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，在该过程中产生小的化学信号并释放到环境中。随着种群的增加，这些信号（称为自诱导物）积累，当它们达到足够的浓度时，被其他细菌检测到。细菌响应这些信号调节基因表达，从而能够以全种群的方式调节其行为[72-79]。许多重要的行为受群体感应调节，包括毒力、运动性和生物膜形成[80-83]。虽然大多数自诱导物介导给定细菌物种内的通信，但称为自诱导物-2（AI-2）的新型信号分子似乎是通用信号分子，促进物种之间的信号传导[25，78]。已知许多种细菌携带LuxS基因（AI-2合酶），包括鼠伤寒沙门氏菌、炭疽杆菌、鼠疫耶尔森氏菌和霍乱弧菌等病原体[84]。因此，AI-2介导的群体感应为控制重要人类病原体的细菌行为提供了可能的机制[33，34，37，46，47，85]。多种细菌物种，包括大肠杆菌、B。anthracis和S.鼠伤寒沙门氏菌具有lsr操纵子，一组负责识别、内化和加工AI-2的基因[20，21]。一旦内化，AI-2被LsrK磷酸化，产生磷酸化AI-2 [5]。在lsr操纵子中编码的两种蛋白质LsrF和LsrG进一步加工磷酸-AI-2，但这些反应的细节在分子水平上尚不清楚[21]。通过内化和修饰信号分子，这些细菌干扰AI-2介导的其他物种的通信，获得竞争优势。本工作将研究大肠杆菌催化AI-2加工反应的机理和产物。coli LsrF和LsrG以及苜蓿中华根瘤菌LsrF的推定功能同源物。将通过质谱和NMR分析细胞提取物，以鉴定这些反应的产物。通过对这些蛋白质的结构分析，已经确定了潜在的催化残基;这些残基将被突变，并通过X射线晶体学研究所得的蛋白质和蛋白质/配体复合物。将在基于NMR的体内试验中筛选突变体的催化活性，该试验监测13 C标记的AI-2的摄取速率。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，因此在开发利用AI-2介导的群体感应作为控制细菌的手段的疗法中可能具有很大的实用性。 公共卫生相关性：细菌，包括许多人类病原体，通过小的化学信号进行通信，以群体范围的方式协调诸如毒力和生物膜形成等行为。这项工作调查的结构和功能的蛋白质参与处理一个物种间的信号分子称为autoinducer-2。了解这种信号分子的加工过程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.