Helicobacter pylori Peptidoglycan Modifications Confer Lysozyme Resistance and Contribute to Survival in the Host

Abstract

The prominent host muramidase lysozyme cleaves bacterial peptidoglycan (PG), and the enzyme is abundant in mucosal secretions. The lytic enzyme susceptibility of Gram-negative bacteria and mechanisms they use to thwart lytic enzyme activity are poorly studied. We previously characterized a Helicobacter pylori PG modification enzyme, an N-deacetylase (PgdA) involved in lysozyme resistance. In this study, another PG modification enzyme, a putative PG O-acetyltransferase (PatA), was identified. Mass spectral analysis of the purified PG demonstrated that a patA strain contained a greatly reduced amount of acetylated muropeptides, indicating a role for PatA in H. pylori PG O-acetylation. The PG modification mutant strains (pgdA, patA, or pgdA patA) were more susceptible to lysozyme killing than the parent, but this assay required high lysozyme levels (up to 50 mg/ml). However, addition of host lactoferrin conferred lysozyme sensitivity to H. pylori, at physiologically relevant concentrations of both host components (3 mg/ml lactoferrin plus 0.3 mg/ml lysozyme). The pgdA patA double mutant strain was far more susceptible to lysozyme/lactoferrin killing than the parent. Peptidoglycan purified from a pgdA patA mutant was five times more sensitive to lysozyme than PG from the parent strain, while PG from both single mutants displayed intermediate sensitivity. Both sensitivity assays for whole cells and for purified PGs indicated that the modifications mediated by PgdA and PatA have a synergistic effect, conferring lysozyme tolerance. In a mouse infection model, significant colonization deficiency was observed for the double mutant at 3 weeks postinoculation. The results show that PG modifications affect the survival of a Gram-negative pathogen.Importance Pathogenic bacteria evade host antibacterial enzymes by a variety of mechanisms, which include resisting lytic enzymes abundant in the host. Enzymatic modifications to peptidoglycan (PG, the site of action of lysozyme) are a known mechanism used by Gram-positive bacteria to protect against host lysozyme attack. However, Gram-negative bacteria contain a thin layer of PG and a recalcitrant outer membrane permeability barrier to resist lysis, so molecular modifications to cell wall structure in order to combat lysis remain largely unstudied. Here we show that two Helicobacter pylori PG modification enzymes (PgdA and PatA) confer a clear protective advantage to a Gram-negative bacterium. They protect the bacterium from lytic enzyme degradation, albeit via different PG modification activities. Many pathogens are Gram negative, so some would be expected to have a similar cell wall-modifying strategy. Understanding such strategies may be useful for combating pathogen growth.

Aconitase Functions as a Pleiotropic Posttranscriptional Regulator in Helicobacter pylori

“…Recently, our laboratory demonstrated that H. pylori apo-AcnB binds to the 3= UTR of the peptidoglycan deacetylase (pgdA) transcript, resulting in transcript stability and increased PgdA expression (5). PgdA confers lysozyme resistance and immune evasion; its expression is increased when H. pylori cells are exposed to oxidative stress or are in contact with host immune cells (19)(20)(21)(22). AcnB plays an important role in the posttranscriptional regulation of PgdA (5).…”

Aconitase-Mediated Posttranscriptional Regulation of Helicobacter pylori Peptidoglycan Deacetylase

“…H. pylori can thrive in the gastric mucosa by employing a battery of diverse antioxidant enzymes that detoxify oxidants and repair essential biomolecules (2,3). Additionally, H. pylori utilizes other mechanisms to persist in the host, including peptidoglycan (PG) modification (4).…”

A Moraxella catarrhalis Two-Component Signal Transduction System Necessary for Growth in Liquid Media Affects Production of Two Lysozyme Inhibitors

“…6D). These lysozyme killing experiments were performed in the presence of human apolactoferrin, another important component of airway fluids (102) that can increase the sensitivity of Gram-negative bacteria to killing by lysozyme (47,72,103) by affecting the permeability of the outer membrane (74). Among the 12 M. catarrhalis strains whose genomes have been sequenced (35,36), LipA proteins have 99 to 100% identity, with LipB proteins being 97 to 100% identical.…”