Characterization of bleach-specific transcriptional regulators in bacteria

说明(申请人提供)：含氯漂白剂是世界上使用最广泛的消毒剂。漂白剂的活性成分次氯酸(HOCl)也是由细胞的天然免疫反应产生的，在哺乳动物的宿主防御中起着重要的抗菌作用。然而，当HOCl在宿主中过量产生时，它是一种毁灭性的氧化剂，对慢性炎症和许多疾病状态的部位的组织损伤负责。尽管HOCl作为一种抗菌剂和在疾病中具有不同的作用，但HOCl毒性背后的生理原因在很大程度上是未知的。此外，生物体已经开发出的防御HOCl2的策略还不清楚。最近，我们发现细菌编码氧化还原调节的蛋白质，这些蛋白质利用氧化敏感的半胱氨酸来特异性地感知和响应HOCl，其功能变化可以提高细菌漂白剂的存活率。这些结果形成了我们工作假说的基础；通过了解细菌如何感知HOCl2并防御HOCl2介导的损害，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害的机制。通过对漂白剂处理细菌的微阵列分析，我们在细菌中发现了两个HOCl特异性的转录调控因子，转录抑制因子NemR和转录激活因子YkgD。这两种调控因子都含有高度保守的半胱氨酸，这些半胱氨酸可能参与HOCl感应，并负责观察到漂白剂对靶基因表达的下调(NEMR)或激活(YkgD)。我们现在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的转录调节因子的氧化还原敏感机制。然后，我们将表征我们发现的影响细菌漂白剂抗性的NemR和YkgD下游靶标的特征。这些研究将极大地增强我们对HOCl的细胞后果的理解，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.