A mechanistic understanding of the effect of <i>Staphylococcus aureus</i> VraS histidine kinase single-point mutation on antibiotic resistance

Bacterial genomic mutations in <i>Staphylococcus aureus</i> have been detected in isolated resistant clinical strains, yet their mechanistic effect on the development of antimicrobial resistance remains unclear. Resistance-associated regulatory systems acquire adaptive mutations under stress conditions that may lead to a gain-of-function effect and contribute to the resistance phenotype. Here, we investigate the effect of a single-point mutation (T331I) in VraS histidine kinase, part of the VraSR two-component system in <i>S. aureus</i>. VraSR senses and responds to environmental stress signals by upregulating gene expression for cell wall synthesis. A combination of enzyme kinetics, microbiological, and transcriptomic analyses revealed the mechanistic effect of the mutation on VraS and <i>S. aureus</i>. Michaelis-Menten kinetics show that the VraS mutation caused an increase in the autophosphorylation rate of VraS and enhanced its catalytic efficiency. The introduction of the mutation through recombineering coupled with CRISPR-Cas9 counterselection to the Newman strain wild-type (WT) genome doubled the minimum inhibitory concentration of three cell wall-targeting antibiotics. The mutation caused an enhanced <i>S. aureus</i> growth rate at sub-lethal doses of the antibiotics, confirming the causative effect of the mutation on bacterial persistence. Transcriptomic analysis showed a genome-wide alteration in gene expression levels and protein-protein interaction network of the mutant compared to the WT strain after exposure to vancomycin. The results suggest that the <i>vraS</i> mutation causes several mechanistic changes at the protein and cellular levels that favor bacterial survival under antibiotic stress and cause the mutation-harboring strains to become the dominant population during infection.IMPORTANCERising antimicrobial resistance (AMR) is a global health problem. Mutations in the two-component system have been linked to drug resistance in <i>Staphylococcus aureus</i>, yet the exact mechanism through which these mutations work is understudied. We investigated the T331I mutation in the <i>vraS</i> gene linked to sensing and responding to cell wall stress. The mutation caused changes at the protein level by increasing the catalytic efficiency of VraS kinase activity. Introducing the mutation to the genome of an <i>S. aureus</i> strain resulted in changes in phenotypic antibiotic susceptibility, growth kinetics, and genome-wide transcriptomic alterations. By a combination of enzyme kinetics, microbiological, and transcriptomic approaches, we highlight how small genetic changes can significantly impact bacterial physiology and survival under antibiotic stress. Understanding the mechanistic basis of antibiotic resistance is crucial to guide the development of novel therapeutic agents to combat AMR.