Redox-regulation in Gram-negative Bacteria

项目总结 细菌经常遇到各种有害的应激源，包括活性氧和氯物质 (RO/CS)。一种特别有效的抗微生物氧化剂是次氯酸(HOCl)，它是在 中性粒细胞介导的吞噬作用和粘膜上皮细胞的酶控制细菌定植。 细菌已经进化出策略来抵消和减少RO/CS的有害影响，例如通过上调 应激特异基因的表达。RO/CS诱导的基因表达变化通常是通过 转录调节因子中氧化还原敏感氨基酸侧链的翻译后修饰 影响它们激活/抑制相应的应激保护靶基因表达的能力。我们的 初步数据表明，致尿性大肠杆菌(UPEC)对亚致死性HOCl-胁迫的反应是上调的 一个操纵子含有三个未鉴定的UPEC特异性基因。我们确认其中一人是HOCL- 敏感的转录抑制因子，在HOCl胁迫期间可逆地失去其抑制因子的活性。它的失活 结果抑制了两个下游靶标，从而增加了对HOCl2的抵抗力 UPEC菌株与共生性和致肠性大肠杆菌的比较。此外，我们的初步数据显示， 亚致死浓度的HOCl2会诱导大量的生物膜基因，刺激生物膜的形成。 本项目的总体目标是全面了解RO/CS应激的原因和影响，并 描述针对RO/CS的特定细菌防御策略，以减轻其损害。我们的工作 假说是RO/CS特异性和可逆地修饰蛋白质中暴露在表面的半胱氨酸残基，这 通过影响基因表达和/或细菌生理，成为氧化还原信号的关键因素。在目标1中，我们 将使用表型和生化策略来评估转录抑制因子 在体外和体内都失活了。此外，我们还将阐明其下游目标之一的功能，即 似乎在很大程度上促进了UPEC对HOCl的抵抗力，并确定了额外的HOCl抵抗力 TnSeq.为了表征HOCl介导的生物膜刺激的好处，我们将分析 CFT073生物膜在亚致死量高氯暴露前后的组成及生物膜细胞的检测 对随后的高氯应激或用于治疗尿路感染的常见抗生素产生更强的抵抗力(目标2)。此外, 我们将通过确定HOCl胁迫如何触发CFT073中生物膜形成的增加来研究 二鸟苷环化酶YdeH在这一过程中所起的作用。在目标3中，我们将结合转录和 表型分析确定UPEC对表面抗菌剂耐药性的新调节基因 AgXX，此前已被证明可生成RO/CS。这些研究将从根本上为我们提供 对RO/CS在UPEC中扮演的角色的新见解。确定、描述和确定UPEC特定的目标 防御系统有可能提高人体自身对抗尿路感染的能力。

PROJECT SUMMARY Bacteria frequently encounter a variety of harmful stressors including reactive oxygen and chlorine species (RO/CS). One particularly potent antimicrobial oxidant is hypochlorous acid (HOCl), which is generated during neutrophil-mediated phagocytosis and by enzymes of the mucosal epithelia to control bacterial colonization. Bacteria have evolved strategies to counteract and reduce the harmful effects of RO/CS, e.g. by upregulating the expression of stress-specific genes. RO/CS-induced changes in gene expression are typically mediated by posttranslational modifications of redox-sensitive amino acid side chains in transcriptional regulators, which affect their ability to activate/repress the expression of the corresponding stress-protective target genes. Our preliminary data show that uropathogenic E. coli (UPEC) respond to sublethal HOCl-stress with the upregulation of an operon harboring three uncharacterized UPEC-specific genes. We identified one of them as a HOCl- sensitive transcriptional repressor that reversibly loses its repressor activity during HOCl-stress. Its inactivation results in the de-repression of the two downstream targets contributing to the increased HOCl resistance of UPEC strains compared to commensal and enteropathogenic E. coli. Moreover, our preliminary data show that sublethal HOCl concentrations cause the induction of numerous biofilm genes and stimulate biofilm formation. The overall goal of this project is to comprehensively understand the cause and effects of RO/CS stress and to characterize specific bacterial defense strategies to RO/CS used to mitigate their damage. Our working hypothesis is that RO/CS specifically and reversibly modify surface-exposed cysteine residues in proteins, which become the key factor in redox signaling by affecting gene expression and/or bacterial physiology. In Aim 1, we will use phenotypic and biochemical strategies to assess the mechanism by which the transcriptional repressor is inactivated in vitro and in vivo. Moreover, we will elucidate the function of one of its downstream targets, which appears to contribute substantially to UPEC’s increased HOCl resistance and identify additional HOCl resistance genes by TnSeq. To characterize the benefits of HOCl-mediated biofilm stimulation, we will analyze the composition of CFT073 biofilms before and after exposure to sublethal HOCl and test whether biofilm cells become more resistant to subsequent HOCl stress or common antibiotics used to treat UTIs (Aim 2). In addition, we will investigate how HOCl stress acts to trigger the increase in biofilm formation in CFT073 by determining the role that the diguanylate cyclase YdeH plays in this process. In Aim 3, we will combine transcriptomic and phenotypic analyses to identify novel regulons contributing to UPEC’s resistance to the surface antimicrobial AgXX, which has previously been shown to generate RO/CS. These studies will provide us with fundamentally new insights into the role that RO/CS play in UPEC. Identifying, characterizing and targeting UPEC-specific defense systems has the potential to increase the body’s own capacity to fight UTIs.