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.

Crystal Structure of the Substrate-Binding Domain from Listeria monocytogenes Bile-Resistance Determinant BilE

“…Infection usually occurs via contaminated food, in particular ready-to-eat varieties [2,3]. The success of Listeria monocytogenes as a food-borne pathogen is aided by its ability to survive and grow in extreme conditions found both in food-preparation environments and in the gastrointestinal tract e.g., at low temperatures (down to −0.4 • C), at high salt concentrations (up to 10% w/v NaCl), or in the presence of bile [4][5][6][7]. Determining the molecular mechanisms by which Listeria monocytogenes survives these stresses could help to understand and control its proliferation and is thus of interest from a food safety and health perspective.…”

“…This is achieved by bile acids (also referred to as bile salts or bile alcohols, Figure S1), a class of amphipathic cholesterol-derived steroids, which are also potent antimicrobials due to their activity against biological membranes [12,14]. Several bile resistance mechanisms have been identified in Listeria monocytogenes and other bacteria including the expression of multidrug resistance efflux pumps and modification of bile acids via the enzyme bile salt hydrolase [6,7,[15][16][17][18].…”

Functional and comparative metagenomic analysis of bile salt hydrolase activity in the human gut microbiome

“…Bile broth assays were carried out as previously described (36). In brief, overnight cultures of L. innocua were inoculated (3%) into broth supplemented with oxgall (Sigma B3883).…”

Rate of environmental change determines stress response specificity

“…For example, preexposure to specific stresses is known to enhance bacterial resistance to different stresses applied subsequently (19)(20)(21). This raises a basic question: How do cells determine when to use the general stress response rather than activating more specific individual pathways?…”