A Nonclassical Mechanism of β-Lactam Resistance in Methicillin-Resistant Staphylococcus aureus and Its Effect on Virulence.

Methicillin-resistant Staphylococcus aureus (MRSA) is a group of pathogenic bacteria that are infamously resistant to β-lactam antibiotics, a property attributed to the gene. Recent studies have reported that mutations associated with the promoter region of demonstrated high levels of β-lactam resistance, suggesting the role of PBP4 as an important non- mediator of β-lactam resistance. The -promoter-associated mutations have been detected in strains with or without . Our previous studies that were carried out in strains devoid of described that -promoter-associated mutations lead to PBP4 overexpression and β-lactam resistance. In this study, by introducing various -promoter-associated mutations in the genome of a MRSA strain, we demonstrate that PBP4 overexpression can supplement -associated resistance in S. aureus and can lead to increased β-lactam resistance. The promoter and regulatory region of is shared with a divergently transcribed gene, , which encodes a multidrug exporter. We demonstrate that the promoter mutations caused an upregulation of and downregulation of , confirming that the resistant phenotype is associated with PBP4 overexpression. PBP4 has also been associated with staphylococcal pathogenesis, however, its exact role remains unclear. Using a Caenorhabditis elegans model, we demonstrate that strains having increased PBP4 expression are less virulent than wild-type strains, suggesting that β-lactam resistance mediated via PBP4 likely comes at the cost of virulence. Our study demonstrates the ability of PBP4 to be an important mediator of β-lactam resistance in not only methicillin-susceptible Staphylococcus aureus (MSSA) background strains as previously demonstrated but also in MRSA strains. When present together, PBP2a and PBP4 overexpression can produce increased levels of β-lactam resistance, causing complications in treatment. Thus, this study suggests the importance of monitoring PBP4-associated resistance in clinical settings, as well as understanding the mechanistic basis of associated resistance, so that treatments targeting PBP4 may be developed. This study also demonstrates that S. aureus strains with increased PBP4 expression are less pathogenic, providing important hints about the role of PBP4 in S. aureus resistance and pathogenesis.