Mutations of the Listeria monocytogenesPeptidoglycan N-Deacetylase and O-Acetylase Result in Enhanced Lysozyme Sensitivity, Bacteriolysis, and Hyperinduction of Innate Immune Pathways

ABSTRACTListeria monocytogenesis 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 L. monocytogenes. L. monocytogenespeptidoglycan is deacetylated by the action of N-acetylglucosamine deacetylase (Pgd) and acetylated by O-acetylmuramic acid transferase (Oat). We characterized Pgd−, Oat−, and double mutants to determine the specific role of L. monocytogenespeptidoglycan acetylation in conferring lysozyme sensitivity during infection of macrophages and mice. Pgd−and Pgd−Oat−double mutants were attenuated approximately 2 and 3.5 logs, respectively, in vivo. 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 in vitrophenotypes was rescued upon infection of LysM−macrophages. The addition of extracellular lysozyme to LysM−macrophages restored cytokine induction, host cell death, and L. monocytogenesgrowth inhibition. This surprising observation suggests that extracellular lysozyme can access the macrophage cytosol and act on intracellular lysozyme-sensitive bacteria.