Probing The Activity Of Fmta, A Novel Esterase In Staphylococcus Aureus, In Vitro And In Vivo Through Mutagenesis.

The versatile bacterium Staphylococcus aureus (S. aureus) resides within the human microbiota, manifesting commensal and pathogenic characteristics. The emergence of methicillin-resistant S. aureus (MRSA) underscores the global challenge of antibiotic resistance. FmtA, a protein exhibiting structural similarities and distinct features relative to Penicillin-binding proteins (PBPs) within the Penicillin penicillin-recognizing proteins (PRPs) umbrella, significantly contributes to the S. aureus antibiotic resistance mechanism. FmtA acts on Wall Teichoic Acid (WTA), an abundant poly-ribitol polymer in S. aureus peptidoglycan, where FmtA removes D-Alanine (D-Ala) from WTA, which is esterified post-synthesis where the D-alanylation level in WTA is an important contributor in S. aureus antibiotic resistance and physiology. Previous research addressed the structural intricacies of FmtA and based on the positioning of key amino acid residues, it was proposed that the enzyme's active site has evolved to specifically bind WTA. It was further hypothesized that FmtA has adapted to carry out D-amino esterase activity against WTA, highlighting its specialized functional evolution. In this study, I investigate the role of specific amino acid residues present in the active site of FmtA, focusing on their interactions with WTA using both in vitro and in vivo approaches. Given the overall negative charge of WTA, derived from its phosphodiester backbone, it is a target for cationic polymers like Branched Polyethyleneimine (BPEI). Previous research has shown that BPEI binding to WTA creates a steric hindrance, disrupting the function of penicillin-binding proteins (PBPs) such as PBP4 in Staphylococcus aureus and PBP2a in Staphylococcus epidermidis. This disruption impacts key bacterial physiological processes, including cell division, virulence, and peptidoglycan biosynthesis. My findings reveal that BPEI can compete with FmtA for WTA binding, though less effectively, leading to partial inhibition of FmtA's enzymatic activity. Additionally, BPEI binding affects the physiological activities of S. aureus, including biofilm formation, which is typically mediated by FmtA. These results suggest that while BPEI hinders FmtA's interaction with WTA and its subsequent enzymatic function, it does not completely block FmtA activity.