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Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes.

ABSTRACT: Listeria monocytogenes is an intracellular bacterial pathogen that causes life-threatening disease. The mechanisms used by L. monocytogenes to invade non-professional phagocytic cells are not fully understood. In addition to the requirement of bacterial determinants, host cell conditions profoundly influence infection. Here, we have shown that inhibition of the RhoA/ROCK pathway by pharmacological inhibitors or RNA interference results in increased L. monocytogenes invasion of murine fibroblasts and hepatocytes. InlF, a member of the internalin multigene family with no known function, was identified as a L. monocytogenes-specific factor mediating increased host cell binding and entry. Conversely, activation of RhoA/ROCK activity resulted in decreased L. monocytogenes adhesion and invasion. Furthermore, virulence of wild-type bacteria during infection of mice was significantly increased upon inhibition of ROCK activity, whereas colonization and virulence of an inlF deletion mutant was not affected, thus supporting a role for InlF as a functional virulence determinant in vivo under specific conditions. In addition, inhibition of ROCK activity in human-derived cells enhanced either bacterial adhesion or adhesion and entry in an InlF-independent manner, further suggesting a host species or cell type-specific role for InlF and that additional bacterial determinants are involved in mediating ROCK-regulated invasion of human cells.

SUBMITTER: Kirchner M

PROVIDER: S-EPMC2575752 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes.

Kirchner Marieluise M Higgins Darren E DE

Molecular microbiology 20080304 3

Listeria monocytogenes is an intracellular bacterial pathogen that causes life-threatening disease. The mechanisms used by L. monocytogenes to invade non-professional phagocytic cells are not fully understood. In addition to the requirement of bacterial determinants, host cell conditions profoundly influence infection. Here, we have shown that inhibition of the RhoA/ROCK pathway by pharmacological inhibitors or RNA interference results in increased L. monocytogenes invasion of murine fibroblasts ...[more]

PMID: 18331468

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Project description:Elevated levels of the second messenger c-di-GMP suppress virulence in diverse pathogenic bacteria, yet mechanisms are poorly characterized. In the foodborne pathogen Listeria monocytogenes, high c-di-GMP levels inhibit mammalian cell invasion. Here, we show that invasion is impaired because of the decreased expression levels of internalin genes whose products are involved in invasion. We further show that at high c-di-GMP levels, the expression of the entire virulence regulon is suppressed, and so is the expression of the prfA gene encoding the master activator of the virulence regulon. Analysis of mechanisms controlling prfA expression pointed to the transcription factor CodY as a c-di-GMP-sensitive component. In high-c-di-GMP strains, codY gene expression is decreased, apparently due to the lower activity of CodY, which functions as an activator of codY transcription. We found that listerial CodY does not bind c-di-GMP in vitro and therefore investigated whether c-di-GMP levels affect two known cofactors of listerial CodY, branched-chain amino acids and GTP. Our manipulation of branched-chain amino acid levels did not perturb the c-di-GMP effect; however, our replacement of listerial CodY with the streptococcal CodY homolog, whose activity is GTP independent, abolished the c-di-GMP effect. The results of this study suggest that elevated c-di-GMP levels decrease the activity of the coordinator of metabolism and virulence, CodY, possibly via lower GTP levels, and that decreased CodY activity suppresses L. monocytogenes virulence by the decreased expression of the PrfA virulence regulon.IMPORTANCEListeria monocytogenes is a pathogen causing listeriosis, a disease responsible for the highest mortality rate among foodborne diseases. Understanding how the virulence of this pathogen is regulated is important for developing treatments to decrease the frequency of listerial infections in susceptible populations. In this study, we describe the mechanism through which elevated levels of the second messenger c-di-GMP inhibit listerial invasion in mammalian cells. Inhibition is caused by the decreased activity of the transcription factor CodY that coordinates metabolism and virulence.

Project description:The emergence of bacterial resistance to therapeutic antibiotics limits options for treatment of common microbial diseases. Subinhibitory antibiotics dosing, often aid in the emergence of resistance, but its impact on pathogen's physiology and pathogenesis is not well understood. Here we investigated the effect of tunicamycin, a cell wall teichoic acid (WTA) biosynthesis inhibiting antibiotic at the subinhibitory dosage on Staphylococcus aureus and Listeria monocytogenes physiology, antibiotic cross-resistance, biofilm-formation, and virulence. Minimum inhibitory concentration (MIC) of tunicamycin to S. aureus and L. monocytogenes was 20-40 ?g/ml and 2.5-5 ?g/ml, respectively, and the subinhibitory concentration was 2.5-5 ?g/ml and 0.31-0.62 ?g/ml, respectively. Tunicamycin pre-exposure reduced cellular WTA levels by 18-20% and affected bacterial cell wall ultrastructure, cell membrane permeability, morphology, laser-induced colony scatter signature, and bacterial ability to form biofilms. It also induced a moderate level of cross-resistance to tetracycline, ampicillin, erythromycin, and meropenem for S. aureus, and ampicillin, erythromycin, vancomycin, and meropenem for L. monocytogenes. Pre-treatment of bacterial cells with subinhibitory concentrations of tunicamycin also significantly reduced bacterial adhesion to and invasion into an enterocyte-like Caco-2 cell line, which is supported by reduced expression of key virulence factors, Internalin B (InlB) and Listeria adhesion protein (LAP) in L. monocytogenes, and a S. aureus surface protein A (SasA) in S. aureus. Tunicamycin-treated bacteria or the bacterial WTA preparation suppressed NF-?B and inflammatory cytokine production (TNF?, and IL-6) from murine macrophage cell line (RAW 264.7) indicating the reduced WTA level possibly attenuates an inflammatory response. These results suggest that at the subinhibitory dosage, tunicamycin-mediated inhibition of WTA biosynthesis interferes with cell wall structure, pathogens infectivity and inflammatory response, and ability to form biofilms but promotes the development of antibiotic cross-resistance.

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.

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Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes.

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Inhibition of ROCK activity allows InlF-mediated invasion and increased virulence of Listeria monocytogenes.

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