Staphylococcus aureus GpsB, Stk, and VraSR form a cell wall surveillance network to maintain cell envelope integrity...

Antibiotic resistance is an alarming threat to public health worldwide. Specifically, drug-resistant Staphylococcus aureus remains a major contributor to global mortality. Therefore, in-depth understanding of antibiotic resistance mechanisms is urgently needed to inform alternative therapeutic strategies. We previously showed that GpsB is a critical regulator of cell wall synthesis in S. aureus . GpsB executes its regulatory role via protein-protein interactions. Yet, how GpsB function is modulated in this organism has remained unclear. Intriguingly, a prior proteomics report indicated that GpsB is phosphorylated at six different residues by the sole serine/threonine kinase Stk. Hence, we explored the physiological significance of GpsB phosphorylation. We identify three phosphosites that are each independently sufficient to drive GpsB phosphoregulation, which likely alters its partner preference. We find that cells lacking gpsB exhibit heightened fosfomycin sensitivity, and that this is exacerbated by stk deletion. Our findings reveal that GpsB collaborates with Stk and the VraSR cell wall stress response system to coordinate the demand-driven availability of cell wall precursors. Collectively, this report establishes GpsB as a pivotal peptidoglycan supply chain sentinel that together with Stk and VraSR forms an integrated cell wall stress monitoring network that maintains cell envelope homeostasis and aids in antibiotic resistance.

The combination of multidrug resistance and an extensive arsenal of virulence factors makes Staphylococcus aureus a formidable pathogen. Therefore, to help devise novel therapeutic strategies a deeper understanding of the molecular mechanisms underlying antibiotic resistance is critical. In this report, we reveal that GpsB plays an important role in cell wall stress response. We find that the Stk-dependent serine/threonine phosphorylation of GpsB influences its interaction partner preference. Additionally, our results show that loss of gpsB sensitizes cells to fosfomycin-induced cell wall stress, and that this effect is further exacerbated by stk deletion. Furthermore, we uncover previously unrecognized GpsB interaction partners involved in cell wall precursor biosynthesis. These findings elucidate the molecular basis underlying the role of GpsB in ensuring cell envelope integrity. Taken together, our study establishes GpsB as a critical mediator of cell wall stress response and antibiotic resistance.