription in S. Typhi

The S. Typhi wild-type strain (Ty2), Δ_rcsB_ mutant (SW513), flhDC mutant (SW491), and flhC Δ_rcsB_ mutant (SW612) were grown in low osmolarity medium (SOB broth) under aerobic conditions. RNA was extracted and transcription of flhC (A) and hilA (B) was determined by real time qRT-PCR. Bars represent the geometric mean of mRNA levels (in percent of wild-type strain levels) ± standard error. Statistical significance between data sets is indicated by asterisks: * (P < 0.05) or ** (P 0.01). ns: not statistically significant.

Figure 4. TviA represses early, middle and late genes of the flagellar regulon in S. Typhi

(A–C) The S. Typhi wild-type strain (Ty2), Δ_viaB_ mutant (STY2),Δ_tviB-vexE_ mutant (SW74), fliZ mutant (SW531), fliZ Δ_viaB_ mutant (SW532), fliZ Δ_tviB-vexE_ mutant (SW533), flhDC mutant (SW491), flhDC Δ_viaB_ mutant (SW492), and flhDC Δ_tviB-vexE_ mutant (SW477) were grown in low osmolarity medium (SOB broth) under aerobic conditions. RNA was extracted and transcription of flhC (early gene) (A), fliZ (middle gene) (B), and fliC (late gene) (C) was determined by real time qRT-PCR. Bars represent the geometric mean of mRNA levels (in percent of wild-type strain levels) ± standard error of three independent experiments. Statistical significance between data sets is indicated by asterisks: * (P < 0.05) or ** (P < 0.01). ns: not statistically significant. (D) The S Typhi wild-type strain (Ty2), Δ_viaB_ mutant (STY2),Δ_tviB-vexE_ mutant (SW74), and the Δ_viaB_ Δ_fliC_ mutant (SW483) were grown in low osmolarity medium (tryptone yeast extract broth) and flagellin expression was determined by Western Blot using FliC-specific antiserum (H antiserum d, αHd). To ensure equal loading of samples, expression of GroEL was determined by Western Blot (αGroEL). Approximate positions of standard proteins are indicated.

Figure 5. TviA represses S. Typhi motility under conditions of low osmolarity

(A) Motility plates containing 0.3 % agar were inoculated with the S. Typhi wild-type strain (Ty2), Δ_viaB_ mutant (STY2), Δ_tviB-vexE_ mutant (SW74), and the Δ_fliC_ mutant (SW359). NaCl was added at a concentration of 300 mM to increase the osmolarity of the medium as indicated. (B) Measurements of the halo size produced by bacterial strains on motility plates. Bars represent the geometric means from three independent experiments. Statistical significance is indicated by asterisks: * (P < 0.05) or ** (P < 0.01).

Figure 6. Repression of SPI-1 encoded invasion gene transcription by TviA is mediated through flhDC and fliZ in S. Typhi

The S. Typhi wild-type strain (Ty2), Δ_viaB_ mutant (STY2), Δ_tviB-vexE_ mutant (SW74), fliZ mutant (SW531), fliZ Δ_viaB_ mutant (SW532), fliZ Δ_tviB-vexE_ mutant (SW533), flhDC mutant (SW491), flhDC Δ_viaB_ mutant (SW492), and flhDC Δ_tviB-vexE_ mutant (SW477) were grown in low osmolarity medium (SOB broth) under aerobic conditions. RNA was extracted and transcription of hilA (A), invG (B), and hilE (C) was determined by real time qRT-PCR. Bars represent the geometric mean of mRNA levels (in percent of wild-type strain levels) ± standard error. Asterisks indicate statistical significance between data sets: * (P < 0.05) or ** (P < 0.01). ns: not statistically significant.

Figure 7. TviA regulates invasion of human epithelial cells by modulating both motility and SPI-1 gene expression in S. Typhi

(A) Motility and T3SS-1 contribute to invasion of the T84 epithelial cell line by S. Typhi. S. Typhi wild-type strain (Ty2), invA mutant (SW222), Δ_fliC_ mutant (SW359), and Δ_fliC_ Δ_invA_ mutant (SW550) were grown under low osmolarity conditions (tryptone yeast extract broth) and monolayers of T84 cell were infected at a multiplicity of infection of 10:1 for 1 h. Recovered bacterial numbers were standardized to the number of the bacteria in the inoculum. (B) TviA modulates invasion of T84 cells by S. Typhi. S. Typhi wild-type strain (Ty2), Δ_viaB_ mutant (STY2), Δ_tviB-vexE_ mutant (SW74), invA mutant (SW222), Δ_viaB invA_ mutant (SW224), Δ_fliC_ mutant (SW359), and Δ_fliC_ viaB mutant (SW483) were grown under low osmolarity conditions (tryptone yeast extract broth) and monolayers of T84 cell were infected as described above. Bars represent the geometric mean of three independent experiments ± standard error. Statistical significance between data sets is indicated by asterisks: * (P < 0.05) or ** (P < 0.01). ns: not statistically significant. pWSK: pWSK29

Figure 8. Effect of TviA and RcsB on osmolarity dependent flhC transcription in S. Typhi

(A) The flhC::lacZYA mutant (SW197, black bars), the Δ_viaB flhC_::lacZYA mutant (SW186, grey bars), and the Δ_viaB_ Δ_rcsB flhC_::lacZYA mutant (SW452, white bars) were grown in tryptone yeast extract medium supplemented with the indicated concentrations of NaCl and β-galactosidase activity was measured. Fold repression mediated by RcsB (B) and TviA (in the presence of RcsB) (C) was calculated by dividing the β-galactosidase activity of the Δ_viaB flhC_::lacZYA mutant and the Δ_viaB_ Δ_rcsB flhC_::lacZYA mutant by the corresponding value of the flhC::lacZYA and the Δ_viaB flhC_::lacZYA, respectively. Bars represent the geometric mean of four independent experiments ± standard error.

Figure 9. TviA-mediated osmoregulation of human epithelial cell invasion by S. Typhi

Invasiveness of S. Typhi towards T84 cells was determined by a gentamicin protection assay as described in Fig. 7A. (A) The S. Typhi wild-type (Ty2, grey bars) and the Δ_viaB_ mutant (SW347, white bars) were grown in tryptone yeast extract broth prior to infection of T84 cells. To adjust the osmolarity of the growth medium, NaCl was added at the indicated concentrations. (B) Fold repression of invasion was calculated by dividing the number of recovered bacteria of the Δ_viaB_ mutant (SW347) by the number of the wild-type strain (Ty2). Bars represent the geometric mean of three independent experiments ± standard error. Statistical significance is indicated by asterisks: ** (P < 0.01). ns: not significant.

Figure 10. Effect of tviA on osmoregulation of motility and the flagellar master regulator flhC in S. Typhimurium

(A) Motility of the S. Typhimurium wild-type strain (IR715), phoN mutant (SW284),Δ_phoN_::tviA mutant (SW474), and the Δ_fliC fljB_ mutant (SW473) was determined using 0.3 % agar plates. NaCl was added at a concentration of 300 mM as indicated to increase the osmolarity of the medium. (B) Measurements of the halo size produced by bacterial strains on motility plates. (C) The phoN flhC::lacZYA (SW317, black bars) and the phoN::tviA flhC::lacZYA (SW316, grey bars) were grown in tryptone yeast extract medium supplemented with the indicated concentrations of NaCl and β-galactosidase activity was measured. (D) Fold repression mediated by TviA was calculated as described in figure 8. Bars represent the geometric mean of three independent experiments ± standard error. Statistical significance is indicated by asterisks: * (P < 0.05), ** (P < 0.01), ns: not significant.

Figure 11. EnvZ mediates osmorelulation of flagella through the availability of tviA in S. Typhi

(A) Osmolarity dependent transcription of tviA in S. Typhi. The tviA::lacZYA mutant (SW620) was cultured in tryptone yeast extract medium supplemented with the indicated concentrations of NaCl and β-galactosidase activity was measured. Bars represent the geometric mean of three independent experiments ± standard error. Statistical significance is indicated by asterisks: * (P < 0.05), ** (P < 0.01), ns: not significant. (B and C) Effect of envZ on motility in S. Typhi. (B) Motility plates containing 0.3 % agar were inoculated with the S. Typhi wild-type strain (Ty2), Δ_viaB_ mutant (STY2), envZ mutant (SW672), and the Δ_fliC_ mutant (SW359). (C) Measurements of the halo size produced by bacterial strains on motility plates. Bars represent the geometric mean of three independent experiments ± standard error. Statistical significance is indicated by asterisks: * (P < 0.05), ns: not significant.

Figure 12. Introduction of tviA into S. Typhimurium renders flagella sensitive to EnvZ-mediated osmorelulation

(A) Regulation of flagellin expression by TviA and EnvZ. The S. Typhimurium wild-type (IR715), the phoN::tviA mutant (SW474), the phoN::tviA envZ mutant (SW679), and the Δ_fliC fljB_ mutant (SPN313) were grown in tryptone yeast extract broth supplement with the indicated concentration of NaCl and FliC as well as GroEL expression was determined by Western Blot using Salmonella H antiserum i (αHi) and anti-GroEL antiserum (αGroEL), respectively. Approximate positions of standard proteins are indicated. (B) Motility of the phoN mutant (SW284), the phoN::tviA mutant (SW474), the phoN::tviA envZ mutant (SW679), and the Δ_fliC fljB_ mutant (SPN313). (C) Measurements of the halo size produced by bacterial strains on motility plates. Bars represent the geometric mean of three independent experiments ± standard error.

Figure 13. Proposed model for a TviA-dependent integration of osmoregulation into the Rcs regulon of S. Typhi

The Rcs phosphorelay system and the EnvZ/OmpR two-component system respond to envelope stress and osmolarity of the medium, respectively, by activating transcription of tviA. TviA acts as an auxiliary protein to the response regulator RcsB, thereby allowing integration of signals from both signaling pathways. RcsB and TviA repress transcription of flhDC and fliZ, thereby limiting expression of flagella and invasion genes under low osmolarity conditions. Please refer to text for details.

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The TviA auxiliary protein renders the Salmonella enterica serotype Typhi RcsB regulon responsive to changes in osmolarity - PubMed (original) (raw)