Characterization of bleach-specific transcriptional regulators in bacteria

说明（由申请人提供）：含氯漂白剂是世界上使用最广泛的消毒剂。漂白剂的有效成分次氯酸（HOCl）也由先天免疫反应细胞产生，在哺乳动物宿主防御中起着重要的抗菌作用。然而，当宿主体内过量产生HOCl时，HOCl是一种破坏性氧化剂，在慢性炎症和许多疾病状态的部位造成组织损伤。尽管HOCl作为抗菌剂和在疾病中有这些不同的作用，但HOCl毒性背后的生理原因在很大程度上是未知的。此外，生物发展出的防御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，该蛋白利用氧化敏感半胱氨酸特异性地感知HOCl并对其做出反应，从而提高细菌漂白剂的存活率。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过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进行微阵列分析，我们在细菌中鉴定出了两种hocl特异性转录调节因子，转录抑制因子NemR和转录激活因子YkgD。这两种调节因子都含有高度保守的半胱氨酸，它们可能参与hocl感应，并负责观察到的漂白反应中靶基因表达的去抑制（NemR）或激活（YkgD）。现在，我们将利用我们在氧化还原调节蛋白方面的广泛专业知识来研究这些新型转录调节因子的氧化还原感应机制。然后，我们将对NemR和YkgD的下游目标进行表征，我们发现这些目标在删除后会影响细菌的漂白剂抗性。这些研究将显著增强我们对HOCl对细胞的影响的理解，并可能揭示在微生物和宿主中操纵HOCl敏感性的潜在方法。

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.