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

群体感应是一种细胞-细胞通讯过程，细菌通过该过程调节行为， 细胞群体密度群体感应涉及信号的产生、分泌和检测 称为自体诱导物的分子。虽然大多数自体诱导物对细菌种类具有高度特异性， 产生它们，一种自诱导物，称为AI-2，由许多物种产生和检测，其功能是 一种跨物种的信号分子已知AI-2调节革兰氏阳性细菌中的多种功能， 但在这些情况下，AI-2信号级联尚未被研究。本提案中的研究旨在 确定AI-2和AI-2信号转导级联在革兰氏阳性病原体中的作用 化脓性链球菌。为了实现这些目标，我将开发遗传工具，并利用它们来识别和 对S.化脓菌AI-2群体感应信号转导通路，确定群体感应靶点 基因，并确定作为S.化脓性链球菌AI-2信号。这是一个漫长 在群体感应领域的长期目标是通过操纵细胞-细胞来控制细菌的毒力 通信本研究的结果将有助于此类新型抗菌剂的开发 治疗具体目标是：1.确定luxS/AI-2群体感应信号转导回路。AI-2 控制S.化脓 毒性转座子诱变，随后筛选溶血素和蛋白酶的产生改变 将用于鉴定AI-2检测和信号转导所需的基因。2.识别调控基因 AI-2随机S.化脓性链球菌启动子融合到gfp基因，针对革兰氏阳性菌优化 细菌，将被构建并引入luxS S。化脓性链球菌改变的GFP生产将是 在外源性添加合成制备的化合物后，通过荧光激活细胞分选（FACS）监测 AI-2。该筛选应识别AI-2激活和AI-2抑制的基因。3.确定结构 色葡萄化脓性链球菌AI-2受体和AI-2分子。与Aim互补的生物化学方法 1将采用放射性标记的AI-2和细胞分级分离来鉴定S.化脓性链球菌Al-2受体基因 将克隆编码该受体的蛋白质，并表达和纯化该蛋白质。X射线晶体学将是 用于测定结合和不结合AI-2的受体的全部或部分结构。

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 th'e receptor with and without bound AI-2.