Transcriptional control by the global regulator Spx

描述(申请人提供)：在致病细菌中，复杂的控制系统已经进化，激活了对免疫细胞用来对抗感染的各种有毒物质的防御。这些系统调节重要的毒力决定因素，这些决定因素是旨在缓解传染病的药物的靶标。低GC革兰氏阳性细菌有一种独特的转录控制系统，由Spx蛋白介导，在对有毒氧化剂的反应中发挥作用，包括吞噬免疫细胞产生的氧化剂。SPX是一种转录调节因子，我们在芽胞形成细菌枯草杆菌中发现，但也在金黄色葡萄球菌、肺炎链球菌、单核细胞增生性李斯特菌和炭疽杆菌中发现。SPX已被证明可以控制金黄色葡萄球菌毒力相关活性的表达，并在宿主细胞入侵期间在单核细胞增生性李斯特菌和炭疽杆菌中产生高水平的SPX。SPX定义了一类结构上类似于砷酸还原酶的调节剂家族。它带有一个N端的CXXC氧化还原二硫键中心，调节其活性。它的控制机制是独特的，因为游离Spx不显示DNA结合活性，但当它以氧化的二硫键形式与RNA聚合酶(RNAP)结合时，它指导酶转录起氧化应激防御功能的特定基因。在应激恢复后，Spx的浓度被蛋白酶ClpXP和Spx结合蛋白YjbH降低到低水平，从而加速Spx的蛋白分解。这项拟议研究的目的是了解枯草杆菌Spx是如何激活转录启动的，以及YjbH是如何调节Spx蛋白分解的。在Spx控制的基因的启动子区域已经发现了特定的核苷酸序列元件。这些序列结合了Spx的复合体和RNAP亚单位(CTD)的C-末端结构域。将确定启动子DNA序列和CTD/Spx与启动子DNA结合的结构要求。此外，还将探索Spx激活的转录复合体中RNAP全酶亚基定位的动态。有效的蛋白质降解需要YjbH，它是一种锌结合蛋白，与Spx相互作用，并加速ClpXP催化的Spx蛋白分解。建议研究氧化剂诱导的Spx的稳定和YjbH活性的氧化还原控制。这项研究将重点放在接头的锌结合部位，以检验锌配位半胱氨酸残基氧化导致YjbH失活和Spx释放的模型。用差示硫醇标记法和质谱仪研究Spx和YjbH体内硫醇氧化的程度和性质。将研究一种与YjbH相互作用的小蛋白因子YirB，以确定它是否是越来越多的小蛋白中的一员，这些小蛋白通过抑制蛋白酶适配蛋白来防止蛋白酶催化的降解。 与公共卫生相关：在致病细菌中已经进化出复杂的控制系统，以动员防御系统来对抗吞噬免疫细胞产生的有毒氧化剂。调节蛋白Spx控制着细菌对氧化攻击反应的重要组成部分，对其作用机制的研究将揭示中和感染微生物的靶标。

DESCRIPTION (provided by applicant): In pathogenic bacteria, complex systems of control have evolved that activate defenses against the various toxic agents used by immune cells to combat infection. These systems regulate important virulence determinants, which are targets of drugs designed to alleviate infectious disease. The low GC Gram-positive bacteria have a unique system of transcriptional control, mediated by the protein Spx, that functions in the response to toxic oxidants, including those produced by phagocytic immune cells. Spx is a transcriptional regulator that we discovered in the spore-forming bacterium, Bacillus subtilis, but is also found in Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, and Bacillus anthracis. Spx has been shown to control the expression of virulence-associated activities in S. aureus, and is produced to high levels in Listeria monocytogenes and B. anthracis during host cell invasion. Spx defines a family of regulators that structurally resemble the enzyme arsenate reductase. It bears an N-terminal CXXC redox disulfide center that regulates its activity. Its mechanism of control is unique in that free Spx does not exhibit DNA-binding activity, but when bound to RNA polymerase (RNAP) in its oxidized disulfide form, it directs the enzyme to transcribe specific genes that function in oxidative stress defense. Spx concentration is reduced to low levels after recovery from stress by the protease ClpXP and the Spx-binding protein YjbH, which accelerates Spx proteolysis. The goal of the proposed research is to learn how B. subtilis Spx activates transcription initiation and how YjbH functions to mediate Spx proteolysis. Specific nucleotide sequence elements have been identified in the promoter regions of genes controlled by Spx. These sequences bind a complex of Spx and the C-terminal domain of RNAP subunit (CTD). The promoter DNA sequence and the structural requirements of CTD/Spx for promoter DNA binding will be determined. Furthermore, the dynamics of subunit positioning in RNAP holoenzyme within the Spx-activated transcription complex will be explored. Efficient proteolysis requires YjbH, a zinc-binding protein that interacts with Spx and accelerates ClpXP-catalyzed Spx proteolysis. Studies are proposed to investigate the oxidant-induced stabilization of Spx and redox control of YjbH activity. This study will focus on the Zn-binding site of the adaptor to test the model that oxidation of Zn-coordinating cysteine residues causes inactivation of YjbH and release of Spx. Differential thiol labeling and mass spectrometry will be performed to investigate the extent and nature of thiol oxidation of Spx and YjbH in vivo. A small protein factor, YirB that interacts with YjbH, will be studied to determine if it is a member of a growing list of small proteins that prevent protease-catalyzed degradation by inhibiting protease adaptor proteins. PUBLIC HEALTH RELEVANCE: Complex systems of control have evolved in disease-causing bacteria to mobilize defenses that counter the toxic oxidizing agents produced by phagocytic immune cells. The regulatory protein Spx controls important components of the bacterial response to oxidative attack, and the study of its mechanism of action will uncover targets for neutralizing infectious microorganisms.