SummaryStaphylococcus species belong to one of the few bacterial genera that are completely lysozyme resistant, which greatly contributes to their persistence and success in colonizing the skin and mucosal areas of humans and animals. In an attempt to discover the cause of lysozyme resistance, we identified a gene, oatA , in Staphylococcus aureus . The corresponding oatA deletion mutant had an increased sensitivity to lysozyme. HPLC and electrospray ionization tandem mass spectrometry analyses of the cell wall revealed that the muramic acid of peptidoglycan of the wildtype strain was O-acetylated at C6-OH, whereas the muramic acid of the oatA mutant lacked this modification. The complemented oatA mutant was lysozyme resistant. We identified the first bacterial peptidoglycan-specific O -acetyltransferase in S. aureus and showed that OatA, an integral membrane protein, is the molecular basis for the high lysozyme resistance in staphylococci.

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.

Staphylococcus aureus peptidoglycan (PG) is completely resistant to the hydrolytic activity of lysozyme. Here we show that modifications in PG by O acetylation, wall teichoic acid, and a high degree of cross-linking contribute to this resistance.The human defense system uses a variety of factors to destroy bacteria that include reactive oxygen substances (7), bacteriolytic enzymes (lysozyme [13] and phospholipase A2 [17]), the complement system, and antimicrobial peptides (9). One important and widespread defense enzyme is lysozyme (5, 10), a component of both phagocytic and secretory granules of neutrophils, monocytes, macrophages, and epithelial cells (9, 13). Pathogenic bacteria, such as Staphylococcus aureus, express a wide variety of virulence factors that enable the organism to cause acute and chronic infections (1, 15). Host-colonizing bacteria must have developed mechanisms to overcome the adaptive and innate immune system. As recently shown, S. aureus is completely resistant to lysozyme, and the primary mechanism for this resistance is the modification of its peptidoglycan (PG) by O acetylation at the C-6 position of the N-acetyl muramic acid (NAM) (3). However, in S. aureus the C-6 position of NAM also carries phosphoester-linked wall teichoic acid (WTA) (19, 21) ( Fig. 1), and the question is whether WTA also contributes to lysozyme resistance. Our results indicate that WTA and the degree of PG cross-linking indeed contribute to resistance against the hydrolytic activity of lysozyme.Characterization of the double mutant SA⌬oatA/tagO. In order to analyze the role of WTA of S. aureus SA113 in lysozyme resistance, we tested a tagO deletion mutant (SA⌬tagO:: erm) that is completely devoid of WTA (21). Surprisingly, this mutant turned out to be as resistant to lysozyme as the wild type (WT) (Fig. 2). This was astonishing insofar as WTA is a much more bulky residue than the O-acetyl group and should therefore hinder the interaction of lysozyme with PG more efficiently. Therefore, we created a double-deletion mutant (SA⌬oatA/tagO) that lacked both WTA and O acetylation. Gene replacement of oatA in the mutant SA⌬tagO::erm was performed as described before (6). As a control, the oatA gene in the double mutant SA⌬oatA/tagO was complemented with the plasmid pRBoatA (3); the complemented mutant restored the lysozyme-resistant phenotype. In liquid medium (B medium or tryptic soy broth), growth of the wild type and that of the tagO mutant were not inhibited at lysozyme concentrations of Ͼ50 mg per ml, growth of the oatA mutant was inhibited by 1 mg per ml, and the double mutant SA⌬oatA/tagO showed the highest lysozyme sensitivity (Ͻ0.05 mg per ml); the MIC of this mutant was 20-fold lower than that of the mutant SA⌬tagO. The increase in the lysozyme inhibition zone of SA⌬oatA/tagO over that of SA⌬oatA is illustrated in Fig. 2.In a cell lysis assay, lysozyme (300 g/ml) was added to exponentially growing cultures. The optical density at 578 nm (OD 578 ) values of SA113 and SA⌬tagO were unaffected in the presence of l...

Human-pathogenic bacteria that are able to cause persistent infections must have developed mechanisms to resist the immune defense system. Lysozyme, a cell wall-lytic enzyme, is one of the first defense compounds induced in serum and tissues after the onset of infection. Recently, we showed that Staphylococcus aureus is resistant to lysozyme by O acetylating its peptidoglycan (PG) by O-acetyltransferase (OatA). We asked the question of which staphylococcal species PG is O acetylated. We applied various methods, such as genome analysis, PCR, Southern blotting, lysozyme sensitivity assay, and verification of O acetylation of PG by high-performance liquid chromatography (HPLC) analysis. PCR analysis using S. aureus-derived oatA primers and Southern blotting did not yield reliable results with other staphylococcal species. Therefore, we used the HPLC-based assay to directly detect PG O acetylation. Our studies revealed that the muramic acid was O acetylated only in pathogenic, lysozyme-resistant staphylococci (e.g., S. aureus, S. epidermidis, S. lugdunensis, and others). All nonpathogenic species were lysozyme sensitive. They can be divided into sensitive species (e.g., S. carnosus, S. gallinarum, and S. xylosus) and hypersensitive species (e.g., S. equorum, S. lentus, and S. arlettae). In all lysozyme-sensitive species, the analyzed PG was de-Oacetylated. When we transformed the oatA gene from lysozyme-resistant S. aureus into S. carnosus, the corresponding transformants also became lysozyme resistant.