New genes involved in cellular responses to quinolone treatment

描述（由申请人提供）：该项目的长期目标是足够详细地了解细菌的应激反应，最终设计出应激反应的小分子抑制剂作为抗菌增强剂。目前的R21申请描述了一个基础科学，原理验证项目，研究一种新的蛋白激酶，当它失活时，会降低大肠杆菌细胞在各种抗菌剂，过氧化氢和高温下的存活率。缺乏这种激酶会使喹诺酮类药物治疗的细菌存活率降低10- 100倍，并导致抑菌化合物氯霉素变成杀菌药。它还显著降低了萘啶酸诱导新的抗性突变体的能力。激酶缺乏是由毒素-抗毒素基因对的缺失抑制的，该基因对被认为既有助于保护免受压力，又有助于细菌凋亡。这种与毒素-抗毒素系统的遗传相互作用导致了激酶通常限制毒素活性的假设；在缺乏激酶的情况下，毒素在应激条件下杀死细胞，从而同时增强许多抗菌剂的作用。该激酶基因还参与Cpx包膜蛋白应激反应途径，其启动子区域上游有两个CpxR结合位点，从而建立了该激酶与应激反应的另一个联系。我们将对这种应激反应激酶的遗传和生物化学进行研究。激酶基因的上游调控将通过Cpx和其他相关双组分调控系统突变的影响来研究，下游效应将通过与毒素-抗毒素系统的遗传相互作用来研究。为了获得激酶在应激反应网络中的作用框架，基因表达谱也将在激酶活性存在/不存在的各种应激下进行。该激酶已被纯化。作为其进一步表征的一部分，将优化酶促反应条件，确定自磷酸化位点，并鉴定其通常磷酸化的蛋白质。提出的工作构成了涉及细菌应激反应、持久性/耐受性和细胞凋亡的调控网络的早期表征。这些研究可能最终导致通过干扰细菌应激反应使许多抗菌剂更有效的方法。

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.