Mutations of the Listeria monocytogenes Peptidoglycan<i>N</i>-Deacetylase and<i>O</i>-Acetylase Result in Enhanced Lysozyme Sensitivity, Bacteriolysis, and Hyperinduction of Innate Immune Pathways

<jats:title>ABSTRACT</jats:title><jats:p><jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">Listeria monocytogenes</jats:named-content>is a Gram-positive intracellular pathogen that is naturally resistant to lysozyme. Recently, it was shown that peptidoglycan modification by N-deacetylation or O-acetylation confers resistance to lysozyme in various Gram-positive bacteria, including<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">L. monocytogenes</jats:named-content>.<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">L. monocytogenes</jats:named-content>peptidoglycan is deacetylated by the action of<jats:italic>N</jats:italic>-acetylglucosamine deacetylase (Pgd) and acetylated by<jats:italic>O</jats:italic>-acetylmuramic acid transferase (Oat). We characterized Pgd<jats:sup>−</jats:sup>, Oat<jats:sup>−</jats:sup>, and double mutants to determine the specific role of<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">L. monocytogenes</jats:named-content>peptidoglycan acetylation in conferring lysozyme sensitivity during infection of macrophages and mice. Pgd<jats:sup>−</jats:sup>and Pgd<jats:sup>−</jats:sup>Oat<jats:sup>−</jats:sup>double mutants were attenuated approximately 2 and 3.5 logs, respectively,<jats:italic>in vivo</jats:italic>. In bone-marrow derived macrophages, the mutants demonstrated intracellular growth defects and increased induction of cytokine transcriptional responses that emanated from a phagosome and the cytosol. Lysozyme-sensitive mutants underwent bacteriolysis in the macrophage cytosol, resulting in AIM2-dependent pyroptosis. Each of the<jats:italic>in vitro</jats:italic>phenotypes was rescued upon infection of LysM<jats:sup>−</jats:sup>macrophages. The addition of extracellular lysozyme to LysM<jats:sup>−</jats:sup>macrophages restored cytokine induction, host cell death, and<jats:named-content xmlns:xlink="http://www.w3.org/1999/xlink" content-type="genus-species" xlink:type="simple">L. monocytogenes</jats:named-content>growth inhibition. This surprising observation suggests that extracellular lysozyme can access the macrophage cytosol and act on intracellular lysozyme-sensitive bacteria.</jats:p>