Mechanistic Insights into VraS Mutations Linked to Bacterial Resistance

与细菌耐药性相关的 VraS 突变的机制见解

基本信息

批准号：
10356943
负责人：
May H. Abdelaziz
金额：
$ 19.7万
依托单位：
UNIVERSITY OF TEXAS TYLER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-02-22 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10356943
关键词：
Address Affinity Antibiotic Resistance Antibiotics Bacterial Antibiotic Resistance Binding Binding Proteins Biochemical Biological Assay Biophysics Catalysis Cell Wall Centers for Disease Control and Prevention (U.S.)Clinical Combating Antibiotic Resistant Bacteria Coupled Daptomycin Data Dimerization Effectiveness Enzymes Financial Hardship Fluorescence Fluorescence Resonance Energy Transfer Future Goals Human Infection Knowledge Link Malignant Neoplasms Measures Methicillin Microbiology Mission Molecular Mutation Mutation Analysis Outcome Oxacillin Patients Phosphotransferases Plant Roots Play Positioning Attribute Public Health Publishing Regulation Relapse Research Resistance Resistance development Role Sampling Scanning Signal Transduction Staphylococcus aureus Stress Structure-Activity Relationship Surface Surface Plasmon Resonance System Teicoplanin Time United States National Institutes of Health Vancomycin Work bacterial resistance biophysical techniques clinical effect clinically relevant dimer drug discovery inhibitor innovation insight interest mutant protein protein interaction protein-histidine kinase rational design reaction rate resistance mechanism response tumor

项目摘要

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.