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. 2012 May;194(9):2286-96.
doi: 10.1128/JB.00104-12. Epub 2012 Feb 24.

VSports - Repression of Salmonella enterica phoP expression by small molecules from physiological bile

L Caetano M Antunes  1 , Melody Wang, Sarah K Andersen, Rosana B R Ferreira, Reinhild Kappelhoff, Jun Han, Christoph H Borchers, B Brett Finlay
Affiliations

Repression of Salmonella enterica phoP expression by small molecules from physiological bile

L Caetano M Antunes et al. J Bacteriol. 2012 May.

Abstract

Infection with Salmonella enterica serovar Typhi in humans causes the life-threatening disease typhoid fever. In the laboratory, typhoid fever can be modeled through the inoculation of susceptible mice with Salmonella enterica serovar Typhimurium. Using this murine model, we previously characterized the interactions between Salmonella Typhimurium and host cells in the gallbladder and showed that this pathogen can successfully invade gallbladder epithelial cells and proliferate. Additionally, we showed that Salmonella Typhimurium can use bile phospholipids to grow at high rates. These abilities are likely important for quick colonization of the gallbladder during typhoid fever and further pathogen dissemination through fecal shedding. To further characterize the interactions between Salmonella and the gallbladder environment, we compared the transcriptomes of Salmonella cultures grown in LB broth or physiological murine bile. Our data showed that many genes involved in bacterial central metabolism are affected by bile, with the citric acid cycle being repressed and alternative respiratory systems being activated. Additionally, our study revealed a new aspect of Salmonella interactions with bile through the identification of the global regulator phoP as a bile-responsive gene VSports手机版. Repression of phoP expression could also be achieved using physiological, but not commercial, bovine bile. The biological activity does not involve PhoPQ sensing of a bile component and is not caused by bile acids, the most abundant organic components of bile. Bioactivity-guided purification allowed the identification of a subset of small molecules from bile that can elicit full activity; however, a single compound with phoP inhibitory activity could not be isolated, suggesting that multiple molecules may act in synergy to achieve this effect. Due to the critical role of phoP in Salmonella virulence, further studies in this area will likely reveal aspects of the interaction between Salmonella and bile that are relevant to disease. .

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Figures

Fig 1
Fig 1
Salmonella displays robust growth in physiological murine bile. Cultures were started by pelleting cells from an overnight Salmonella culture by centrifugation, resuspending them in one volume of PBS, diluting this solution 1:50 in PBS, and inoculating LB broth or bile with this suspension at a 1:100 dilution. This resulted in an initial bacterial concentration of approximately 8 × 105 cells per ml (indicated by the dashed line). Samples were incubated at 37°C with shaking for 24 h, and aliquots were removed, diluted, and spotted on LB broth plates for bacterial enumeration after 2 and 24 h of growth. Average results of three independent cultures with standard errors of means are shown.
Fig 2
Fig 2
Salmonella growth in physiological murine bile modulates transcript levels of phoP and phoP-regulated genes. Salmonella was grown in LB broth or bile for 24 h, RNA was isolated, cDNA was synthesized, and RT-PCR was performed using primers specific for the genes indicated. Expression levels during growth in LB broth were normalized to 1, and expression in bile was adjusted accordingly. All results were normalized using the expression levels of gapA. Average expression levels of three independent cultures with standard errors of means are shown. P values were >0.05 (no asterisk), <0.03 (*), or <0.003 (**).
Fig 3
Fig 3
phoP expression is strongly repressed by Salmonella growth in physiological murine bile. A Salmonella strain containing a phoP::gfp plasmid was grown overnight in LB broth with carbenicillin and subcultured 1:100 in LB broth or bile containing carbenicillin. After incubation at 37°C with shaking for 4 h, cultures were diluted in PBS, and gfp expression was analyzed through flow cytometry. (A) Histogram showing the relative proportions of cells (y axis) expressing gfp at various levels (x axis) during growth in LB broth or bile, as indicated. (B) Data from panel A expressed as average fluorescence intensity per cell. The data shown are the average from three independent cultures. Bars represent the standard errors of means. The P value was <0.0001 (***).
Fig 4
Fig 4
The repression of phoP expression by physiological murine bile is independent of PhoPQ signaling. The phoP::gfp reporter plasmid was inserted into wild-type Salmonella as well as a phoP::Tn10 mutant. Strains were grown overnight in LB broth with carbenicillin and subcultured 1:100 in LB broth or bile containing carbenicillin. After incubation at 37°C with shaking for 4 h, cultures were diluted in PBS, and GFP expression was analyzed through flow cytometry. (A) Histogram showing the relative proportions of cells (y axis) expressing gfp at various levels (x axis) during growth of the phoP::Tn10 Salmonella strain in LB broth or bile, as indicated. (B) Same as in panel A, except that levels of gfp production by the wild-type and phoP::Tn10 (phoP) Salmonella strains were analyzed during growth in physiological murine bile. (C) Data from panel A expressed as average fluorescence intensity per cell. (D) Data from panel B expressed as average fluorescence intensity per cell. The data shown are the average from three independent cultures. Bars represent the standard errors of means. P values were <0.005 (**) and <0.0001 (***).
Fig 5
Fig 5
Commercial ox bile does not significantly repress phoP while still repressing hilA expression. Salmonella strains containing phoP::gfp or hilA::gfp reporter plasmids were grown overnight in LB broth with carbenicillin and subcultured 1:200 in LB broth containing carbenicillin with or without 3% (wt/vol) bovine bile (pH adjusted). After incubation at 37°C with shaking for 4 h, cultures were diluted in PBS, and gfp expression was analyzed by flow cytometry. (A) Histogram showing the relative proportions of cells (y axis) expressing gfp at various levels (x axis) during growth of the hilA::gfp Salmonella strain in LB broth with or without bovine bile, as indicated. (B) Same as in panel A, but with the phoP::gfp Salmonella reporter strain. (C) Data from panel A expressed as average fluorescence intensity per cell. (D) Data from panel B expressed as average fluorescence intensity per cell. The data shown are the average from three independent cultures. Bars represent the standard errors of means. P values were ≤0.0001 (***).
Fig 6
Fig 6
Physiological bovine bile alone does not support Salmonella growth. Bile or LB broth samples were kept undiluted, diluted with PBS, or mixed, as indicated, always at a 1:1 ratio. Streptomycin was added to all cultures at a final concentration of 100 μg/ml, and the pH of all solutions was adjusted to approximately 7.3. Overnight cultures of Salmonella Typhimurium SL1344 were then subcultured 1:200 in each of the culture media and incubated at 37°C with shaking; growth was monitored at the indicated time points by measuring the optical density of the cultures at 600 nm. The data shown are the average from three independent cultures. Bars represent the standard errors of means.
Fig 7
Fig 7
Physiological bovine bile represses phoP expression. LB broth was diluted 1:1 with either water or fresh bovine bile. Carbenicillin was added, and the pH of the solutions was adjusted. Overnight cultures of the Salmonella phoP::gfp reporter strain were then used to inoculate these solutions at a 1:200 dilution. Cultures were incubated at 37°C with shaking for 4 h, diluted in PBS, and analyzed by flow cytometry. (A) Histogram showing the relative proportions of cells (y axis) expressing GFP at various levels (x axis) during growth of the phoP::gfp Salmonella strain in LB broth-water (LB) or LB broth-bile (Bile) mixtures, as indicated. (B) Data from panel A expressed as average fluorescence intensity per cell. The data shown are the average from three independent cultures. Bars represent the standard errors of means. The P value was ≤0.0002 (***).
Fig 8
Fig 8
The repression of phoP expression by bile is not due to osmolarity. Overnight cultures of the Salmonella phoP::gfp reporter strain were subcultured at 1:200 in LB broth containing various concentrations of NaCl, as indicated. Cultures were allowed to grow for 4 h at 37°C with shaking, diluted, and analyzed for gfp expression through flow cytometry. The data shown are the average from three independent cultures. Bars represent the standard errors of means.
Fig 9
Fig 9
The repression of phoP by bile is due to specific molecules with hydrophobic properties. Bovine bile was extracted as described below. Extracts were evaporated and resuspended in LB broth containing carbenicillin. Overnight cultures of the Salmonella phoP::gfp reporter strain were subcultured 1:200 in all solutions, which were then incubated for 4 h at 37°C with shaking, diluted, and analyzed for gfp production through flow cytometry. The expression level during growth in LB broth (negative control) was set to 100%, and the activity of test fractions was normalized accordingly. (A) Bovine bile was applied to C18 resin cartridges, and the flowthrough (FT) was collected. Cartridges were washed with water, and the bound fraction was eluted sequentially with 50% and 100% methanol. Fractions were dried and assayed for phoP inhibition as described above. (B) The 100% fraction from panel A was dried, resuspended in 25% methanol, and subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) using a linear gradient of 20% to 100% methanol over 90 min (dashed line), at a flow rate of 1 ml per minute. Fractions were collected every minute, dried, and assayed for phoP inhibition as described above. (C) Fractions 53 and 54 from panel B were combined, dried, resuspended in 25% methanol, and subjected to RP-HPLC again using a linear gradient of 60% to 70% methanol over 30 min (dashed line), at a flow rate of 1 ml per minute. Fractions were collected every minute, dried, and assayed for phoP inhibition as described above. The data shown are the average from three (A) or two (B and C) independent cultures. Bars represent the standard errors of means.
Fig 10
Fig 10
The most abundant compound of the bioactive fraction is taurodeoxycholic acid. Fractions 18, 19, and 21 were dried, dissolved in 200 μl of methanol, diluted 1:500 with 75% methanol, and injected (3 μl) into a 10- by 0.21-cm C18 UPLC column and run on UPLC-QTOF MS in ESI(−) mode. (A) Base peak ion chromatograms of fractions 18, 19, and 21. Retention times are shown. The main compound detected in all fractions was m/z 498.2900756 (m/z determined from fraction 19). amu, atomic mass units. (B) m/z 498.2900756 from fraction 19 was subjected to collision-induced fragmentation MS, and chromatograms showing daughter ions of this compound (bottom panel) as well as a taurodeoxycholic acid standard (top panel) are presented. (C) Peak area of m/z 498.2900756 from fractions 18, 19, and 21 as an example of the criteria used to select potential culprits of the phoP inhibitory activity in the active fractions.
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References

    1. Antunes LC, et al. 2011. Metabolomics reveals phospholipids as important nutrient sources during Salmonella growth in bile in vitro and in vivo. J. Bacteriol. 193:4719–4725 - PMC - PubMed
    1. Antunes LC, et al. 2010. Inhibition of Salmonella host cell invasion by dimethyl sulfide. Appl. Environ. Microbiol. 76:5300–5304 - PMC - PubMed
    1. Bakken K, Vogelsang TM. 1950. The pathogenesis of Salmonella typhimurium infection in mice. Acta Pathol. Microbiol. Scand. 27:41–50 - PubMed
    1. Beier D, Gross R. 2006. Regulation of bacterial virulence by two-component systems. Curr. Opin. Microbiol. 9:143–152 - PubMed
    1. Chatterjee A, Dutta PK, Chowdhury R. 2007. Effect of fatty acids and cholesterol present in bile on expression of virulence factors and motility of Vibrio cholerae. Infect. Immun. 75:1946–1953 - PMC - PubMed

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