Virulence gene regulators of enteric bacterial pathogens: Determining the structural and functional mechanisms of small molecule and polypeptide inhibitors

肠道细菌病原菌毒力基因调控：结构和功能的确定 小分子和多肽抑制剂的作用机制 摘要： AraC/XylS家族是细菌转录因子家族中最大的家族之一，约有16K成员 分布在81%的已测序细菌物种中。家庭成员存在于致病属中 包括不动杆菌、大肠杆菌、克雷伯氏菌、军团菌、假单胞菌、沙门氏菌、志贺氏菌、弧菌和 耶尔西尼亚。任何给定家庭成员的调节蛋白通常包括三个类别之一：新陈代谢， 应激反应，或致病机制。那些参与新陈代谢或压力反应的人通常有很好的 特征化的配体。例如，AraC调节与阿拉伯糖代谢有关的基因的表达 其活性受阿拉伯糖的调节。相比之下，小分子配体还没有被确定为 AraC家族中的绝大多数毒力调节因子(以下称为AraC-vRs)，它导致了 人们普遍认为，该家族的AraC-VR分支已经失去了对配体的反应能力。 我们和其他人发表的研究以及我们的初步研究表明，这一假设是不正确的。 此外，一大类内源性编码的多肽，即AraC负性调节因子的ANR，具有 已发现通过一种未知的机制抑制AraC-VRS。 这个项目的长期目标是确定毒力的结构和分子机制。 基因调控。这项提案的具体目标是确定AraC家庭成员包括 Rns是产肠毒素大肠杆菌(ETEC)的主要毒力调节因子，可被小分子脂肪抑制 酸和2)AraC负性调节因子(ANRs)。我们的中心假设是RNS必须二聚化才能 结合DNA并调节转录，这些抑制剂通过不同的机制阻止这一点。这个 这些研究的动机是，他们将确定分子和结构要求 抑制毒力基因的表达，并将通过三个具体的目的进行测试：1)确定结构 配体结合和二聚化调节RNS活性的机制；2)测试我们的假设 RNS同源RegA以相同的方式调节；3)确定ANRs抑制RNS的机制 活性，并澄清这是否有别于小分子脂肪酸的抑制机制。 这个项目的创新之处在于，调控这些蛋白质的基本分子机制并不是 明白了。我们的多学科团队，拥有微生物学、生物化学和结构生物学的专业知识， 具有独特的地位，能够开展拟议的研究以确定这些机制。这项研究是 意义重大，不仅是因为它将回答有关AraC蛋白在肠道中如何发挥作用的悬而未决的问题 病原体，但因为我们希望证明来自各种肠道的AraC家族蛋白 病原体有一个共同的机制，那就是被脂肪酸抑制。这将开辟新的可能性 旨在对抗全球死亡率和发病率的治疗战略。

Virulence gene regulators of enteric bacterial pathogens: Determining the structural and functional mechanisms of small molecule and polypeptide inhibitors Summary: The AraC/XylS family is one of the largest families of bacterial transcription factors with ~16K members distributed amongst 81% of sequenced bacterial species. Family members are present in pathogenic genera including Acinetobacter, Escherichia, Klebsiella, Legionella, Pseudomonas, Salmonella, Shigella, Vibrio, and Yersinia. The regulon of any given family member typically encompasses one of three categories: metabolism, stress response, or pathogenesis. Those involved in metabolism or stress response often have well characterized ligands. For example, AraC regulates the expression of genes involved in arabinose metabolism and its activity is modulated by arabinose. In contrast, small molecule ligands have not been identified for the vast majority of virulence regulators within the AraC family (hereafter referred to as AraC-VRs), which has led to the commonly held belief that the AraC-VR branch of the family has lost the ability to respond to ligands. Published work by us and others –and our preliminary studies– suggest that this assumption is incorrect. Additionally, a large family of endogenously encoded polypeptides, ANRs for AraC negative regulators, have been discovered that inhibit AraC-VRs though an unknown mechanism. The long-term goal of this project is to define the structural and molecular mechanisms underlying virulence gene regulation. The specific objectives of this proposal are to determine how AraC family members including Rns, a primary virulence regulator in enterotoxigenic E. coli (ETEC), are inhibited by 1) small molecule fatty acids and 2) AraC negative regulators (ANRs). Our central hypothesis is that Rns must dimerize in order to bind to DNA and regulate transcription and that these inhibitors block this by distinct mechanisms. The motivating rationale for these studies is that they will identify the molecular and structural requirements for inhibiting virulence gene expression, and will be tested by three specific aims: 1) Determine the structural mechanism by which ligand binding and dimerization regulates Rns activity; 2) Test our hypothesis that the Rns homolog RegA is regulated in the same manner; 3) Determine the mechanism by which ANRs inhibit Rns activity, and clarify if this is distinct from the inhibitory mechanism of small molecule fatty acids. This project is innovative in that the basic molecular mechanisms by which these proteins are regulated are not understood. Our multidisciplinary team, with expertise in microbiology, biochemistry, and structural biology, is uniquely positioned to undertake the proposed studies to determine these mechanisms. This research is significant, not only because it will answer outstanding questions of how AraC proteins function in enteric pathogens, but because we expect to demonstrate that AraC family proteins from a wide variety of enteric pathogens share a common mechanism of being inhibited by fatty acids. This will open up new possibilities for therapeutic strategies to combat global mortality and morbidity.