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New Insights on the Role of the pLMST6 Plasmid in Listeria monocytogenes Biocide Tolerance and Virulence.

ABSTRACT: Listeria monocytogenes the causative agent of listeriosis is an important public health concern and food safety challenge. Increased tolerance of this bacterium to benzalkonium chloride (BC), an antibacterial agent widely used in industrial settings, is a growing issue. Plasmid pLMST6 harboring the gene of the multidrug efflux pump protein EmrC has been recently linked to enhanced BC tolerance and meningitis due to L. monocytogenes ST6 strains. In this study, occurrence and contribution of this plasmid to BC tolerance was examined using PCR, plasmid curing and transformation, RT-qPCR and proteome analysis, respectively. Furthermore, the substrate specificity of the pLMST6 associated EmrC efflux pump and the impact of the plasmid on L. monocytogenes virulence were investigated. pLMST6 was detected in 7 (1.6%) of 439 L. monocytogenes strains isolated from different sources. A phenotypic role of this plasmid in conferring increased BC tolerance was confirmed by showing that plasmid cure increases BC susceptibility whereas plasmid complementation and transformation increased BC tolerance in different L. monocytogenes genetic backgrounds and L. innocua. RT-qPCR showed that BC stress exposure strongly induces the expression of mRNAs associated with pLMST6 genes for EmrC and a TetR transcription regulator. A full proteome analysis in a plasmid harboring L. monocytogenes strain revealed that the pLMST6 encoded putative TetR family transcription regulator protein is the most upregulated protein in response to BC stress exposure. An investigation into the EmrC efflux pump's substrate spectrum showed that while pLMST6 confers increased tolerance to other quaternary ammonium compounds (QACs) based disinfectants it has no impact on the sensitivity of L. monocytogenes to non-QAC disinfectants as well as on antibiotics such as ampicillin, tetracycline and gentamicin. A reduction in the survival of zebrafish embryos infected with pLMST6 plasmid harboring L. monocytogenes strains was observed when compared with plasmid cured variants of the same strains suggesting that some pLMST6 harbored genes might contribute to increased virulence capacity. Overall these results confirm the phenotypic contribution of pLMST6 plasmid in promoting and dissemination of BC tolerance in L. monocytogenes as well as provide new insights on different molecular levels of pLMST6 associated genes in response to BC stress.

SUBMITTER: Kropac AC

PROVIDER: S-EPMC6629823 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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New Insights on the Role of the pLMST6 Plasmid in Listeria monocytogenes Biocide Tolerance and Virulence.

Kropac Alexandra C AC Eshwar Athmanya K AK Stephan Roger R Tasara Taurai T

Frontiers in microbiology 20190709

Listeria monocytogenes the causative agent of listeriosis is an important public health concern and food safety challenge. Increased tolerance of this bacterium to benzalkonium chloride (BC), an antibacterial agent widely used in industrial settings, is a growing issue. Plasmid pLMST6 harboring the gene of the multidrug efflux pump protein EmrC has been recently linked to enhanced BC tolerance and meningitis due to L. monocytogenes ST6 strains. In this study, occurrence and contrib ...[more]

PMID: 31338084

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Project description:BackgroundListeria monocytogenes, an intracellular foodborne pathogen, infects immunocompromised hosts. The primary route of transmission is through contaminated food. In the gastrointestinal tract, it traverses the epithelial barrier through intracellular or paracellular routes. Strategies to prevent L. monocytogenes entry can potentially minimize infection in high-risk populations. Listeria adhesion protein (LAP) aids L. monocytogenes in crossing epithelial barriers via the paracellular route. The use of recombinant probiotic bacteria expressing LAP would aid targeted clearance of Listeria from the gut and protect high-risk populations from infection.Methodology/principal findingsThe objective was to investigate the ability of probiotic bacteria or LAP-expressing recombinant probiotic Lactobacillus paracasei (Lbp(LAP)) to prevent L. monocytogenes adhesion, invasion, and transwell-based transepithelial translocation in a Caco-2 cell culture model. Several wild type probiotic bacteria showed strong adhesion to Caco-2 cells but none effectively prevented L. monocytogenes infection. Pre-exposure to Lbp(LAP) for 1, 4, 15, or 24 h significantly (P<0.05) reduced adhesion, invasion, and transepithelial translocation of L. monocytogenes in Caco-2 cells, whereas pre-exposure to parental Lb. paracasei had no significant effect. Similarly, Lbp(LAP) pre-exposure reduced L. monocytogenes translocation by as much as 46% after 24 h. Lbp(LAP) also prevented L. monocytogenes-mediated cell damage and compromise of tight junction integrity. Furthermore, Lbp(LAP) cells reduced L. monocytogenes-mediated cell cytotoxicity by 99.8% after 1 h and 79% after 24 h.Conclusions/significanceWild type probiotic bacteria were unable to prevent L. monocytogenes infection in vitro. In contrast, Lbp(LAP) blocked adhesion, invasion, and translocation of L. monocytogenes by interacting with host cell receptor Hsp60, thereby protecting cells from infection. These data show promise for the use of recombinant probiotics in preventing L. monocytogenes infection in high-risk populations.

Project description:While the stress-responsive alternative sigma factor sigma(B) has been identified in different species of Bacillus, Listeria, and Staphylococcus, the sigma(B) regulon has been extensively characterized only in B. subtilis. We combined biocomputing and microarray-based strategies to identify sigma(B)-dependent genes in the facultative intracellular pathogen Listeria monocytogenes. Hidden Markov model (HMM)-based searches identified 170 candidate sigma(B)-dependent promoter sequences in the strain EGD-e genome sequence. These data were used to develop a specialized, 208-gene microarray, which included 166 genes downstream of HMM-predicted sigma(B)-dependent promoters as well as selected virulence and stress response genes. RNA for the microarray experiments was isolated from both wild-type and Delta sigB null mutant L. monocytogenes cells grown to stationary phase or exposed to osmotic stress (0.5 M KCl). Microarray analyses identified a total of 55 genes with statistically significant sigma(B)-dependent expression under the conditions used in these experiments, with at least 1.5-fold-higher expression in the wild type over the sigB mutant under either stress condition (51 genes showed at least 2.0-fold-higher expression in the wild type). Of the 55 genes exhibiting sigma(B)-dependent expression, 54 were preceded by a sequence resembling the sigma(B) promoter consensus sequence. Rapid amplification of cDNA ends-PCR was used to confirm the sigma(B)-dependent nature of a subset of eight selected promoter regions. Notably, the sigma(B)-dependent L. monocytogenes genes identified through this HMM/microarray strategy included both stress response genes (e.g., gadB, ctc, and the glutathione reductase gene lmo1433) and virulence genes (e.g., inlA, inlB, and bsh). Our data demonstrate that, in addition to regulating expression of genes important for survival under environmental stress conditions, sigma(B) also contributes to regulation of virulence gene expression in L. monocytogenes. These findings strongly suggest that sigma(B) contributes to L. monocytogenes gene expression during infection.

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New Insights on the Role of the pLMST6 Plasmid in Listeria monocytogenes Biocide Tolerance and Virulence.

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New Insights on the Role of the pLMST6 Plasmid in <i>Listeria monocytogenes</i> Biocide Tolerance and Virulence.

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