The degradation of PBP2a promoted by 8-octyl berberine through the AAA+ protease system inhibits peptidoglycan biosynthesis of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is resistant to various antibiotics, and identifying new antibiotics is urgently needed. Based on the structural modification of berberine (BBR), we discovered that 8-octyl berberine (OBBR) had stronger antibacterial activity, especially against MRSA, but its mechanism on MRSA has not been determined. This study revealed that, compared with vancomycin and BBR, OBBR exhibited superior in vitro and in vivo bactericidal activity against MRSA. More importantly, treatment with OBBR didn't develop resistance within 20 round of passages. Mechanistically, via membrane integrity assays and TEM, we observed that OBBR causes significant destruction of the MRSA cell wall and membrane. Transcriptomic and metabolomic analyses revealed that OBBR influenced the peptidoglycan biosynthesis pathway, and the expression of penicillin-binding protein 2a (PBP2a), a peptidoglycan synthesizing transpeptidase, was downregulated in MRSA after OBBR treatment. Further experiments (BOMD, CETSA, DARTs, SPR, GST pull-down and Co-IP) revealed that OBBR can result in an alteration of the spatial conformation of PBP2a and a strong binding interaction between them, and facilitate PBP2a degradation by interacting with ClpC of the AAA+ protease system. Overall, the potential mechanism of the anti-MRSA effect of OBBR is to promote PBP2a protein degradation via the AAA+ protease system rather than inhibiting the transpeptide activity of the PBP2a protein. Therefore, we propose a different bactericidal mechanism from that of β-lactam antibiotics and propose a novel drug candidate against MRSA in clinical settings.