Characterization of receptors of the bacterial signaling molecule autoinducer-2

描述（由申请人提供）：细菌通过一种被称为群体感应的过程进行交流，在这个过程中，微小的化学信号被产生并释放到环境中。随着细菌数量的增加，这些信号（称为自诱导剂）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，从而能够在群体范围内调节其行为[1-8]。群体感应调节许多重要行为，包括毒力、运动性和生物膜形成[9- 11,13]。虽然大多数自诱导剂介导特定细菌物种内的通信，但一种新的信号分子，称为自诱导剂-2 (AI-2)，似乎是一种通用的信号分子，促进物种之间的信号传递[6,19]。已知超过55种细菌携带AI-2合成酶LuxS基因，包括鼠伤寒沙门氏菌、炭疽芽孢杆菌、鼠疫耶尔森菌和霍乱弧菌等病原体。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[29,30,50 -53]。了解AI-2的化学特性对于设计群体感应的激动剂和拮抗剂至关重要，但先前的研究表明，两种不同的细菌可以识别化学形式不同的AI-2[24,32]。因此，在特定物种中识别AI-2受体并在其受体的背景下表征AI-2是至关重要的。这项工作旨在扩展已知的AI-2受体集，并确定生物医学相关细菌物种识别的AI-2的化学形式。候选AI-2受体将通过基因组分析（包括序列相似性搜索和折叠预测）在蜡样芽孢杆菌、638肠杆菌、绿脓杆菌和铜绿假单胞菌等多种细菌中进行鉴定。这些候选蛋白将被克隆、过表达、纯化，并通过哈维弧菌生物发光试验检测AI-2的结合。一旦AI-2受体被生物化学鉴定，它们将被进一步纯化和结晶。AI-2/受体复合物的x射线结构将被确定，允许对特定物种识别的AI-2的化学形式进行表征。

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 [1-8]. Many important behaviors are regulated by quorum sensing, including virulence, motility, and biofilm formation [9-11, 13]. 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 [6, 19]. More than 55 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 [20]. Thus, AI-2 mediated quorum sensing offers a possible mechanism for controlling bacterial behavior of significant human pathogens [29, 30, 50-53]. Knowledge of the chemical identity of AI-2 is essential for the design of agonists and antagonists of quorum sensing, but previous work has shown that two different bacterial species recognize chemically distinct forms of AI-2 [24, 32]. Thus, it is crucial to identify AI-2 receptors in a given species and characterize AI-2 in the context of its receptor. This work seeks to both extend the set of known AI-2 receptors and determine the chemical form of AI-2 recognized by biomedically relevant bacterial species. Candidate AI-2 receptors will be identified in several bacterial species, including Bacillus cereus, Enterobacter sp. 638, Sinorhizobium meliloti and Pseudomonas aeruginosa, through genomic analysis including sequence similarity searches and fold prediction. These candidate proteins will be cloned, overexpressed, purified, and assayed for AI-2 binding via a Vibrio harveyi bioluminescence assay. Once AI-2 receptors have been identified biochemically, they will be further purified and crystallized. X-ray structures will be determined for AI-2/receptor complexes, allowing characterization of the chemical form of AI- 2 recognized by a particular species.