Contribution of three bile-associated loci, bsh, pva, and btlB, to gastrointestinal persistence and bile tolerance of Listeria monocytogenes

Abstract

Listeria monocytogenes must resist the deleterious actions of bile in order to infect and subsequently colonize the human gastrointestinal tract. The molecular mechanisms used by the bacterium to resist bile and the influence of bile on pathogenesis are as yet largely unexplored. This study describes the analysis of three genes--bsh, pva, and btlB--previously annotated as bile-associated loci in the sequenced L. monocytogenes EGDe genome (lmo2067, lmo0446, and lmo0754, respectively). Analysis of deletion mutants revealed a role for all three genes in resisting the acute toxicity of bile and bile salts, particularly glycoconjugated bile salts at low pH. Mutants were unaffected in the other stress responses examined (acid, salt, and detergents). Bile hydrolysis assays demonstrate that L. monocytogenes possesses only one bile salt hydrolase gene, namely, bsh. Transcriptional analyses and activity assays revealed that, although it is regulated by both PrfA and sigma(B), the latter appears to play the greater role in modulating bsh expression. In addition to being incapable of bile hydrolysis, a sigB mutant was shown to be exquisitely sensitive to bile salts. Furthermore, increased expression of sigB was detected under anaerobic conditions and during murine infection. A gene previously annotated as a possible penicillin V amidase (pva) or bile salt hydrolase was shown to be required for resistance to penicillin V but not penicillin G but did not demonstrate a role in bile hydrolysis. Finally, animal (murine) studies revealed an important role for both bsh and btlB in the intestinal persistence of L. monocytogenes.

FIG. 1.

Genomic organization of bsh, pva, and btlB regions. This figure was drawn approximately to scale by using L. monocytogenes EGDe and L. innocua Clip 11262 genome sequence data. The numbers are National Center for Biotechnology Information annotation numbers. Arrows indicate orientation of genes. Hairpins depict putative terminators. Black open reading frames are orthologs. Open reading frames present in L. monocytogenes but absent from L. innocua are in white. Those present in L. innocua but not L. monocytogenes are in gray. Hatched arrows represent the three genes examined in the present study.

FIG. 2.

Role of L. monocytogenes LO28 bsh, pva, and btlB genes in bile tolerance. Overnight cultures were inoculated into BHI supplemented with 30% oxgall (A) or BHI adjusted to various pHs and supplemented with either 5 mM TDCA (B) or 5 mM GDCA (C). Viable plate counts were performed at intervals (A) or after 16 h (B and C) by serial dilution in Ringer solution and enumeration on BHI agar. The horizontal lines in panels B and C represent the initial inoculum. The error bars represent the standard deviations of triplicate experiments.

FIG. 3.

Growth of L. monocytogenes LO28 wild-type (squares) and pva mutant (triangles) in BHI broth supplemented with 50 ng of penicillin G (open symbols) or 50 ng of penicillin V (closed symbols)/ml. Cell growth was measured spectrophotometrically by the determining optical density at 595 nm (OD₅₉₅). The error bars represent the standard deviations of triplicate experiments.

FIG. 4.

BSH activity of L. monocytogenes LO28 and EGDe bsh, pva, and btlB mutants. Strains were patch inoculated onto BHI or MRS agar supplemented with 0.5% (wt/vol) GDCA and incubated anaerobically for 48 h. Strains with BSH activity are surrounded with white precipitate halos of unconjugated bile acids.

FIG. 5.

(A) Analysis of expression and regulation of bsh, pva, and btlB genes by using RT-PCR. cDNA was synthesized from RNA isolated from L. monocytogenes LO28 wild-type and prfA and sigB mutant cells grown anaerobically to exponential and stationary phases in BHI broth at 37°C. (B) Expression of sigB in cells grown to exponential phase either aerobically or anaerobically in BHI broth at 37°C. (C) Expression of sigB during murine infection. cDNA was synthesized from RNA that was extracted from intestines 2 h after oral infection with 10¹⁰ L. monocytogenes LO28 cells. In all cases, control PCRs were performed with 16S RNA primers to confirm identical template DNA levels. The number of PCR cycles are shown in parentheses.

FIG. 6.

(A) BSH activity of prfA and sigB mutants. Strains were patched onto MRS agar supplemented with 0.5% (wt/vol) GDCA and then incubated anaerobically for 48 h. White precipitate halos indicate BSH activity. (B) Bile tolerance of the wild type and of prfA and sigB mutants. Overnight cultures were inoculated into BHI (pH 6) supplemented with 5 mM GDCA and incubated anaerobically at 37°C. Viable plate counts were performed after 4 h. The error bars represent the standard deviations of triplicate experiments.

FIG. 7.

Role of bsh, pva, and btlB in persistence in the murine gastrointestinal tract. Excretion of rifampin-marked strains was measured in fecal samples collected 24, 48, and 72 h after oral infection of BALB/c mice. The error bars represent the standard deviations of triplicate experiments.