The presence of peptidoglycan O-acetyltransferase in various staphylococcal species correlates with lysozyme resistance and pathogenicity

Abstract

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-O-acetylated. When we transformed the oatA gene from lysozyme-resistant S. aureus into S. carnosus, the corresponding transformants also became lysozyme resistant.

FIG. 1.

Agar diffusion-based assay with 4 mg of lysozyme. (A) S. lugdunensis, (B) S. epidermidis, (C) S. haemolyticus, (D) S. carnosus, (E) S. gallinarum, (F) S. xylosus, (G) S. lentus, (H) S. equorum, and (I) S. arlettae. Growth inhibition caused by lysozyme was tested on TSA plates. Overnight cultures were diluted in TSA soft agar to 0.5 × 10⁶ CFU/ml⁻¹ and poured onto TSA plates. Lysozyme was applied into wells (diameter, 5 mm). Plates were incubated overnight at 37°C. Lysozyme formed white, characteristic precipitation zone. Micrococcus luteus is shown as an example of a lysozyme-hypersensitive bacterium.

FIG. 2.

Growth curve and lysozyme-induced cell lysis. Overnight cultures were inoculated into fresh BM broth. After 5 h of incubation, lysozyme was added at a concentration of 300 μg/ml (indicated by an arrow). Incubation continued for another 19 h. (A) S. lentus alone ▪ and with lysozyme □ and S. arlettae alone ▴ and with lysozyme ▵ are shown. (B) S. carnosus alone ▪ and with lysozyme □ and S. carnosus carrying pTX15-oatA in the absence ▴ and presence of lysozyme ▵ are shown.

FIG. 3.

Fragment of pTX15 encoding xylose-inducible oatA. Sequences up- and downstream of the oatA gene are also shown. T_oatA, terminator region.

FIG. 4.

Southern blot showing oatA homologues detected in staphylococcal species. EcoRI-digested chromosomal DNA was probed with oatA from S. aureus SA113 (wild type). Molecular sizes (in kilobases) of HindIII-digested phage λ DNA are shown to the left of the blot. The oatA signal and SA0834, the paralogue of oatA (56% identity with oatA at the DNA sequence level), are indicated by arrows. Ma. caseolyticus, Macrococcus caseolyticus.

FIG. 5.

HPLC-based detection of O-linked acetate from the peptidoglycan of staphylococci. The acetate peak (retention time, 15.2 min) is indicated by a black arrow. (A) Acetate (40 μmol/ml), (B) S. aureus SA113, (C) oatA deletion mutant, (D) S. epidermidis, (E) S. lugdunensis, (F) S. hyicus, (G) S. carnosus, (H) S. equorum, and (I) S. arlettae.