Generation of Markerless Gene Deletion Mutants in Listeria monocytogenes Using a Mutated pheS for Counterselection.

Gene alteration/deletion by allelic exchange is the preferred strategy for gene manipulation in bacteria. Here we present the fundamentals for an efficient allelic exchange gene deletion method in the bacterial pathogen Listeria monocytogenes. Combining vector generation by Gibson assembly with a counterselection system based on the mutated phenylalanine synthetase (pheS*) makes the generation of gene deletion mutants straightforward and time efficient.

Bone Marrow-Derived Macrophage (BMDM ) Infection by Listeria monocytogenes.

Listeria monocytogenes is a gram-positive bacterium adapted to life as both an environmental saprophyte and a pathogenic parasite of mammalian hosts, with a transcriptomic program tailored for each niche. Study of the L. monocytogenes pathogenic lifestyle requires conditions that mimic the mammalian niche. Of the myriad experimental models used to achieve such conditions, the bone marrow-derived macrophage (BMDM) is a relatively simple and reliable primary immune cell model for L. monocytogenes infections. Here we describe the extraction, preparation, and storage of BMDMs and their use in L. monocytogenes infection experiments.

A Metzincin and TIMP-Like Protein Pair of a Phage Origin Sensitize Listeria monocytogenes to Phage Lysins and Other Cell Wall Targeting Agents.

Infection of mammalian cells by Listeria monocytogenes (Lm) was shown to be facilitated by its phage elements. In a search for additional phage remnants that play a role in Lm's lifecycle, we identified a conserved locus containing two XRE regulators and a pair of genes encoding a secreted metzincin protease and a lipoprotein structurally similar to a TIMP-family metzincin inhibitor. We found that the XRE regulators act as a classic CI/Cro regulatory switch that regulates the expression of the metzincin and TIMP-like genes under intracellular growth conditions. We established that when these genes are expressed, their products alter Lm morphology and increase its sensitivity to phage mediated lysis, thereby enhancing virion release. Expression of these proteins also sensitized the bacteria to cell wall targeting compounds, implying that they modulate the cell wall structure. Our data indicate that these effects are mediated by the cleavage of the TIMP-like protein by the metzincin, and its subsequent release to the extracellular milieu. While the importance of this locus to Lm pathogenicity remains unclear, the observation that this phage-associated protein pair act upon the bacterial cell wall may hold promise in the field of antibiotic potentiation to combat antibiotic resistant bacterial pathogens.