Al-2-Dependent Quorum Sensing in the Gram-Positive Bacterium Streptococcus pyogen

描述（由申请人提供）：群体感应是一种细胞间的通讯过程，细菌通过该过程调节细胞种群密度的行为。群体感应涉及到被称为自诱导剂的信号分子的产生、分泌和检测。虽然大多数自诱导剂对产生它们的细菌种类具有高度特异性，但一种称为AI-2的自诱导剂可被许多物种产生和检测，并作为种间信号分子发挥作用。已知AI-2在革兰氏阳性细菌中调节许多功能，但在这些情况下，AI-2信号级联尚未被研究。本课题旨在明确AI-2及其信号转导级联在革兰氏阳性病原菌化脓性链球菌中的作用。为了实现这些目标，我将开发遗传工具，并使用它们来识别和表征化脓性葡萄球菌AI-2群体感应信号转导电路，识别群体感应靶基因，并确定化脓性葡萄球菌AI-2信号的化学部分。通过操纵细胞间的通讯来控制细菌的毒力一直是群体感应领域的一个长期目标。这项研究的结果将有助于这种新型抗菌疗法的发展。具体目的是：(1)确定luxS/AI-2群体感应信号转导电路。AI-2控制溶血素（SLS）和半胱氨酸蛋白酶（SpeB）的产生，这是化脓链球菌毒力所必需的。转座子诱变，随后筛选溶血素和蛋白酶的改变，将用于识别AI-2检测和信号转导所需的基因。(2)鉴定AI-2调控的基因。将构建一个随机的与gfp基因融合的化脓性葡萄球菌启动子文库，并将其引入到革兰氏阳性菌中。在外源性添加合成的AI-2后，将通过荧光活化细胞分选（FACS）监测改变的GFP产生。该筛选应识别ai -2激活基因和ai -2抑制基因。(3)确定化脓链球菌AI-2受体和AI-2分子的结构。与目的1相辅相成的生化方法将采用放射性标记的AI-2和细胞分离来鉴定化脓性葡萄球菌Al-2受体。编码受体的基因将被克隆，蛋白质将被表达和纯化。x射线晶体学将用于确定有或没有结合AI-2的受体的全部或部分结构。

DESCRIPTION (provided by applicant): Quorum sensing is a cell-cell communication process by which bacteria regulate behaviors as a function of cell population density. Quorum sensing involves the production, secretion, and detection of signal molecules termed autoinducers. While most autoinducers are highly specific for the species of bacteria that produces them, one autoinducer, termed AI-2, is produced and detected by many species, and functions as an interspecies signaling molecule. AI-2 is known to regulate numerous functions in Gram-positive bacteria, but in these cases, the AI-2 signaling cascade has not been investigated. The research in this proposal aims to define the role of AI-2 and the AI-2 signal transduction cascade in the Gram-positive pathogen Streptococcus pyogenes. To achieve these goals, I will develop genetic tools and use them to identify and characterize the S. pyogenes AI-2 quorum sensing signal transduction circuit, identify quorum sensing target genes, and determine the chemical moiety that functions as the S. pyogenes AI-2 signal. It has been a long-standing goal in the quorum sensing field to control bacterial virulence by manipulating cell-cell communication. Results from this research will contribute to the development of such novel antimicrobial therapies. The specific aims are: (1) Identify the luxS/AI-2 quorum sensing signal transduction circuit. AI-2 controls production of SLS (hemolysin) and SpeB (cysteine protease) which are required for S. pyogenes virulence. Transposon mutageneses, followed by screens for altered production of hemolysin and protease will be used to identify genes required for AI-2 detection and signal transduction. (2) Identify genes regulated by AI-2. A library of random S. pyogenes promoters fused to a gfp gene, optimized for Gram-positive bacteria, will be constructed and introduced into a luxS S. pyogenes strain. Altered GFP production will be monitored by fluorescence activated cell sorting (FACS) following exogenous addition of synthetically prepared AI-2. This screen should identify AI-2-activated and AI-2-repressed genes. (3) Determine the structure of the S. pyogenes AI-2 receptor and AI-2 molecule. A biochemical approach complementary to that in aim 1 will employ radiolabeled AI-2 and cell fractionation to identify the S. pyogenes Al-2 receptor. The gene encoding the receptor will be cloned and the protein expressed and purified. X-ray crystallography will be used to determine the structure of all or a portion of the receptor with and without bound AI-2.