Molecular Basis of Resistance to Muramidase and Cationic Antimicrobial Peptide Activity of Lysozyme in Staphylococci

Abstract

It has been shown recently that modification of peptidoglycan by O-acetylation renders pathogenic staphylococci resistant to the muramidase activity of lysozyme. Here, we show that a Staphylococcus aureus double mutant defective in O-acetyltransferase A (OatA), and the glycopeptide resistance-associated two-component system, GraRS, is much more sensitive to lysozyme than S. aureus with the oatA mutation alone. The graRS single mutant was resistant to the muramidase activity of lysozyme, but was sensitive to cationic antimicrobial peptides (CAMPs) such as the human lysozyme-derived peptide ₁₀₇R-A-W-V-A-W-R-N-R ₁₁₅ (LP9), polymyxin B, or gallidermin. A comparative transcriptome analysis of wild type and the graRS mutant revealed that GraRS controls 248 genes. It up-regulates global regulators ( rot, sarS, or mgrA), various colonization factors, and exotoxin-encoding genes, as well as the ica and dlt operons. A pronounced decrease in the expression of the latter two operons explains why the graRS mutant is also biofilm-negative. The decrease of the dlt transcript in the graRS mutant correlates with a 46.7% decrease in the content of esterified d-alanyl groups in teichoic acids. The oatA /dltA double mutant showed the highest sensitivity to lysozyme; this mutant completely lacks teichoic acid–bound d-alanine esters, which are responsible for the increased susceptibility to CAMPs and peptidoglycan O-acetylation. Our results demonstrate that resistance to lysozyme can be dissected into genes mediating resistance to its muramidase activity ( oatA) and genes mediating resistance to CAMPs (graRS and dlt). The two lysozyme activities act synergistically, as the oatA /dltA or oatA /graRS double mutants are much more susceptible to lysozyme than each of the single mutants.

Author Summary

In humans, lysozyme plays an important role in the suppression of bacterial infections. However, some bacterial pathogens, such as Staphylococcus aureus, are completely resistant to lysozyme. Here we demonstrate that lysozyme acts on S. aureus in two ways: as a muramidase (cell wall lytic enzyme) and as a cationic antimicrobial peptide (CAMP). S. aureus has developed resistance mechanisms against both activities by modifying distinct cell wall structures. Modification of the peptidoglycan by O-acetylation (OatA) renders the cells resistant to the muramidase activity. Modification of teichoic acids by d-alanine esterification (Dlt) renders the cells resistant to lysozyme's CAMPs and other CAMPs. Transcriptome analysis of the glycopeptide resistance-associated (GraRS) two-component system revealed that this global regulator controls 248 genes such as other global regulators, colonization factors, or exotoxin-encoding genes. Since GraRS also upregulates the dlt operon, it was not surprising that in the graRS mutant teichoic acid d-alanylation is markedly decreased, which explains its increased sensitivity to CAMPs. By comparative analysis of mutants we were able to dissect genes that were responsive to the dual activities of lysozyme. Here we show how efficiently S. aureus is protected from the human defense system, which enables this pathogen to cause persistent infections.