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Comparative Study
. 2018 Feb 20;86(3):e00490-17.
doi: 10.1128/IAI.00490-17. Print 2018 Mar.

Comparison of Salmonella enterica Serovars Typhi and Typhimurium Reveals Typhoidal Serovar-Specific Responses to Bile

Rebecca Johnson  1 , Matt Ravenhall  2 , Derek Pickard  3 , Gordon Dougan  3 , Alexander Byrne  1 , Gad Frankel  4
Affiliations
Comparative Study

Comparison of Salmonella enterica Serovars Typhi and Typhimurium Reveals Typhoidal Serovar-Specific Responses to Bile

Rebecca Johnson (V体育官网入口) et al. Infect Immun. .

Abstract

Salmonella enterica serovars Typhi and Typhimurium cause typhoid fever and gastroenteritis, respectively VSports手机版. A unique feature of typhoid infection is asymptomatic carriage within the gallbladder, which is linked with S Typhi transmission. Despite this, S Typhi responses to bile have been poorly studied. Transcriptome sequencing (RNA-Seq) of S Typhi Ty2 and a clinical S Typhi isolate belonging to the globally dominant H58 lineage (strain 129-0238), as well as S Typhimurium 14028, revealed that 249, 389, and 453 genes, respectively, were differentially expressed in the presence of 3% bile compared to control cultures lacking bile. fad genes, the actP-acs operon, and putative sialic acid uptake and metabolism genes (t1787 to t1790) were upregulated in all strains following bile exposure, which may represent adaptation to the small intestine environment. Genes within the Salmonella pathogenicity island 1 (SPI-1), those encoding a type IIII secretion system (T3SS), and motility genes were significantly upregulated in both S Typhi strains in bile but downregulated in S Typhimurium. Western blots of the SPI-1 proteins SipC, SipD, SopB, and SopE validated the gene expression data. Consistent with this, bile significantly increased S Typhi HeLa cell invasion, while S Typhimurium invasion was significantly repressed. Protein stability assays demonstrated that in S Typhi the half-life of HilD, the dominant regulator of SPI-1, is three times longer in the presence of bile; this increase in stability was independent of the acetyltransferase Pat. Overall, we found that S Typhi exhibits a specific response to bile, especially with regard to virulence gene expression, which could impact pathogenesis and transmission. .

Keywords: H58 clade; RNA-Seq; SPI-1 regulation; bile responses; cell invasion; typhoid fever. V体育安卓版.

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FIG 1
FIG 1
Comparison of pathways differentially regulated by bile between S. Typhi and S. Typhimurium. Overrepresented gene ontology (GO) terms within upregulated and downregulated genes following growth in 3% bile for each strain.
FIG 2
FIG 2
Gene expression in response to bile differs among Salmonella strains. Comparison of genes upregulated and downregulated in response to bile in S. Typhimurium (Tm), S. Typhi Ty2 (Ty2), and S. Typhi 129-0238 (H58).
FIG 3
FIG 3
Effects of bile on SPI-1 expression and activity. (A) Heatmap showing log2-fold changes in gene expression for S. Typhimurium (Tm), S. Typhi Ty2 (Ty2), and S. Typhi 129-0238 (H58) across the SPI-1 pathogenicity island and for non-SPI-1-carried effectors. Asterisks (*) indicate genes significantly affected by bile across all three strains. (B) Western blots of SipC, SipD, and SopE of S. Typhimurium 14028 (Tm), S. Typhi Ty2 (Ty2), and two H58 clinical isolates (ERL12148 and 129-0238) grown in LB with or without 3% bile; SopE panels are not shown for S. Typhimurium 14028, as this strain lacks SopE. DnaK was used as a loading control. A representative blot for two independent repeats is shown. Numbers below blots indicate fold changes in density in 3% bile compared to LB; all bands were normalized to their respective DnaK control prior to comparison. (C) Strains grown in LB or 3% bile to late exponential phase were added to HeLa cells at an MOI of 100 for 30 min. The percentages of intracellular bacteria at 2 h postinfection relative to the inoculum added are shown. n = 3; error bars show SD. Invasion rates of strains were compared by t test (**, P < 0.01; ***, P < 0.001).
FIG 4
FIG 4
Effects of bile on hilA and hilD transcription in Salmonella. The reporter activity (β-galactosidase units) of hilA::lacZ and hilD::lacZ in S. Typhimurium 14028 (A, B) and S. Typhi Ty2 (C, D) following growth to late exponential phase in LB in the presence or absence of bile. n = 3; error bars show SD. Reporter activity between strains was compared by t test (*, P < 0.05; ***, P < 0.001).
FIG 5
FIG 5
HilD autoregulation in S. Typhi. The reporter activity of an S. Typhi Ty2 hilD::lacZ chromosomal transcriptional reporter strain complemented with HilD (pWSK29-Spec HilD-4HA [HilD]) or an empty vector control (pWSK29-Spec [EV]) was determined by β-galactosidase assay following growth in LB. n = 3; error bars show SD. Reporter activity between strains was compared by one-way ANOVA (***, P < 0.001).
FIG 6
FIG 6
Bile promotes HilD stability in S. Typhi. WT S. Typhi Ty2 constitutively expressing C-terminally 4HA-tagged HilD from S. Typhi Ty2 (A) or S. Typhimurium 14028 (B) was grown in LB with or without bile. Thirty micrograms per milliliter chloramphenicol was added to stop protein synthesis, and samples were collected every 10 min. HilD levels were determined via Western blotting using an anti-HA antibody, and DnaK was used as a loading control. A representative blot for three independent repeats is shown. Half-life measurements are averaged from three independent repeats, and standard deviations are shown.
FIG 7
FIG 7
Proposed model of how bile influences SPI-1 expression in S. Typhi. (A) HilD is at the top of the SPI-1-regulatory hierarchy, where it regulates its own expression and the expression of HilA. HilD also regulates expression of the additional regulators HilC and RtsA, which also control HilA expression. (B) In the absence of bile, the turnover of HilD is high and the expression of hilD is at a basal level, and as a result the expression of hilA is low. (C) In the presence of bile, HilD is more stable, leading to enhanced expression of hilD, hilA, and thus SPI-1.
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References

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