PhoP-PhoQ-regulated loci are required for enhanced bile resistance in Salmonella spp - PubMed (original) (raw)

PhoP-PhoQ-regulated loci are required for enhanced bile resistance in Salmonella spp

J C van Velkinburgh et al. Infect Immun. 1999 Apr.

Abstract

As enteric pathogens, Salmonella spp. are resistant to the actions of bile. Salmonella typhimurium and Salmonella typhi strains were examined to better define the bile resistance phenotype. The MICs of bile for wild-type S. typhimurium and S. typhi were 18 and 12%, respectively, and pretreatment of log-phase S. typhimurium with 15% bile dramatically increased bile resistance. Mutant strains of S. typhimurium and S. typhi lacking the virulence regulator PhoP-PhoQ were killed at significantly lower bile concentrations than wild-type strains, while strains with constitutively active PhoP were able to survive prolonged incubation with bile at concentrations of >60%. PhoP-PhoQ was shown to mediate resistance specifically to the bile components deoxycholate and conjugated forms of chenodeoxycholate, and the protective effect was not generalized to other membrane-active agents. Growth of both S. typhimurium and S. typhi in bile and in deoxycholate resulted in the induction or repression of a number of proteins, many of which appeared identical to PhoP-PhoQ-activated or -repressed products. The PhoP-PhoQ regulon was not induced by bile, nor did any of the 21 PhoP-activated or -repressed genes tested play a role in bile resistance. However, of the PhoP-activated or -repressed genes tested, two (prgC and prgH) were transcriptionally repressed by bile in the medium independent of PhoP-PhoQ. These data suggest that salmonellae can sense and respond to bile to increase resistance and that this response likely includes proteins that are members of the PhoP regulon. These bile- and PhoP-PhoQ-regulated products may play an important role in the survival of Salmonella spp. in the intestine or gallbladder.

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Figures

FIG. 1

FIG. 1

Bile survival assay of S. typhimurium (A) and S. typhi (B). Strains grown to the stationary phase in LB broth were diluted and incubated in the presence of 30% bile. Aliquots were removed at various times, diluted or washed, and plated to determine the number of surviving cells. Symbols: □, wild type; ◊, PhoP−; ○, PhoPc.

FIG. 2

FIG. 2

PhoP-PhoQ-independent effect of bile on prgC and prgH transcription. Strains grown to the stationary (A) or logarithmic (B) phase in the presence or absence of bile (3% for PhoP− and 15% for PhoP+) were assayed for alkaline phosphatase activity. The pagG results are representative of PhoP-PhoQ-regulated fusions unaffected by bile. Stippled bars show results from cultures grown without bile, and black bars show those with bile. The means of three independent experiments with associated standard errors are shown.

FIG. 3

FIG. 3

Comparison of S. typhimurium proteins affected by bile or deoxycholate in the growth medium and proteins regulated by PhoP-PhoQ. Membrane extracts (A) and whole-cell extracts (B) were separated by SDS–10% PAGE. Lanes 1 to 5 show protein profiles of the following strains, respectively: wild type, wild type plus 3% bile, wild type plus 1% deoxycholate, PhoPc, and PhoP−. Molecular weight (MW) markers are indicated to the left of the panels. Open arrows denote proteins affected by bile or deoxycholate but not by PhoP-PhoQ. Closed arrows denote proteins affected by bile or deoxycholate and by PhoP-PhoQ. Only those protein species most obviously affected are noted.

FIG. 4

FIG. 4

Comparison of S. typhi proteins affected by bile or deoxycholate in the growth medium and proteins regulated by PhoP-PhoQ. Membrane extracts (A) and whole-cell extracts (B) were separated by SDS–10% PAGE. Lanes 1 to 5 show protein profiles of the following strains, respectively: wild type, wild type plus 3% bile, wild type plus 1% deoxycholate, PhoPc, and PhoP−. Molecular weight (MW) markers are indicated to the left of the panels. Open arrows denote proteins affected by bile or deoxycholate but not by PhoP-PhoQ. Closed arrows denote proteins affected by bile or deoxycholate and by PhoP-PhoQ. Only those protein species most obviously affected are noted.

FIG. 5

FIG. 5

Examination by 2-D gel electrophoresis of S. typhimurium proteins affected by bile. (A) Wild-type cells grown in LB broth. (B) Cells grown in LB broth plus 3% bile. Protein species in circles denote those repressed by bile, and protein species in squares denote those activated by bile. Only those proteins most obviously affected by bile are noted. The arrowhead indicates the isoelectric focusing standard (molecular mass, 33 kDa; pI 5.2). Molecular mass standards (in kilodaltons) are noted to the left of each panel.

FIG. 6

FIG. 6

Examination by 2-D gel electrophoresis of S. typhi proteins affected by bile. (A) Wild-type cells grown in LB broth. (B) Cells grown in LB broth plus 3% bile. Protein species in circles denote those repressed by bile, and protein species in squares denote those activated by bile. Only those proteins most obviously affected by bile are noted. The arrowhead indicates the isoelectric focusing standard (molecular mass, 33 kDa; pI 5.2). Molecular mass standards (in kilodaltons) are noted to the left of each panel.

FIG. 7

FIG. 7

S. typhimurium adaptation to bile. Wild-type S. typhimurium grown logarithmically with no bile or with 15% bile was washed, diluted, and incubated with 24% bile. The percentage of survival was determined at various times. While the results presented are from a single experiment, the experiment was repeated three times, with nearly identical outcomes. Symbols: □, no bile; ◊, 15% bile.

FIG. 8

FIG. 8

Comparison of logarithmic-phase S. typhimurium proteins affected by 3 and 15% bile. Membrane extracts were separated by SDS–10% PAGE. Lanes 1 to 5 show protein profiles of the following strains, respectively: wild type, wild type plus 3% bile, wild type plus 15% bile, PhoPc, and PhoP−. Molecular weight (MW) markers are indicated to the left of the panels. Open arrows denote obvious proteins affected by both 3 and 15% bile. Closed arrows denote obvious proteins affected by only 15% bile or 3% bile. The asterisks denote examples of proteins that are visible in lane 3 (15% bile) but not lane 2 (3% bile) and that may represent mediators of the adaptation effect seen in organisms grown with 15% bile.

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