PBP4 is required for serum-induced cell wall thickening and antibiotic tolerance in Staphylococcus aureus

The bacterial pathogen Staphylococcus aureus responds to the host environment by synthesising a thick peptidoglycan cell wall which protects the bacterium from membrane-targeting antimicrobials and the immune response. However, the proteins required for this response were previously unknown. Here, we demonstrate by three independent approaches that the penicillin binding protein PBP4 is crucial for serum-induced cell wall thickening. Firstly, mutants lacking various non-essential cell wall synthesis enzymes were tested, revealing that a mutant lacking pbp4 was unable to generate a thick cell wall in serum. This resulted in reduced serum-induced tolerance of the pbp4 mutant towards the last resort antibiotic daptomycin relative to wildtype cells. Secondly, we found that serum-induced cell wall thickening occurred in each of a panel of 134 clinical bacteraemia isolates, except for one strain with a naturally-occurring mutation that confers a S140R substitution in the active site of PBP4. Finally, inhibition of PBP4 with cefoxitin prevented serum-induced cell wall thickening and the resulting antibiotic tolerance in the USA300 strain and in clinical MRSA isolates. Together, this provides a rationale for combining daptomycin with cefoxitin, a PBP4 inhibitor, to potentially improve treatment outcomes for patients with invasive MRSA infections.