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Recombinant broad-range phospholipase C from Listeria monocytogenes exhibits optimal activity at acidic pH.

ABSTRACT: The broad-range phospholipase C (PLC) from Listeria monocytogenes has been expressed using an intein expression system and characterized. This zinc metalloenzyme, similar to the homologous enzyme from Bacillus cereus, targets a wide range of lipid substrates. With monomeric substrates, the length of the hydrophobic acyl chain has significant impact on enzyme efficiency by affecting substrate affinity (Km). Based on a homology model of the enzyme to the B. cereus protein, several active site residue mutations were generated. While this PLC shares many of the mechanistic characteristics of the B. cereus PLC, a major difference is that the L. monocytogenes enzyme displays an acidic pH optimum regardless of substrate status (monomer, micelle, or vesicle). This unusual behavior might be advantageous for its role in the pathogenicity of L. monocytogenes.

SUBMITTER: Huang Q

PROVIDER: S-EPMC4829451 | biostudies-literature | 2016 Jun

REPOSITORIES: biostudies-literature

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Project description:In this study, we investigated the requirement of the Listeria monocytogenes broad-range phospholipase C (PC-PLC) during infection of human epithelial cells. L. monocytogenes is a facultative intracellular bacterial pathogen of humans and a variety of animal species. After entering a host cell, L. monocytogenes is initially surrounded by a membrane-bound vacuole. Bacteria promote their escape from this vacuole, grow within the host cell cytosol, and spread from cell to cell via actin-based motility. Most infection studies with L. monocytogenes have been performed with mouse cells or an in vivo mouse model of infection. In all mouse-derived cells tested, the pore-forming cytolysin listeriolysin O (LLO) is absolutely required for lysis of primary vacuoles formed during host cell entry. However, L. monocytogenes can escape from primary vacuoles in the absence of LLO during infection of human epithelial cell lines Henle 407, HEp-2, and HeLa. Previous studies have shown that the broad-range phospholipase C, PC-PLC, promotes lysis of Henle 407 cell primary vacuoles in the absence of LLO. Here, we have shown that PC-PLC is also required for lysis of HEp-2 and HeLa cell primary vacuoles in the absence of LLO expression. Furthermore, our results indicated that the amount of PC-PLC activity is critical for the efficiency of vacuolar lysis. In an LLO-negative derivative of L. monocytogenes strain 10403S, expression of PC-PLC has to increase before or upon entry into human epithelial cells, compared to expression in broth culture, to allow bacterial escape from primary vacuoles. Using a system for inducible PC-PLC expression in L. monocytogenes, we provide evidence that phospholipase activity can be increased by elevated expression of PC-PLC or Mpl, the enzyme required for proteolytic activation of PC-PLC. Lastly, by using the inducible PC-PLC expression system, we demonstrate that, in the absence of LLO, PC-PLC activity is not only required for lysis of primary vacuoles in human epithelial cells but is also necessary for efficient cell-to-cell spread. We speculate that the additional requirement for PC-PLC activity is for lysis of secondary double-membrane vacuoles formed during cell-to-cell spread.

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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Recombinant broad-range phospholipase C from Listeria monocytogenes exhibits optimal activity at acidic pH.

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