# Mechanistic Insights into VraS Mutations Linked to Bacterial Resistance

> **NIH NIH R21** · UNIVERSITY OF TEXAS TYLER · 2021 · $183,750

## Abstract

ABSTRACT
Antibiotic resistance is “one of the biggest public health challenges of our time” according to the
Centers for Disease Control and Prevention. VraS is a bacterial histidine kinase that is part of VraTSR, a
three-component regulatory system which plays a pivotal role in relaying and responding to
environmental stress signals across the bacterial cell wall. VraS was proven to be a key bacterial
defense system that neutralizes the effect of cell wall inhibitor antibiotics like methicillin, oxacillin,
vancomycin, and more recent agents like daptomycin and teicoplanin. Inhibiting VraS thwarts resistance
in Staphylococcus aureus by enhancing the effectiveness of current antibiotics. Several VraS mutants
have been isolated in antibiotic resistant S. aureus strains, but there is no clear understanding of how
these mutants are linked to VraS activation and in turn, the development of resistance.
 The overall objective of this proposal is to determine the effects of seven VraS clinically relevant
mutations on key aspects that regulate its function including catalytic profile, stability, dimerization, and
its binding to the response regulator VraR. The central hypothesis is that mutations will result in
constitutively active forms of VraS. This objective will be accomplished by achieving two specific aims.
The first aim is to identify the catalytic profile and stability of VraS mutants. One mutant, T331I, has an
autophosphorylation rate that is approximate 12 times that of the wild type VraS demonstrating its
enhanced catalytic profile. In the proposed project, six other types of VraS mutants will be expressed
and their catalytic parameters (autophosphorylation rates, substrate affinity and catalytic efficiency) will
be measured using a coupled kinase assay. Differential scanning fluorimetry will be used to assess the
mutants’ stability. The working hypothesis is that some mutations like T331I will activate VraS through
modulating one or more of these parameters. The second aim is to evaluate the effect of mutations on
VraS protein–protein interactions. The working hypothesis is that some mutations will alter these
interactions, enhancing VraS functionality. The dimerization affinity of VraS and its mutants will be
measured using competitive Fluorescence Resonance Energy Transfer binding assays. The binding
affinity between VraS or its mutants and VraR will be evaluated using surface plasmon resonance.
 The proposed study is innovative because VraS interactions and catalysis are understudied. The
seven clinically relevant mutations that will be the focus of this study have not been investigated before.
The expected outcome of the proposed research is a better understanding of VraS and identification of
key mutations that cause S. aureus to activate VraS and neutralize currently used antibiotics. This will
facilitate future structural studies and microbiological assays to detect the mutations effects on resistance
and efficacy of inhibition.

## Key facts

- **NIH application ID:** 10218523
- **Project number:** 1R21AI154189-01A1
- **Recipient organization:** UNIVERSITY OF TEXAS TYLER
- **Principal Investigator:** May H. Abdelaziz
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $183,750
- **Award type:** 1
- **Project period:** 2021-02-22 → 2023-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10218523

## Citation

> US National Institutes of Health, RePORTER application 10218523, Mechanistic Insights into VraS Mutations Linked to Bacterial Resistance (1R21AI154189-01A1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10218523. Licensed CC0.

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