Lai, L.-Y. | bioRxiv Science

bioRxiv2023

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum β-lactam resistance in Staphylococcus aureus

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 {beta}-lactams (NGB) such as Methicillin, Nafcillin, Oxacillin etc. Resistance to NGBs, which is alternatively known as broad-spectrum {beta}- 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 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 towards NGBs at levels comparable to that of MRSAs. Our study, provides a fresh new perspective about alternative mechanisms of NGBs resistance, challenging our current overall understanding of high-level, broad-spectrum {beta}-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach towards diagnosis and treatment of {beta}-lactam resistant S. aureus infections.

microbiology↗

bioRxiv2022

A non-classical mechanism of β-lactam resistance in Methicillin-Resistant Staphylococcus aureus (MRSA) and its effect on virulence

Methicillin-Resistant Staphylococcus aureus (MRSA) are pathogenic bacteria that are infamously resistant to {beta}-lactam antibiotics, a property attributed to the mecA gene. Recent studies have reported that mutations associated with the promoter region of pbp4 demonstrated high levels of {beta}-lactam resistance, suggesting the role of PBP4 as an important non-mecA mediator of {beta}-lactam resistance. The pbp4 promoter-associated mutations have been detected in strains with or without mecA. Our previous studies that were carried out in strains devoid of mecA described that pbp4 promoter-associated mutations lead to PBP4 overexpression and {beta}-lactam resistance. In this study, by introducing various pbp4 promoter-associated mutations in the genome of an MRSA strain, we demonstrate that PBP4 overexpression can supplement mecA-associated resistance in S. aureus and can lead to increased {beta}-lactam resistance. The promoter and regulatory region of pbp4 is shared with a divergently transcribed gene, abcA, which encodes for a multidrug exporter. We demonstrate that the promoter mutations caused an upregulation of pbp4 and downregulation of abcA, confirming that the resistant phenotype is associated with PBP4 overexpression only. PBP4 has also been associated with staphylococcal pathogenesis, however, its exact role remains unclear. Using a C. elegans model, we demonstrate that strains having increased PBP4 expression are less virulent compared to wild-type strains, suggesting that {beta}-lactam resistance mediated via PBP4 likely comes at the cost of virulence. ImportanceOur study demonstrates the ability of PBP4 to be an important mediator of {beta}-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 {beta}-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.

microbiology↗

bioRxiv2022

In-vivo detection of cyclic-di-AMP in Staphylococcus aureus

Cyclic-di-AMP (CDA) is a signaling molecule that controls various cellular functions including antibiotic tolerance and osmoregulation in Staphylococcus aureus. In this study, we developed a novel biosensor (bsuO P6-4) for in-vivo detection of CDA in S. aureus. Our study showed that bsuO P6-4 could detect a wide concentration range of CDA in both laboratory and clinical strains making it suitable for use in both basic and clinical research applications.

microbiology↗