Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections. Due to the rapid evolution of antibiotic resistance that leads to treatment failure, it is important to understand the underlying mechanisms. Here, the cell wall structures of several laboratory vancomycin-intermediate S. aureus (VISA) strains were analyzed. Among the VISA strains were S. aureus VC40, which accumulated 79 mutations, including most importantly 2 exchanges in the histidine-kinase VraS, and developed full resistance against vancomycin (MIC, 64 μg/ml); a revertant S. aureus VC40R, which has an additional mutation in vraR (MIC, 4 μg/ml); and S. aureus VraS(VC40), in which the 2 vraS mutations were reconstituted into a susceptible background (MIC, 4 μg/ml). A ultraperformance liquid chromatography (UPLC) analysis showed that S. aureus VC40 had a significantly decreased cross-linking of the peptidoglycan. Both S. aureus VC40 and S. aureus VraS(VC40) displayed reduced autolysis and an altered autolysin profile in a zymogram. Most striking was the significant increase in d-alanine and N-acetyl-d-glucosamine (GlcNAc) substitution of the wall teichoic acids (WTAs) in S. aureus VC40. Nuclear magnetic resonance (NMR) analysis revealed that this strain had mostly β-glycosylated WTAs in contrast to the other strains, which showed only the α-glycosylation peak. Salt stress induced the incorporation of β-GlcNAc anomers and drastically increased the vancomycin MIC for S. aureus VC40R. In addition, β-glycosylated WTAs decreased the binding affinity of AtlA, the major autolysin of S. aureus, to the cell wall, compared with α-glycosylated WTAs. In conclusion, there is a novel connection between wall teichoic acids, autolysis, and vancomycin susceptibility in S. aureus. IMPORTANCE Infections with methicillin-resistant Staphylococcus aureus are commonly treated with vancomycin. This antibiotic inhibits cell wall biosynthesis by binding to the cell wall building block lipid II. We set out to characterize the mechanisms leading to decreased vancomycin susceptibility in a laboratory-generated strain, S. aureus VC40. This strain has an altered cell wall architecture with a thick cell wall with low cross-linking, which provides decoy binding sites for vancomycin. The low cross-linking, necessary for this resistance mechanism, decreases the stability of the cell wall against lytic enzymes, which separate the daughter cells. Protection against these enzymes is provided by another cell wall polymer, the teichoic acids, which contain an unusually high substitution with sugars in the β-conformation. By experimentally increasing the proportion of β-N-acetyl-d-glucosamine in a closely related isolate through the induction of salt stress, we could show that the β-conformation of the sugars plays a vital role in the resistance of S. aureus VC40.

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