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

群体感应是一种细胞间的交流过程，细菌通过这个过程来调节行为，作为 细胞密度。群体感应涉及信号的产生、分泌和检测 称为自体诱导剂的分子。虽然大多数自体诱导剂是高度特异的细菌种类， 产生它们，一种自动诱导剂，称为AI-2，由许多物种产生和检测，其功能是 一种跨物种信号分子。已知AI-2调节革兰氏阳性细菌的多种功能， 但在这些情况下，AI-2信号级联还没有得到研究。这项提案中的研究旨在 明确AI-2及其信号转导通路在革兰氏阳性菌中的作用 化脓性链球菌。为了实现这些目标，我将开发基因工具，并使用它们来识别和 鉴定化脓性链球菌AI-2群体感应信号转导电路，识别群体感应靶标 基因，并确定作为化脓性链球菌AI-2信号的化学部分。已经很长时间了 通过操纵细胞-细胞来控制细菌毒力的群体传感领域的目标 沟通。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群体感应信号转导电路。AI-2 控制化脓性链球菌所需的SLS(溶血素)和SpeB(半胱氨酸蛋白酶)的产生 致命性。转座子诱变，随后筛选溶血素和蛋白酶的改变产生 将用于识别AI-2检测和信号转导所需的基因。2.识别受调控的基因 被AI-2。针对革兰氏阳性的GFP基因融合的化脓性链球菌随机启动子文库 细菌，并将其导入化脓性链球菌。修改后的GFP生产将是 外源添加人工合成材料后的荧光激活细胞分类(FACS)监测 AI-2。；这个屏幕应该识别AI-2激活和AI-2抑制的基因。3.确定结构 化脓性链球菌AI-2受体和AI-2分子。一种补充AIM的生化方法 1将使用放射性标记的AI-2和细胞分级来鉴定化脓性链球菌Al-2受体。基因 编码该受体的蛋白质将被克隆、表达和纯化。X射线结晶学将是 用于确定与AI-2结合或不结合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.