New genes involved in cellular responses to quinolone treatment

描述(申请人提供)：该计划的长期目标是足够详细地了解细菌的应激反应，最终设计应激反应的小分子抑制剂作为抗菌增强剂。目前的R21应用描述了一个基础科学的、原理证明的项目，以研究一种新的蛋白激酶，当被灭活时，用各种抗菌剂、过氧化氢和高温处理的大肠杆菌细胞的存活率降低。该激酶的缺失使喹诺酮类药物的细菌存活率降低了10-100倍，并导致抑菌化合物氯霉素变得杀菌。它还极大地降低了萘啶酸诱导新的耐药突变的能力。一对毒素-抗毒素基因的缺失被认为有助于保护人们免受压力和促进细菌凋亡，从而抑制了这种蛋白激酶的缺失。这种与毒素-抗毒素系统的遗传相互作用导致了一种假说，即激酶通常会限制毒素的活性；在没有激酶的情况下，毒素会在应激条件下杀死细胞，从而同时增强许多抗菌剂的作用。该激酶基因还通过在其启动子区域上游有两个CpxR结合位点而参与CPX包膜蛋白应激反应途径，从而建立了该激酶与应激反应的另一联系。这种应激反应激酶的遗传学和生物化学都将被研究。将通过CPX和其他相关的双组分调控系统的突变来研究激酶基因的上游调控，并通过与毒素-抗毒素系统的遗传相互作用来研究下游的影响。为了获得该激酶在应激反应网络中所起作用的框架，还将在存在或不存在该激酶活性的情况下，在各种胁迫下进行基因表达谱分析。该激酶已被提纯。作为其进一步表征的一部分，将优化酶反应条件，确定自动磷酸化位点(S)，并鉴定其正常磷酸化的蛋白质。这项拟议的工作构成了参与细菌应激反应、持久性/耐受性和细胞凋亡的调控网络的早期特征。这样的研究可能最终会导致通过干扰细菌应激反应来使许多抗菌剂更有效的方法。 细菌对抗菌素治疗的耐药性、耐受性和持久性对我们治愈感染的能力构成越来越大的威胁。参与细菌应激反应的保护性基因帮助细菌逃避抗菌素治疗并存活下来。这些保护性的应激反应网络将被研究，长期目标是开发用于抗菌增强的小分子抑制剂。

DESCRIPTION (provided by applicant): The long-term goal of this program is to understand bacterial stress responses in enough detail to eventually design small-molecule inhibitors of stress response as antimicrobial potentiators. The present R21 application describes a basic-science, proof-of-principle project to study a new protein kinase that when inactivated decreases survival of E. coli cells treated with a variety of antimicrobials, hydrogen peroxide, and high temperature. A deficiency of the kinase reduces bacterial survival to quinolone treatment by 10- to 100-fold and causes the bacteriostatic compound chloramphenicol to become bactericidal. It also dramatically lowers the ability of nalidixic acid to induce new resistant mutants. The kinase deficiency is suppressed by deletion of a toxin-antitoxin gene pair thought to contribute both to protection from stress and to bacterial apoptosis. This genetic interaction with toxin-antitoxin systems leads to the hypothesis that the kinase normally limits toxin activity; in the absence of the kinase, toxins kill cells during stressful conditions, thereby enhancing the action of many antimicrobials at the same time. The kinase gene is also implicated in the Cpx envelope protein stress response pathway by having two CpxR binding sites upstream of its promoter region, establishing another link of the kinase to stress responses. Both the genetics and biochemistry of this stress-response kinase will be studied. The upstream regulation of the kinase gene will be studied through effects of mutations in the Cpx and other related two-component regulatory systems, and downstream effects will be studied through genetic interactions with the toxin-antitoxin systems. To obtain a framework for the role of the kinase in stress response networks, gene expression profiling will also be carried out with a variety of stresses in the presence/absence of the kinase activity. The kinase has been purified. As a part of its further characterization, the enzymatic reaction conditions will be optimized, the autophosphorylation site(s) will be determined, and proteins it normally phosphorylates will be identified. The proposed work constitutes an early characterization of regulatory networks involved in bacterial stress response, persistence/tolerance, and apoptosis. Such studies may eventually lead to ways for making many antimicrobials more effective by interfering with bacterial stress responses. Bacterial resistance, tolerance, and persistence to antimicrobial treatment is a growing threat for our ability to cure infections. Protective genes involved in bacterial stress responses help bacteria evade and survive antimicrobial treatment. These protective stress response networks will be studied with the long-term goal of developing small-molecule inhibitors for antimicrobial enhancement.