Antibiotic-resistant Staphylococcus aureus is a major concern to public health. Methicillin-resistant S. aureus strains are completely resistant to all ␤-lactams antibiotics. One of the main factors involved in methicillin resistance in S. aureus is the penicillin-binding protein, PBP2a. This protein is insensitive to inactivation by ␤-lactam antibiotics such as methicillin. Although other proteins are implicated in high and homogeneous levels of methicillin resistance, the functions of these other proteins remain elusive. Herein, we report for the first time on the putative function of one of these proteins, FmtA. This protein specifically interacts with ␤-lactam antibiotics forming covalently bound complexes. The serine residue present in the sequence motif Ser-X-X-Lys (which is conserved among penicillin-binding proteins and ␤-lactamases) is the active-site nucleophile during the formation of acyl-enzyme species. FmtA has a low binding affinity for ␤-lactams, and it experiences a slow acylation rate, suggesting that this protein is intrinsically resistant to ␤-lactam inactivation. We found that FmtA undergoes conformational changes in presence of ␤-lactams that may be essential to the ␤-lactam resistance mechanism. FmtA binds to peptidoglycan in vitro. Our findings suggest that FmtA is a penicillin-binding protein, and as such, it may compensate for suppressed peptidoglycan biosynthesis under ␤-lactam induced cell wall stress conditions.

In this this study, a cloning plasmid named pMD19-FnBPB-ClfA was constructed, by PCR specific amplifications of genes from region D, fibronectin-binding protein B (FnBPB) and region A in clumping factor A (ClfA), all from Staphylococcus aureus. Splicing by overlap extension using PCR tandem gene FnBPB-ClfA was performed and then gene segments of pMD19-FnBPB-ClfA were inserted into a prokaryotic expression vector named pET-32a (+). They were ultimately transferred into the host strain BL21 (DE3), resulting in the expression plasmid pET-FnBPB-ClfA. SDS-PAGE demonstrated an extrinsic protein belt consistent with the desired protein at 51 kDa, when the expression constructed was induced with 1 mmol/L isopropyl β-D-1-Thiogalactopyranoside (IPTG). Western-blot identification demonstrated consensus between the expressed protein and the endogenous protein. After purification and emulsification with Freund's adjuvant, the expressed protein was used to immunize mice. After three subsequent immunizations in the same mice, we gained a highly effective antiserum. Through ELISA, tube agglutination, phagocytosis of opsonized experiment, and antibodies against S. aureus adhesion ability test, it was demonstrated that the fusion gene was successfully expressed in prokaryotic cells, the expressed protein was adherence active, the prepared immune antiserum was capable of preventing S. aureus from adhering to bovine fibrinogens and the antiserum had functions of phagocytosis and opsonization.