Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation β-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum β-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC ) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA -deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus . The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum β-lactam resistance in S. aureus . It thus suggests reconsideration of the current approach toward diagnosis and treatment of β-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus , high-level, broad-spectrum resistance to β-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA . This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to β-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA , as seen by increased bacterial survival in the presence of β-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and G GDEF d omain p rotein containing p hosphodiesterase ( gdpP ) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of β-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

Staphylococcus aureus remains to be one of the leading causes of global mortality. The most common class of antibiotics used to treat S. aureus infections are next-generation β-lactams (NGBs), as they are highly efficacious and have low adverse effects. NGB resistance in S. aureus is classically attributed to penicillin-binding protein-2a (PBP2a), but previous studies from our group have also implicated an altered expression of penicillin-binding protein-4 (PBP4) with NGB resistance. PBP4 is the sole low-molecular-mass (LMM) PBP present in S. aureus; it is also the only known LMM PBP with transpeptidase activity, giving it the unique ability to bring about peptidoglycan cross-linking. In this article, we review some of the recent findings from our group, which reveal that mutations associated with PBP4 lead to altered protein expression and NGB resistance in both methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) backgrounds. We discuss the clinical relevance of PBP4-associated mutations, particularly in methicillin-resistant lacking mec (MRLM) isolates, as well as the synergistic effect of altered PBP4 and GdpP functions. Finally, this review summarizes the potential role played by PBP4 in S. aureus virulence. Together, we highlight the increasing relevance of PBP4 as a mediator of NGB resistance and discuss its potential as an important factor during infection diagnosis and therapy.

Sensory induction of mecA expression plays a pivotal role in mediating broad-spectrum β-lactam resistance of MRSA. In contemporary MRSA isolates, sensory induction of β-lactam resistance originates at the membrane-associated BlaR1, which, upon detection of β-lactam drugs, triggers a signal transduction cascade that promotes mecA induction. We hereby show that phosphorylation of BlaR1, mediated through the serine-threonine kinase, Stk1, stabilizes its membrane-spanning state and retention, allowing for proper drug sensing and subsequent signal transduction events to occur, culminating in mecA-mediated β-lactam resistance. Our results demonstrate that targeting Stk1 can potentiate synthetic lethality to β-lactams in the majority of naturally isolated strains of MRSA. We additionally present the structural and kinetic basis for a Stk1-inhibitor complex that can enable rational design of Stk1-directed anti-MRSA therapeutics in the future. Our results reveal a novel role of the STK signaling pathway in bacterial protein stabilization in the cytosolic membrane.

Staphylococcus aureus remains to be one of the leading causes of global mortality. The most common class of antibiotics used to treat S. aureus infections are Next-Generation β-lactams (NGBs), as they are highly efficacious and have low adverse effects. NGB resistance in S. aureus is classically attributed to Penicillin-Binding Protein-2a (PBP2a), but previous studies from our group have also implicated altered expression of Penicillin-Binding Protein-4 (PBP4) with high-level NGB resistance. PBP4 is the only low-molecular mass (LMM) PBP present in S. aureus; it is also the only known LMM PBP with transpeptidase activity, giving it the unique ability to bring about peptidoglycan cross-linking. In this article, we review some of the recent findings from our group, which reveal that mutations associated with PBP4 lead to altered protein expression and NGB resistance in both MSSA and MRSA backgrounds. We discuss the clinical relevance of PBP4-associated mutations, particularly in Methicillin Resistant Lacking mec (MRLM) isolates, as well as the combined effect of altered expression of PBP4 and GdpP. Finally, this review summarizes the potential role played by PBP4 in S. aureus virulence. Together, we highlight the increasing relevance of PBP4 as a mediator of NGB resistance and discuss its potential to be an important factor during infection diagnosis and therapy.