Bile stress response in Listeria monocytogenes LO28: adaptation, cross-protection, and identification of genetic loci involved in bile resistance

Abstract

Bile is one of many barriers that Listeria monocytogenes must overcome in the human gastrointestinal tract in order to infect and cause disease. We demonstrated that stationary-phase cultures of L. monocytogenes LO28 were able to tolerate concentrations of bovine, porcine, and human bile and bile acids well in excess of those encountered in vivo. Strain LO28 was relatively bile resistant compared with other clinical isolates of L. monocytogenes, as well as with Listeria innocua, Salmonella enterica serovar Typhimurium LT2, and Lactobacillus sakei. While exponential-phase L. monocytogenes LO28 cells were exquisitely sensitive to unconjugated bile acids, prior adaptation to sublethal levels of bile acids or heterologous stresses, such as acid, heat, salt, or sodium dodecyl sulfate (SDS), significantly enhanced bile resistance. This adaptive response was independent of protein synthesis, and in the cases of bile and SDS adaptation, occurred in seconds. In order to identify genetic loci involved in the bile tolerance phenotype of L. monocytogenes LO28, transposon (Tn917) and plasmid (pORI19) integration banks were screened for bile-sensitive mutants. The disrupted genes included a homologue of the capA locus required for capsule formation in Bacillus anthracis; a gene encoding the transcriptional regulator ZurR; a homologue of an Escherichia coli gene, lytB, involved in isoprenoid biosynthesis; a gene encoding a homologue of the Bacillus subtilis membrane protein YxiO; and a gene encoding an amino acid transporter with a putative role in pH homeostasis, gadE. Interestingly, all of the identified loci play putative roles in maintenance of the cell envelope or in stress responses.

FIG. 1.

(A) Survival of L. monocytogenes LO28 in bovine bile (oxgall). Overnight cultures were inoculated (3%) into BHI broth containing 0.3 (○), 10 (▵), 20 (▪), or 30% (•) oxgall or BHI alone (□). The cultures were incubated anaerobically at 37°C. Viable-cell counts were performed at intervals by serial dilution in one-quarter-strength Ringer's solution and enumeration on BHI. (B) Comparative bile tolerances of various bacteria. Overnight cultures were inoculated into broth containing 30% oxgall. Viable-cell counts (% survival) were performed after 5 min and are expressed as percentages of counts obtained for control cultures, i.e., cells grown in broth without bile. The error bars represent standard deviations of triplicate experiments. ND, not detected.

FIG. 2.

(A) Effects of individual bile acids on growth. Overnight cultures were inoculated into BHI supplemented with 5 mM GCA, TCA, GDCA, TDCA, or cholic acid (CA). The cultures were incubated anaerobically for 16 h at 37°C without shaking. Cell growth was measured spectrophotometrically by determining the optical density at 600 nm (OD600nm). (B) pH dependency of TDCA (open bars) and GDCA (solid bars) toxicities. Viable-plate counts were performed after 18 h of anaerobic incubation in BHI broth set at different initial pHs and containing 5 mM TDCA or GDCA. The error bars represent standard deviations of triplicate experiments. ND, not detected.

FIG. 3.

Survival of stationary-phase (○) and exponential-phase (□) L. monocytogenes in BHI broth supplemented with 0.3% bile salts (sodium cholate-sodium deoxycholate [1:1]). The cultures were adapted to 0.08% bile for 30 min in the absence (▪) or presence (▵) of chloramphenicol (10 μg/ml).

FIG. 4.

Sites (vertical arrowheads) of pORI19 (BSM1) or Tn917 (BSM2 to -5) insertion in L. monocytogenes LO28 bile-sensitive mutants. For all genes, the National Center for Biotechnology Information annotation number for the corresponding gene in L. monocytogenes EGDe is given. Genes disrupted in LO28 are indicated by solid arrows. Adjacent open reading frames in EGDe are represented by shaded arrows. Horizontal arrowheads represent putative promoter regions. Putative terminator regions are depicted by lollipops.

FIG. 5.

Survival of wild-type L. monocytogenes LO28 (□) and BSM1 (capA; ▴), BSM2 (gadE; ▪), BSM3 (yxiO; ○), BSM4 (zurR; ▵), and BSM5 (lytB; •) in high levels of bile. Overnight cultures were inoculated (3%) into BHI broth supplemented with 30% (wt/vol) oxgall. Viable-cell counts were performed at intervals by serial dilution in one-quarter-strength Ringer's solution and enumeration on BHI.