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Investigation of the mechanisms by which Listeria monocytogenes grows in porcine gallbladder bile.

ABSTRACT: The food-borne pathogen Listeria monocytogenes is known to colonize the lumen of the gallbladder in infected mice and to grow rapidly in this environment (J. Hardy et al., Science 303:851-853, 2004). However, relatively little is known about the mechanisms utilized by the pathogen to survive and grow in this location. We utilized gallbladder bile (GB bile) isolated directly from porcine gallbladders as an ex vivo model of gallbladder growth. We demonstrate that GB bile is generally nontoxic for bacteria and can readily support growth of a variety of bacterial species including L. monocytogenes, Lactococcus lactis, Salmonella enterica serovar Typhimurium, and Escherichia coli. Significantly, L. monocytogenes grew at the same rate as the nonpathogenic species Listeria innocua, indicating that the pathogen does not possess specialized mechanisms that enable growth in this environment. However, when we reduced the pH of GB bile to pH 5.5 in order to mimic the release of bile within the small intestine, the toxicity of GB bile increased significantly and specific resistance mechanisms (Sigma B, BSH, and BilE) were essential for survival of the pathogen under these conditions. In order to identify genetic loci that are necessary for growth of L. monocytogenes in the gallbladder, a mariner transposon bank was created and screened for mutants unable to replicate in GB bile. This led to the identification of mutants in six loci, including genes encoding enzymes involved in purine metabolism, amino acid biosynthesis, and biotin uptake. Although GB bile does not represent a significant impediment to bacterial growth, specific metabolic processes are required by L. monocytogenes in order to grow in this environment.

SUBMITTER: Dowd GC

PROVIDER: S-EPMC3019883 | biostudies-other | 2011 Jan

REPOSITORIES: biostudies-other

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Investigation of the mechanisms by which Listeria monocytogenes grows in porcine gallbladder bile.

Dowd Georgina C GC Joyce Susan A SA Hill Colin C Gahan Cormac G M CG

Infection and immunity 20101011 1

The food-borne pathogen Listeria monocytogenes is known to colonize the lumen of the gallbladder in infected mice and to grow rapidly in this environment (J. Hardy et al., Science 303:851-853, 2004). However, relatively little is known about the mechanisms utilized by the pathogen to survive and grow in this location. We utilized gallbladder bile (GB bile) isolated directly from porcine gallbladders as an ex vivo model of gallbladder growth. We demonstrate that GB bile is generally nontoxic for ...[more]

PMID: 20937762

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Project description:Listeria monocytogenes is a foodborne Gram-positive bacterium causing listeriosis in both animals and humans. It can persist and grow in various environments including conditions countered during saprophytic or intra-host lifestyles. Sigma (σ) subunit of RNA polymerase is a transcriptional factor responsible for guiding the core RNA polymerase and initiating gene expression under normal growth or physiological changes. In L. monocytogenes, there is one housekeeping sigma factor, σA, and four alternative sigma factors σB, σC, σH, and σL. Generally, σA directs expression of genes required for normal growth while alternative σ factors alter gene expression in response to specific conditions (e.g., stress). In this study, we aimed to determine the exclusive role of σA in L. monocytogenes by comparing a wild type strain with its isogenic mutant lacking genes encoding all alternative sigma factors (i.e., sigB, sigC, sigH, and sigL). We further investigated their survival abilities in 6% porcine bile (pH 8.2) mimicking gallbladder bile and their transcriptomics profiles in rich medium (i.e., BHI) and 1% porcine bile. Surprisingly, the results showed that survival abilities of wild type and ΔsigBΔsigCΔsigHΔsigL (or ΔsigBCHL) quadruple mutant strains in 6% bile were similar suggesting a compensatory role for σA. RNA-seq results revealed that bile stimulon of L. monocytogenes wild type contained 66 genes (43 and 23 genes were up- and down-regulated, respectively); however, only 29 genes (five up- and 24 down-regulated by bile) were differentially expressed in ΔsigBCHL. We have shown that bile exposure mediates increased transcription levels of dlt and ilv operons and decreased transcription levels of prfA and heat shock genes in wild type. Furthermore, we identified σA-dependent bile inducible genes that are involved in phosphotransferase systems, chaperones, and transporter systems; these genes appear to contribute to L. monocytogenes cellular homeostasis. As a result, σA seemingly plays a compensatory role in the absence of alternative sigma factors under bile exposure. Our data support that the bile stimulon is prone to facilitate resistance to bile prior to initiated infection.

Project description:Listeria monocytogenes is ubiquitously found in nature and can easily enter food-processing facilities due to contaminations of raw materials. Several countermeasures are used to combat contamination of food products, for instance, the use of disinfectants that contain quaternary ammonium compounds, such as benzalkonium chloride (BAC) and cetyltrimethylammonium bromide (CTAB). In this study, we assessed the potential of the commonly used wild-type strain EGD-e to adapt to BAC and CTAB under laboratory growth conditions. All BAC-tolerant suppressors exclusively carried mutations in fepR, encoding a TetR-like transcriptional regulator, or its promoter region, likely resulting in the overproduction of the efflux pump FepA. In contrast, CTAB tolerance was associated with mutations in sugR, which regulates the expression of the efflux pumps SugE1 and SugE2. L. monocytogenes strains lacking either FepA or SugE1/2 could still acquire tolerance toward BAC and CTAB. Genomic analysis revealed that the overproduction of the remaining efflux system could compensate for the deleted one, and even in the absence of both efflux systems, tolerant strains could be isolated, which all carried mutations in the diacylglycerol kinase-encoding gene lmo1753 (dgkB). DgkB converts diacylglycerol to phosphatidic acid, which is subsequently reused for the synthesis of phospholipids, suggesting that alterations in membrane composition could be the third adaptation mechanism. IMPORTANCE Survival and proliferation of Listeria monocytogenes in the food industry are ongoing concerns, and while there are various countermeasures to combat contamination of food products, the pathogen still successfully manages to withstand the harsh conditions present in food-processing facilities, resulting in reoccurring outbreaks, subsequent infection, and disease. To counteract the spread of L. monocytogenes, it is crucial to understand and elucidate the underlying mechanism that permits their successful evasion. We present various adaptation mechanisms of L. monocytogenes to withstand two important quaternary ammonium compounds.

Project description:Listeria monocytogenes is a gram-positive, food-borne pathogen responsible for invasive infections with high overall mortality. Early host defenses encountered by L. monocytogenes following ingestion include low pH of the stomach and bile present in the small intestinal lumen. We hypothesized that “epidemic” strains are better able to withstand exposure to low pH and bile encountered in the gastrointestinal tract as compared to most “environmental” strains. Furthermore, we hypothesized that epidemic and environmental strains would have distinct transcriptional programs upon exposure to these conditions. Our treatments included 1 hr exposure to acid (pH 5.5 and 3.5) and bile (0.3%) stress. Strains were pre-exposed to pH 5.5 (1 hr) before being treated with pH 3.5. We used a collection of 12 previously characterized epidemic and environmental strains and each strainXtreatment combination included 3 biological replicates for each microarray experiment. All microarray experiments were two color competitive hybridizations that paired experimental conditions with the same strain at neutral pH for acid stress and pH 5.5 for bile stress. Transcriptomes of environmental strains exposed to acid and bile stress showed remarkably greater number of genes with differences of ≥2-fold expression levels as compared to epidemic strains (5 and 7, respectively). Environmental strains were characterized by up-regulation of several stress related genes and down-regulation of several cell envelope biosynthesis and virulence related genes, suggesting that complex regulatory networks orchestrate the cellular changes in the environmental strains to overcome stressful environments. The transcriptome of epidemic strains, in contrast, showed muted responses to these stress conditions implying their pre-adaptability to acid and bile stress encountered during natural infection that may enable epidemic strains to survive and become “primed” for subsequent colonization and infection in the lower gastrointestinal tract. Keywords: stress response, comparative transcriptomics, acid-adaptation, differential virulence, acid-stress response, bile-stress response

Dataset Information

Investigation of the mechanisms by which Listeria monocytogenes grows in porcine gallbladder bile.

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Investigation of the mechanisms by which Listeria monocytogenes grows in porcine gallbladder bile.

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