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 i s 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 i s 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.

Public Health Relevance

Infections caused by antibiotic resistant Staphylococcus aureus continue to be a significant public health threat with a high financial burden. This project will determine the effects of clinically relevant mutations of the histidine kinase VraS on key aspects that regulate its function. The expected findings will meet NIH's mission to reduce illness and research objectives of the National Action Plan for Combating Antibiotic-Resistant Bacteria by uncovering how this bacterial system is activated by mutations linked to antibiotic resistance.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Exploratory/Developmental Grants (R21)
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Macromolecular Structure and Function A Study Section (MSFA)
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Huntley, Clayton C
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University of Texas Tyler
Schools of Pharmacy
United States
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