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

• 批准号: 10218523
• 负责人: May H. Abdelaziz
• 金额: $ 18.38万
• 依托单位: UNIVERSITY OF TEXAS TYLER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-02-22 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10218523
• 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; 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; design; dimer; drug discovery; inhibitor/antagonist; innovation; insight; interest; mutant; protein protein interaction; protein-histidine kinase; reaction rate; resistance mechanism; response; tumor

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.

摘要 抗生素耐药性是“我们这个时代最大的公共卫生挑战之一” 疾病控制和预防中心。VraS是一种细菌组氨酸激酶，是VraTSR的一部分，是一种 三元监管体系，起着承上启下的作用 穿过细菌细胞壁的环境压力信号。VraS被证明是一种关键的细菌 一种防御系统，能中和细胞壁抑制剂抗生素如甲氧西林、苯唑西林、 万古霉素，以及最近的达托霉素和替考拉宁等药物。抑制VraS挫败耐药性 在金黄色葡萄球菌中，通过提高现有抗生素的有效性。几个VraS突变体 已经在抗药性金黄色葡萄球菌菌株中分离出来，但还不清楚是如何 这些突变与VraS的激活和抗性的发展有关。 这项建议的总体目标是确定七种临床相关的Vras的效果 调节其功能的关键方面的突变，包括催化图谱、稳定性、二聚化和 它与反应调节因子Vrar的结合。中心假设是突变会导致 构成上活跃的Vras形式。这一目标将通过实现两个具体目标来实现。 第一个目的是鉴定VraS突变体的催化特性和稳定性。一个突变体T331I具有一个 自动磷酸化速率大约是野生型VraS的12倍，证明了它的 增强的催化特性。在拟议的项目中，将表达其他六种类型的VraS突变体 它们的催化参数(自动磷酸化速率、底物亲和力和催化效率)将 用偶联激酶法进行测定。将使用差示扫描荧光法来评估 变种人的稳定性。工作假设是，某些突变，如T331I，会通过 调制这些参数中的一个或多个。第二个目标是评估突变对人类健康的影响 VraS蛋白质-蛋白质相互作用。有效的假设是，某些突变会改变这些 交互，增强VraS功能。VraS及其突变体的二聚化亲和力将是 用竞争性荧光共振能量转移结合分析法测定。装订 VraS或其突变体与Vrar之间的亲和力将通过表面等离子体共振进行评估。 这项拟议的研究具有创新性，因为对VraS相互作用和催化的研究还不够深入。这个 这项研究的重点是七个临床相关的突变，这些突变以前从未被调查过。 拟议研究的预期结果是更好地了解VraS和确定 导致金黄色葡萄球菌激活VraS并中和当前使用的抗生素的关键突变。这将是 促进未来的结构研究和微生物检测，以检测突变对耐药性的影响 和抑制的效果。