Staphylococcus aureus sensor histidine kinase VraS mutations linked to antibiotic resistance affect enzymatic...

Staphylococcus aureus (S. aureus) is a highly pathogenic bacterium that utilizes the VraSR two-component system to regulate cell wall synthesis and facilitate resistance against antibiotics. Resistant S. aureus isolates were shown to harbor several single-nucleotide polymorphisms in VraS; however, their effect on VraS functionality is not clear. We have investigated the effect of seven mutations in the VraS intracellular domain reported in clinically resistant strains on autophosphorylation rate, stability, and VraS-VraR equilibrium binding affinity (K_D). The expression of wild-type VraS and mutants was optimized, and the proteins were purified using affinity chromatography. A coupled kinase assay was used to assess the autophosphorylation kinetic constants. The stability of the purified proteins was assessed using differential scanning fluorimetry, and surface plasmon resonance was used to measure the K_D of the constructs to VraR. The results show that several mutations enhanced the catalytic efficiency of VraS and led to an increase in protein stability. All the mutants retained the same affinity to VraR as the wild type, except D242G, which showed a 17-fold decrease in affinity. Molecular dynamics simulation of a generated dimeric VraS homology model shows that the M192I mutant may have an increased possibility of forming the Michaelis complex. This study investigated the effect of VraS mutations on the enzymatic activity, stability, and affinity to its cognate response regulator, which can translate to a modified bacterial response to stress. The results highlight the importance of studying bacterial kinase mutations as an underlying mechanism of antibiotic resistance in S. aureus.