The Lysozyme-Induced Peptidoglycan N -Acetylglucosamine Deacetylase PgdA (EF1843) Is Required for Enterococcus faecalis Virulence

Abstract

f Lysozyme is a key component of the innate immune response in humans that provides a first line of defense against microbes. The bactericidal effect of lysozyme relies both on the cell wall lytic activity of this enzyme and on a cationic antimicrobial peptide activity that leads to membrane permeabilization. Among Gram-positive bacteria, the opportunistic pathogen Enterococcus faecalis has been shown to be extremely resistant to lysozyme. This unusual resistance is explained partly by peptidoglycan Oacetylation, which inhibits the enzymatic activity of lysozyme, and partly by D-alanylation of teichoic acids, which is likely to inhibit binding of lysozyme to the bacterial cell wall. Surprisingly, combined mutations abolishing both peptidoglycan O-acetylation and teichoic acid alanylation are not sufficient to confer lysozyme susceptibility. In this work, we identify another mechanism involved in E. faecalis lysozyme resistance. We show that exposure to lysozyme triggers the expression of EF1843, a protein that is not detected under normal growth conditions. Analysis of peptidoglycan structure from strains with EF1843 loss-and gain-of-function mutations, together with in vitro assays using recombinant protein, showed that EF1843 is a peptidoglycan N-acetylglucosamine deacetylase. EF1843-mediated peptidoglycan deacetylation was shown to contribute to lysozyme resistance by inhibiting both lysozyme enzymatic activity and, to a lesser extent, lysozyme cationic antimicrobial activity. Finally, EF1843 mutation was shown to reduce the ability of E. faecalis to cause lethality in the Galleria mellonella infection model. Taken together, our results reveal that peptidoglycan deacetylation is a component of the arsenal that enables E. faecalis to thrive inside mammalian hosts, as both a commensal and a pathogen.

Eep Confers Lysozyme Resistance to Enterococcus faecalis via the Activation of the Extracytoplasmic Function Sigma Factor SigV

“…When the eep gene was complemented back into the eep deletion mutant using a low-copy-number plasmid, the complement strain behaved similarly to the wild type, suggesting that the phenotype we observed was Eep dependent. Consistent with previous observations, the pgdA deletion mutant did not show any change in lysozyme susceptibility compared to that of the wild type (20), even though it is known to be regulated by SigV at the transcriptional level (4, 5). FA2-2 and mutants lacking eep and sigV in this genetic background were used as controls in this study to eliminate any strain bias in the lysozyme resistance mechanism.…”

“…Our present studies also confirmed the absence of a link between PgdA and lysozyme resistance in E. faecalis V583. PgdA does, however, contribute to lysozyme susceptibility in Streptococcus pneumoniae and virulence in animal models of infection (31)(32)(33), and recent evidence suggests that PgdA is linked to virulence in E. faecalis, as a mutant in pgdA was attenuated in a Galleria mellonella infection model (20). What role PgdA might be playing in a mammalian host during infection remains to be elucidated.…”

Clostridium difficile Extracytoplasmic Function σ Factor σ V Regulates Lysozyme Resistance and Is Necessary for Pathogenesis in the Hamster Model of Infection

“…difficile contains 10 putative polysaccharide deacetylases, of which only 1, encoded by pdaV, is significantly induced by V in a lysozyme-dependent manner. PdaV belongs to the PgdA family of polysaccharide deacetylases, several of which have been implicated in increased lysozyme resistance (19,20,24). We found that expression of pdaV in the csfV mutant increased lysozyme resistance and peptidoglycan deacetylation.…”

“…In B. subtilis, V is required for expression of oatA, which encodes a peptidoglycan O-acetyltransferase that increases lysozyme resistance (22,32,33). In E. faecalis, V is required for expression of a peptidoglycan deacetylase, PgdA (19,21). We found that in response to lysozyme, C. difficile V induces expression of a peptidoglycan deacetylase, which results in deacetylation of the C. difficile peptidoglycan.…”

“…Increased lysozyme resistance is an important virulence factor for numerous Gram-positive bacteria, including Enterococcus faecalis, Staphylococcus aureus, Streptococcus pneumoniae, and Listeria monocytogenes (15)(16)(17)(18)(19)(20). To resist killing by lysozyme, bacteria have evolved several mechanisms to modify the acetylation patterns of peptidoglycan, resulting in increased lysozyme resistance (14).…”