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Characterization of Listeria monocytogenes Peptidoglycan N-Deacetylase and O-Acetylase Mutations and the Role of Lysozyme Resistance During Infection

  • Author(s): Rae, Chris Steven
  • Advisor(s): Portnoy, Dan
  • et al.
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 an O-acetylmuramic acid transferase (Oat). We characterized Pgd-minus, Oat-minus and double mutants to determine the specific role of L. monocytogenes peptidoglycan acetylation in conferring lysozyme sensitivity and during infection of macrophages and mice. Pgd-minus and Pgd-minus/Oat-minus 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 AIM-2-dependent pyroptosis. Each of the in vitro phenotypes was rescued upon infection of LysM-minus macrophages. The addition of extracellular lysozyme to LysM-minus macrophages restored cytokine induction, host-cell death and L. monocytogenes growth inhibition. This surprising observation suggested that extracellular lysozyme can access the macrophage cytosol and act on intracellular, lysozyme-sensitive, bacteria.

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