Characterization of bleach-specific transcriptional regulators in bacteria

描述（由申请人提供）：氯基漂白剂是世界上使用最广泛的消毒剂。漂白剂的活性成分，次氯酸（HOCl）也是由先天免疫反应的细胞产生的，在哺乳动物宿主防御中起着重要的抗菌作用。然而，当在宿主体内过量产生时，HOCl是一种破坏性的氧化剂，导致慢性炎症和许多疾病状态部位的组织损伤。尽管HOCl作为抗微生物剂和在疾病中具有这些不同的作用，但HOCl毒性背后的生理原因在很大程度上是未知的。此外，生物体开发的防御HOCl的策略尚不清楚。最近，我们发现细菌编码氧化还原调节蛋白，其使用氧化敏感的半胱氨酸来特异性地感知和响应HOCl，并具有增强细菌漂白剂存活的功能变化。这些结果构成了我们工作假设的基础;通过了解细菌如何感知HOCl并防御HOCl介导的损伤，我们将深入了解漂白剂处理的生物体中发生的生理变化，并揭示预防和修复漂白剂造成的损伤的机制。通过对漂白剂处理过的细菌的微阵列分析，我们已经确定了两个HOCl特异性的转录调节因子，转录抑制因子NemR和转录激活因子YkgD。这两种调节剂都含有高度保守的半胱氨酸，其可能参与HOCl传感并负责响应漂白剂而观察到的靶基因表达的去抑制（NemR）或激活（YkgD）。现在，我们将利用我们广泛的专业知识与氧化还原调节蛋白质，以调查这些新的转录调节剂的氧化还原传感机制。然后，我们将表征NemR和YkgD的选定下游靶标，我们发现这些靶标在缺失时会影响细菌的漂白剂抗性。这些研究将显著增强我们对HOCl的细胞后果的理解，并可能揭示操纵微生物和宿主中HOCl敏感性的潜在手段。 公共卫生相关性：次氯酸（HOCl）是家用漂白剂的活性成分，是一种高效的商业消毒剂，是哺乳动物免疫系统细胞产生的主要抗菌成分，可杀死入侵的细菌。然而，在慢性炎症部位过度产生的HOCl导致组织损伤和多种疾病。我们现在将阐明细菌开发的感知和防御HOCl压力的机制，最终目标是设计增加细菌漂白剂敏感性和减轻漂白剂在宿主中造成的损害的策略。

DESCRIPTION (provided by applicant): Chlorine-based bleach is the most widely used disinfectant in the world. The active ingredient of bleach, hypochlorous acid (HOCl) is also made by cells of the innate immune response, where it plays an important antimicrobial role in the mammalian host defense. However, when produced in excess in the host, HOCl is a devastating oxidant, responsible for tissue damage at sites of chronic inflammation and numerous disease states. Despite these various roles of HOCl as an antimicrobial agent and in disease, the physiological reasons behind the toxicity of HOCl are largely unknown. Moreover, the strategies that organisms have developed to defend against HOCl are unclear. Very recently, we discovered that bacteria encode redox regulated proteins, which use oxidation-sensitive cysteines to specifically sense and respond to HOCl with functional changes that enhance bacterial bleach survival. These results formed the basis for our working hypothesis; by understanding how bacteria sense HOCl and defend against HOCl-mediated damage, we will obtain an in-depth view of the physiological changes that occur in bleach-treated organisms, and uncover mechanisms that prevent and repair the damage that bleach inflicts. Through microarray analysis of bleach treated bacteria, we have identified two HOCl-specific transcriptional regulators in bacteria, the transcriptional repressor NemR, and the transcriptional activator YkgD. Both of these regulators contain highly conserved cysteines, which are likely involved in HOCl-sensing and responsible for the observed de-repression (NemR) or activation (YkgD) of target gene expression in response to bleach. We will now draw on our extensive expertise with redox-regulated proteins to investigate the redox-sensing mechanism of these novel transcriptional regulators. We will then characterize select downstream targets of NemR and YkgD that we have found to affect bacterial bleach resistance when deleted. These studies will significantly enhance our understanding about the cellular consequences of HOCl and may reveal potential means to manipulate HOCl sensitivity in both microbes and hosts. PUBLIC HEALTH RELEVANCE: Hypochlorous acid (HOCl), the active ingredient of household bleach, is a highly effective commercial disinfectant, and produced as key antimicrobial component by cells of the mammalian immune system to kill off invading bacteria. Overproduced at sites of chronic inflammation, however, HOCl leads to tissue damage and a variety of diseases. We will now elucidate the mechanisms that bacteria developed to sense and defend against HOCl-stress with the ultimate goal to devise strategies that increase bacterial bleach sensitivity and mitigate the damage that bleach causes in the host.