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

革兰氏阳性菌化脓性链球菌中 Al-2 依赖性群体感应

• 批准号: 7248925
• 负责人: MICHAEL J FEDERLE
• 金额: $ 9万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-15 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7248925
• 关键词: Bacteria; Behavior; Binding; Biochemical; Cell Communication; Cell Fractionation; Cell Signaling Process; Cell physiology; Cells; Chemicals; Cysteine Protease; Detection; Development; Endopeptidases; Fluorescence-Activated Cell Sorting; Gene Targeting; Genes; Genetic; Goals; Gram-Positive Bacteria; Green Fluorescent Proteins; Hemolysin; Libraries; Monitor; Mutagenesis; Peptide Hydrolases; Population Density; Production; Proteins; Radiolabeled; Research; Role; Signal Transduction; Signaling Molecule; Streptococcus; Streptococcus pyogenes; Structure; Virulence; X-Ray Crystallography; antimicrobial; novel; pathogen; promoter; quorum sensing; radiotracer; receptor; tool

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.

描述(由申请人提供)：群体感应是一种细胞间的交流过程，细菌通过该过程来调节作为细胞种群密度函数的行为。群体感应涉及信号分子的产生、分泌和检测，称为自体诱导剂。虽然大多数自体诱导剂对产生它们的细菌物种具有高度特异性，但有一种自体诱导剂AI-2被许多物种产生和检测，并作为跨物种信号分子发挥作用。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信号级联尚未被研究。本研究旨在明确AI-2及其信号转导通路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，我将开发基因工具，并利用它们来鉴定和鉴定化脓性链球菌AI-2群体感应信号转导电路，识别群体感应靶基因，并确定作为化脓性链球菌AI-2信号的化学部分。通过控制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此类新型抗菌疗法的开发。具体目标是：(1)确定LUXS/AI-2群体感应信号转导电路。AI-2控制化脓性链球菌毒力所需的溶血素(SLS)和半胱氨酸蛋白酶(SpeB)的产生。转座子突变，然后是溶血素和蛋白酶的改变产生的筛选将被用来识别AI-2检测和信号转导所需的基因。(2)鉴定AI-2调控基因。将构建一个与GFP基因融合的随机化脓性链球菌启动子文库，该文库针对革兰氏阳性菌进行了优化，并将其导入到一株豪华化脓性葡萄球菌中。在外源添加合成的AI-2后，将通过荧光激活细胞分选(FACS)来监测改变的GFP产生。这个屏幕应该识别AI-2激活和AI-2抑制的基因。(3)确定化脓性链球菌AI-2受体和AI-2分子的结构。一种补充目标1的生化方法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。将克隆该受体的编码基因，并表达和纯化该蛋白。X射线结晶学将被用来确定结合和不结合AI-2的受体的全部或部分结构。