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Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

ABSTRACT: The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here we demonstrate that depleting ?E-catenin, which indirectly links E-cadherin to F-actin, did not decrease L. monocytogenes invasion of epithelial cells in tissue culture. Instead, invasion increased due to increased bacterial adhesion to epithelial monolayers with compromised cell-cell junctions. Furthermore, expression of a mutant E-cadherin lacking the intracellular domain was sufficient for efficient L. monocytogenes invasion of epithelial cells. Importantly, direct biotin-mediated binding of bacteria to surface lipids in the plasma membrane of host epithelial cells was sufficient for uptake. Our results indicate that the only requirement for L. monocytogenes invasion of epithelial cells is adhesion to the host cell surface, and that E-cadherin-mediated coupling of the bacterium to F-actin is not required.

SUBMITTER: Ortega FE

PROVIDER: S-EPMC5662255 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

Ortega Fabian E FE Rengarajan Michelle M Chavez Natalie N Radhakrishnan Prathima P Gloerich Martijn M Bianchini Julie J Siemers Kathleen K Luckett William S WS Lauer Peter P Nelson W James WJ Theriot Julie A JA

Molecular biology of the cell 20170906 22

The intestinal epithelium is the first physiological barrier breached by the Gram-positive facultative pathogen Listeria monocytogenes during an in vivo infection. Listeria monocytogenes binds to the epithelial host cell receptor E-cadherin, which mediates a physical link between the bacterium and filamentous actin (F-actin). However, the importance of anchoring the bacterium to F-actin through E-cadherin for bacterial invasion has not been tested directly in epithelial cells. Here ...[more]

PMID: 28877987

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Project description:BackgroundListeria adhesion protein (Lap), an alcohol acetaldehyde dehydrogenase (lmo1634) promotes bacterial paracellular translocation through epithelial cell junctions during gastrointestinal phase of infection. Secreted Lap is critical for pathogenesis and is mediated by SecA2 system; however, if strain dependent variation in Lap secretion would affect L. monocytogenes paracellular translocation through epithelial barrier is unknown.MethodsAmounts of Lap secretion were examined in clinical isolates of L. monocytogenes by cell fractionation analysis using Western blot. Quantitative reverse transcriptase PCR (qRT-PCR) was used to verify protein expression profiles. Adhesion and invasion of isolates were analyzed by in vitro Caco-2 cell culture model and paracellular translocation was determined using a trans-well model pre-seeded with Caco-2 cells.ResultsWestern blot revealed that expression of Lap in whole cell preparation of isolates was very similar; however, cell fractionation analysis indicated variable Lap secretion among isolates. The strains showing high Lap secretion in supernatant exhibited significantly higher adhesion (3.4 - 4.8% vs 1.5 - 2.3%, P < 0.05), invasion and paracellular translocation in Caco-2 cells than the low secreting isolates. In cell wall fraction, Lap level was mostly uniform for both groups, while Lap accumulated in cytosol in low secreting strains indicating that Lap distribution in cellular compartments is a strain-dependent phenomenon, which may be controlled by the protein transport system, SecA2. ΔsecA2 mutants showed significantly reduced paracellular translocation through epithelial barrier (0.48 ± 0.01 vs 0.24 ± 0.02, P < 0.05). qRT-PCR did not show any discernible variation in lap transcript levels in either high or low secreting isolates.ConclusionThis study revealed that secreted Lap is an important determinant in Lap-mediated L. monocytogenes translocation through paracellular route and may serve as an indicator for pathogenic potential of an isolate.

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: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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Adhesion to the host cell surface is sufficient to mediate Listeria monocytogenes entry into epithelial cells.

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Adhesion to the host cell surface is sufficient to mediate <i>Listeria monocytogenes</i> entry into epithelial cells.

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