Treatment of serious Staphylococcus aureus infection is complicated by the development of intermediate level resistance to vancomycin (termed VISA) during prolonged bacteraemia. The genetic mechanisms behind the emergence of these strains is highly heterogeneous and typically involves two-component regulatory systems (TCRS) that modulate cell wall metabolism including WalKR, which has a critical role in coordinating autolysis. In Bacillus spp., WalKR is negatively regulated by physical interaction with auxiliary proteins YycH and YycI, however the role of these proteins in S. aureus is yet to be described.
We recently performed whole genome sequencing and identified a yycI mutation that was predicted to lead to YycI truncation in a clinical VISA strain. Using allelic exchange, we created markerless yycH, yycI and yycHI deletion strains, each of which showed reduced susceptibility to vancomycin. Importantly, vancomycin-susceptible phenotypes were returned upon gene complementation. Using RNA-Seq, we revealed downregulation of the WalKR regulon including autolysin genes atlA and sle1. These data were corroborated by a reduced autolytic rate for each mutant, which in contrast to Bacillus spp., suggested a role for YycHI in activating WalKR. Using a bacterial two-hybrid system, we showed that YycH and YycI interacted, and that YycHI formed a ternary protein complex with the sensor histidine kinase WalK, which appears to be important for regulation of the TCRS. Mutation to YycH or YycI associated with clinical VISA strains had a deleterious impact upon YycHI/WalK complex formation, which likely impacts on WalKR activation.
Taken together, we have described a novel antibiotic resistance strategy in S. aureus, whereby yycHI mutations are selected for in vivo, leading to reduced WalKR activation, impaired cell wall turnover and ultimately reduced vancomycin efficacy.