Mechanistic Insights into VraS Mutations Linked to Bacterial Resistance

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， 三个组成部分的调节系统，在中继和响应 环境压力信号穿过细菌细胞壁。VraS被证明是一种关键的细菌 防御系统，中和细胞壁抑制剂抗生素的作用，如甲氧西林，苯唑西林， 万古霉素和最近的药物如达托霉素和替考拉宁。抑制VraS阻碍耐药性 在金黄色葡萄球菌通过提高现有的抗生素的有效性。几种VraS突变体 在抗生素耐药的S.金黄色葡萄球菌菌株，但目前还没有明确的了解如何 这些突变体与VraS活化相关，进而与抗性的发展相关。 本提案的总体目标是确定7种临床相关VraS的影响 调节其功能的关键方面的突变，包括催化特征，稳定性，二聚化， 其与响应调节器VraR的结合。核心假设是突变会导致 VraS的组成型活性形式。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突变体的催化特征和稳定性。一种突变体T331 I具有 自磷酸化速率约为野生型VraS的12倍，表明其 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（自磷酸化速率、底物亲和力和催化效率）将 可以使用偶联激酶测定来测量。差示扫描荧光法将用于评估 变种人的稳定性工作假设是，一些突变如T331 I将通过以下途径激活VraS： 调节这些参数中的一个或多个。第二个目的是评估突变对 VraS蛋白-蛋白相互作用。工作假设是，一些突变会改变这些 交互，增强VraS功能。VraS及其突变体的二聚化亲和力将是 使用竞争性荧光共振能量转移结合测定来测量。的结合 VraS或其突变体与VraR之间的亲和力将使用表面等离子体共振进行评价。 拟议的研究是创新的，因为VraS相互作用和催化是欠研究。的 将成为本研究重点的七种临床相关突变以前未被研究。 拟议研究的预期成果是更好地了解VraS和识别 导致S.金黄色葡萄球菌激活VraS并中和目前使用的抗生素。这将 促进未来的结构研究和微生物测定，以检测突变对耐药性的影响 和抑制功效。

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.