Structural and functional studies of autoinducer-2 processing proteins

描述(由申请人提供)：细菌通过一种称为群体感应的过程进行交流，在这种过程中，微小的化学信号被产生并释放到环境中。随着种群数量的增加，这些信号(称为自动诱导子)会积累起来，当它们达到足够的浓度时，就会被其他细菌检测到。细菌根据这些信号调节基因表达，从而能够在整个种群范围内调节它们的行为[72-79]。许多重要的行为都受到群体感应的调节，包括毒力、运动性和生物膜的形成[80-83]。虽然大多数自体诱导剂在给定的细菌物种内调节通讯，但一种新的信号分子，称为自体诱导器-2(AI-2)，似乎是一个通用的信号分子，促进物种之间的信号传递[25，78]。已知许多种类的细菌携带AI-2合酶LuxS基因，包括鼠伤寒沙门氏菌、炭疽杆菌、鼠疫耶尔森菌和霍乱弧菌[84]等病原体。因此，AI-2介导的群体感应为控制重要的人类病原体的细菌行为提供了一种可能的机制[33，34，37，46，47，85]。包括大肠杆菌、炭疽杆菌和鼠伤寒沙门氏菌在内的各种细菌都有LSR操纵子，这是一组负责识别、内化和处理AI-2的基因[20，21]。一旦内化，AI-2被LsrK磷酸化，产生磷酸化的AI-2[5]。LSR操纵子中编码的两种蛋白质LsrF和LsrG进一步处理磷酸化AI-2，但这些反应的细节在分子水平上尚不清楚[21]。这些细菌通过内化和修饰信号分子，干扰AI-2介导的其他物种的交流，获得竞争优势。本工作将研究E.coliLsrF和LsrG催化的AI-2加工反应的机制和产物，以及来自苜蓿中华根瘤菌的一个可能的功能同源物。细胞提取物将通过质谱学和核磁共振进行分析，以鉴定这些反应的产物。通过对这些蛋白质的结构分析，确定了潜在的催化残基；这些残基将发生突变，并通过X射线结晶学研究得到的蛋白质和蛋白质/配体复合体。将在基于核磁共振的体内试验中筛选突变的催化活性，以监测13C标记的AI-2的摄取速度。本文提出的工作旨在从结构和功能上表征负责降解P-AI-2的蛋白质，因此可能对开发利用AI-2介导的群体感应作为控制细菌的手段的疗法非常有用。 与公共卫生相关：细菌，包括许多人类病原体，通过微小的化学信号进行交流，以种群范围的方式协调毒力和生物膜形成等行为。这项工作研究了蛋白质的结构和功能，这些蛋白质参与了一种名为AutoInducer-2的跨物种信号分子的处理。了解这种信号分子的处理是利用这种通信来控制细菌行为的重要一步。

DESCRIPTION (provided by applicant): Bacteria communicate through a process known as quorum sensing, in which small chemical signals are produced and released into the environment. As the population increases, these signals (called autoinducers) accumulate and, when they reach sufficient concentration, are detected by other bacteria. Bacteria modulate gene expression in response to these signals, and thus are able to regulate their behavior in a population-wide manner [72-79]. Many important behaviors are regulated by quorum sensing, including virulence, motility, and biofilm formation [80-83]. While most autoinducers mediate communication within a given bacterial species, a novel signal molecule, termed autoinducer-2 (AI-2), appears to be a universal signal molecule, facilitating signaling between species [25, 78]. Many species of bacteria are known to carry the gene for LuxS, the AI-2 synthase, including pathogens such as Salmonella typhimurium, Bacillus anthracis, Yersinia pestis, and Vibrio cholerae [84]. Thus, AI-2 mediated quorum sensing offers a possible mechanism for controlling bacterial behavior of significant human pathogens [33, 34, 37, 46, 47, 85]. A variety of bacterial species, including Escherichia coli, B. anthracis, and S. typhimurium have the lsr operon, a set of genes responsible for recognizing, internalizing, and processing AI-2 [20, 21]. Once internalized, AI-2 is phosphorylated by LsrK, giving rise to phospho-AI-2 [5]. Two proteins encoded in the lsr operon, LsrF and LsrG, further process phospho-AI-2, but the details of these reactions are not understood at the molecular level [21]. By internalizing and modifying the signal molecule, these bacteria interfere with the AI-2 mediated communication of other species, gaining a competitive advantage. This work will investigate the mechanisms and products of the AI-2 processing reactions catalyzed by E. coli LsrF and LsrG and a putative functional homolog of LsrF from Sinorhizobium meliloti. Cell extracts will be analyzed by mass spectrometry and NMR to identify the products of these reactions. Potential catalytic residues have been identified through structural analysis of these proteins; these residues will be mutated and the resulting proteins and protein/ligand complexes studied by x-ray crystallography. Mutants will be screened for catalytic activity in NMR-based in vivo assays that monitor rates of uptake of 13C-labeled AI-2. The work proposed here seeks to characterize, structurally and functionally, the proteins responsible for degrading P-AI-2 and thus could be of great utility in developing therapies that exploit AI-2 mediated quorum sensing as a means to control bacteria. PUBLIC HEALTH RELEVANCE: Bacteria, including many human pathogens, communicate via small chemical signals, coordinating behaviors such as virulence and biofilm formation in a population-wide manner. This work investigates the structure and function of proteins involved in processing an interspecies signal molecule called autoinducer-2. Understanding the processing of this signal molecule is an important step towards exploiting this communication to control bacterial behavior.