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

Abstract

Bacterial genomic mutations in Staphylococcus aureus 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 S. aureus. 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 S. aureus. 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 S. aureus 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 vraS 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 Staphylococcus aureus, yet the exact mechanism through which these mutations work is understudied. We investigated the T331I mutation in the vraS 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 S. aureus 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.

Keywords: Staphylococcus aureus; VraS; antibiotic resistance; kinase; mutation.

Fig 1

Autophosphorylation of VraS. (A) The autophosphorylation reaction rate of VraS WT and T331I mutant (5 µM) as a function of the rate of NADH disappearance, compared to a blank reaction at 2 mM ATP. The reaction reaches a plateau with T331I as the NADH in the assay mixture is depleted. (B) Surface representation of the crystallized VraS catalytic domain (PDB 4GT8). The inset shows T331 (red sticks) proximity to the bound ATP (in colored sticks).

Fig 2

Relationship between the drug concentrations and the bacterial burden. Dose-response curves of the tested antibiotics in the Newman WT and T331I mutant strain. Results obtained are in Table 1, and the parameters for the model are in Table S1.

Fig 3

Growth curves of the T331I mutant (red lines) and WT (New) strains (black lines) in the presence of different antibiotic concentrations. PC indicates positive control growth without antibiotics. The data represent the average ± SD of at least three biological replicas.

Fig 4

Fold change in the expression levels of vraR, pbp2, and blaZ after mutation and/or antibiotic exposure (black: no antibiotic control, magenta: vancomycin, green: methicillin, and purple: daptomycin). The data represent the mean ± SE (n  =  3), and statistical significance between control versus treated samples for each strain and between WT and T331I under the same treatment was calculated via one-way ANOVA with multiple comparisons.

Fig 5

Volcano plots of differential gene expression between T331I and WT strains with and without vancomycin stress. (A) T331I compared to WT, (B) WT with vancomycin compared to WT, (C) T331I with vancomycin compared to T331I, and (D) T331I with vancomycin compared to WT with vancomycin. Colored dots (red, upregulated; green, downregulated; and blue, not significantly changed) based on P-value (<0.05) and log2(fold change) and logFC cutoff of 1.3. The vertical lines represent the twofold change in gene expression selected to generate the data in Table 2.

Fig 6

The predicted change in the protein-protein interaction network in S. aureus after mutation and/or vancomycin stress as visualized using Cytoscape 3.10.1. The inset shows a zoomed view of the interactions of the VraSR system, and the red lines highlight VraS PPIs. (A) PPIs for WT strain after exposure to vancomycin, (B) PPIs for T331I compared to WT, and (C) PPIs of T331I after vancomycin compared to T331I.