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

Abstract

ABSTRACT 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.