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

Listeria monocytogenes 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 L. monocytogenes. L. monocytogenes peptidoglycan 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. monocytogenes peptidoglycan 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 vitro phenotypes was rescued upon infection of LysM–macrophages. The addition of extracellular lysozyme to LysM–macrophages restored cytokine induction, host cell death, and L. monocytogenes growth inhibition. This surprising observation suggests that extracellular lysozyme can access the macrophage cytosol and act on intracellular lysozyme-sensitive bacteria.