Influence of Wall Teichoic Acid on Lysozyme Resistance inStaphylococcus aureus

Abstract

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...

Teichoic acids are temporal and spatial regulators of peptidoglycan cross-linking in Staphylococcus aureus

“…S. aureus is known for its intrinsic ability to resist lysozyme due to modifications of its PGN, such as O-acetylation of N-acetylmuramic acid residues and attachment of teichoic acids, which prevent access of the enzyme to its substrate (10,31). We treated cell walls from both the wild-type and the TagO mutant with hydrofluoric acid, which removes teichoic acids and O-acetyl groups, and incubated the resulting "naked" PGN with lysozyme.…”

“…TAs contribute to a variety of processes, including resistance to environmental stresses, such as heat (4) or low osmolarity (5), to antimicrobial peptides (6), antimicrobial fatty acids (7), cationic antibiotics (8), and lytic enzymes produced by the host, including lysozymes (9,10). TAs also act as receptors for phage particles (11), and they can bind cationic groups (particularly magnesium ions), providing a reservoir of ions close to the bacterial surface that may be important for the activity of different enzymes (12).…”

Reduced Vancomycin Susceptibility inStaphylococcus aureus, Including Vancomycin-Intermediate and Heterogeneous Vancomycin-Intermediate Strains: Resistance Mechanisms, Laboratory Detection, and Clinical Implications

“…Some data suggest that the teichoic acids have a role to help protect the cell envelope as a mechanical barrier to host defense molecules and antibiotics, and also, the positive charge of D-alanine residues repels positively charged molecules such as defensins (54,248,249). Wall teichoic acids also contribute to lysozyme resistance in S. aureus by preventing lysozyme binding to peptidoglycan (19). The dltABCD operon is controlled by the regulator GraRS (also called ApsRS), which senses and responds to defensins and other antimicrobial peptides and regulates the alanylation of teichoic acids in response to the presence of antimicrobial compounds, indicating that the structure of teichoic acids can change in response to challenges (111,180,181).…”

Pathways and roles of wall teichoic acid glycosylation in Staphylococcus aureus

“…Of note, WTAs are exposed at the S. aureus cell surface and they are required for PBP2a-mediated ␤-lactam antibiotic resistance Farha et al, 2013;Maki et al, 1994). Moreover, S. aureus mutants lacking WTA are susceptible to antimicrobial fatty acids from human skin (Kohler et al, 2009) and WTA contributes to S. aureus lysozyme resistance (Bera et al, 2007) suggesting a general protective role of WTA against antimicrobials. Because of its importance in vivo and in resistance to ␤-lactam antibiotics S. aureus WTA represents a very attractive target for the development of new antibiotics.…”