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通讯过程，细菌通过该过程根据细胞群体密度调节行为。群体感应涉及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于产生它们的细菌物种具有高度特异性，但一种称为 AI-2 的自诱导剂可以由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 可以调节革兰氏阳性菌的多种功能，但在这些情况下，AI-2 信号级联尚未得到研究。本提案中的研究旨在明确 AI-2 和 AI-2 信号转导级联在革兰氏阳性病原体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链球菌 AI-2 群体感应信号转导电路，识别群体感应目标基因，并确定充当化脓性链球菌 AI-2 的化学部分。 2 信号。通过操纵细胞间通讯来控制细菌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开发此类新型抗菌疗法。具体目标是：（1）识别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 SLS（溶血素）和 SpeB（半胱氨酸蛋白酶）的产生。转座子诱变，然后筛选改变的溶血素和蛋白酶的产生，将用于鉴定 AI-2 检测和信号转导所需的基因。 (2)鉴定受AI-2调控的基因。将构建与 gfp 基因融合、针对革兰氏阳性菌进行优化的随机化脓性链球菌启动子文库，并将其引入 luxS 化脓性链球菌菌株中。外源添加合成制备的 AI-2 后，将通过荧光激活细胞分选 (FACS) 监测改变的 GFP 产量。该筛选应识别 AI-2 激活和 AI-2 抑制基因。 (3)确定化脓性链球菌AI-2受体和AI-2分子的结构。与目标 1 中的方法互补的生化方法将采用放射性标记的 AI-2 和细胞分级分离来鉴定化脓性链球菌 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结合或未结合AI-2的受体的全部或部分结构。