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

Silvia Herbert,Ambrose Cheung,Christiane Nerz,Dirk Kraus,Michael Meehl,Friedrich Götz,Agnieszka Bera,Andreas Peschel,Christiane Goerke,Jeffrey N Weiser

doi:10.1371/journal.ppat.0030102

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-R115 (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.

Highlights

In humans, lysozyme is found in a wide variety of fluids, such as tears, breast milk, and respiratory and saliva secretions, as well as in cells of the innate immune system, including neutrophils, monocytes, macrophages, and epithelial cells [1,2]
We demonstrate that lysozyme acts on S. aureus in two ways: as a muramidase and as a cationic antimicrobial peptide (CAMP)
Transcriptome analysis of the glycopeptide resistance-associated (GraRS) twocomponent system revealed that this global regulator controls 248 genes such as other global regulators, colonization factors, or exotoxin-encoding genes

Summary

Introduction

Lysozyme is found in a wide variety of fluids, such as tears, breast milk, and respiratory and saliva secretions, as well as in cells of the innate immune system, including neutrophils, monocytes, macrophages, and epithelial cells [1,2]. Lysozyme is an important protein in the innate defense response against invading microorganisms and acts on bacteria by hydrolyzing the ß-1,4 glycosidic bonds between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlucNAc), resulting in degradation of peptidoglycan (PG), and subsequent cell lysis [3,4]. Most bacterial species are sensitive to lysozyme, but some important human pathogens, such as Staphylococcus aureus, Neisseria gonorrhoeae, and Proteus mirabilis, are resistant. We were able to prove that O-acetyltransferase A (OatA) of S. aureus is responsible for O-acetylation of the PG, and this leads to resistance to the muramidase activity of lysozyme [10]. We showed that the MurNAc was Oacetylated only in pathogenic, lysozyme-resistant staphylococci (e.g., S. aureus, S. epidermidis, S. lugdunensis, and others). All nonpathogenic species (e.g., S. carnosus, S. gallinarum, or S. xylosus) were lysozyme sensitive and lacked PG-specific Oacetylation. OatA can be regarded as a general virulence factor [11]

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