Structural and functional studies of autoinducer-2 processing proteins

自诱导剂 2 加工蛋白的结构和功能研究

• 批准号: 8231966
• 负责人: Stephen T. Miller
• 金额: $ 25.59万
• 依托单位: SWARTHMORE COLLEGE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-01 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8231966
• 关键词: Alanine; Bacillus anthracis; Bacteria; Bacterial Proteins; Behavior; Binding; Biochemical; Biological Assay; Cell Density; Cell Extracts; Cell physiology; Chemicals; Communication; Complex; Crystallography; Detection; Environment; Escherichia coli; Gene Expression; Gene Expression Regulation; Genes; Homologous Gene; Human; In Vitro; Label; Ligands; Mass Spectrum Analysis; Mediating; Melilotus; Methods; Microbial Biofilms; Molecular; Monitor; Mutate; Operon; Population; Process; Production; Protein Analysis; Proteins; Reaction; Role; Salmonella typhimurium; Signal Transduction; Signaling Molecule; Sinorhizobium meliloti; Structure; System; Techniques; Testing; VAI-2; Vibrio cholerae; Virulence; Work; Yersinia pestis; analog; base; cell motility; enzyme substrate complex; in vivo; mutant; novel; pathogen; protein structure function; quorum sensing; research study; response; therapy development; uptake

DESCRIPTION (provided by applicant): Bacteria communicate through a process known as quorum sensing, in which small chemical signals are produced and released into the environment. As the population increases, these signals (called autoinducers) accumulate and, when they reach sufficient concentration, are detected by other bacteria. Bacteria modulate gene expression in response to these signals, and thus are able to regulate their behavior in a population-wide manner [72-79]. Many important behaviors are regulated by quorum sensing, including virulence, motility, and biofilm formation [80-83]. While most autoinducers mediate communication within a given bacterial species, a novel signal molecule, termed autoinducer-2 (AI-2), appears to be a universal signal molecule, facilitating signaling between species [25, 78]. Many species of bacteria are known to carry the gene for LuxS, the AI-2 synthase, including pathogens such as Salmonella typhimurium, Bacillus anthracis, Yersinia pestis, and Vibrio cholerae [84]. Thus, AI-2 mediated quorum sensing offers a possible mechanism for controlling bacterial behavior of significant human pathogens [33, 34, 37, 46, 47, 85]. A variety of bacterial species, including Escherichia coli, B. anthracis, and S. typhimurium have the lsr operon, a set of genes responsible for recognizing, internalizing, and processing AI-2 [20, 21]. Once internalized, AI-2 is phosphorylated by LsrK, giving rise to phospho-AI-2 [5]. Two proteins encoded in the lsr operon, LsrF and LsrG, further process phospho-AI-2, but the details of these reactions are not understood at the molecular level [21]. By internalizing and modifying the signal molecule, these bacteria interfere with the AI-2 mediated communication of other species, gaining a competitive advantage. This work will investigate the mechanisms and products of the AI-2 processing reactions catalyzed by E. coli LsrF and LsrG and a putative functional homolog of LsrF from Sinorhizobium meliloti. Cell extracts will be analyzed by mass spectrometry and NMR to identify the products of these reactions. Potential catalytic residues have been identified through structural analysis of these proteins; these residues will be mutated and the resulting proteins and protein/ligand complexes studied by x-ray crystallography. Mutants will be screened for catalytic activity in NMR-based in vivo assays that monitor rates of uptake of 13C-labeled AI-2. The work proposed here seeks to characterize, structurally and functionally, the proteins responsible for degrading P-AI-2 and thus could be of great utility in developing therapies that exploit AI-2 mediated quorum sensing as a means to control bacteria. PUBLIC HEALTH RELEVANCE: Bacteria, including many human pathogens, communicate via small chemical signals, coordinating behaviors such as virulence and biofilm formation in a population-wide manner. This work investigates the structure and function of proteins involved in processing an interspecies signal molecule called autoinducer-2. Understanding the processing of this signal molecule is an important step towards exploiting this communication to control bacterial behavior.

描述（由申请人提供）：细菌通过称为群体感应的过程进行交流，其中产生微小的化学信号并将其释放到环境中。随着种群数量的增加，这些信号（称为自诱导剂）会积累，当它们达到足够的浓度时，就会被其他细菌检测到。细菌响应这些信号调节基因表达，因此能够在群体范围内调节其行为[72-79]。许多重要的行为都受到群体感应的调节，包括毒力、运动性和生物膜形成[80-83]。虽然大多数自诱导剂介导特定细菌物种内的通讯，但一种称为自诱导剂-2 (AI-2) 的新型信号分子似乎是一种通用信号分子，促进物种之间的信号传导 [25, 78]。已知许多细菌物种携带 LuxS（AI-2 合酶）基因，包括鼠伤寒沙门氏菌、炭疽杆菌、鼠疫耶尔森氏菌和霍乱弧菌等病原体 [84]。因此，AI-2介导的群体感应为控制重要人类病原体的细菌行为提供了一种可能的机制[33,34,37,46,47,85]。多种细菌，包括大肠杆菌、炭疽杆菌和鼠伤寒沙门氏菌，都具有 lsr 操纵子，这是一组负责识别、内化和处理 AI-2 的基因 [20, 21]。一旦内化，AI-2 就会被 LsrK 磷酸化，产生磷酸化 AI-2 [5]。 lsr 操纵子中编码的两种蛋白质 LsrF 和 LsrG 进一步加工磷酸化 AI-2，但这些反应的细节在分子水平上尚不清楚 [21]。通过内化和修改信号分子，这些细菌干扰 AI-2 介导的其他物种的通讯，从而获得竞争优势。这项工作将研究大肠杆菌 LsrF 和 LsrG 以及来自苜蓿中华根瘤菌的 LsrF 的假定功能同源物催化的 AI-2 加工反应的机制和产物。细胞提取物将通过质谱和核磁共振进行分析，以鉴定这些反应的产物。通过对这些蛋白质的结构分析已鉴定出潜在的催化残基；这些残基将发生突变，并通过 X 射线晶体学研究产生的蛋白质和蛋白质/配体复合物。将在基于 NMR 的体内测定中筛选突变体的催化活性，以监测 13C 标记的 AI-2 的摄取率。这里提出的工作旨在从结构和功能上表征负责降解 P-AI-2 的蛋白质，因此在开发利用 AI-2 介导的群体感应作为控制细菌的手段的疗法方面可能非常有用。 公共卫生相关性：细菌，包括许多人类病原体，通过微小的化学信号进行交流，以全人群的方式协调毒力和生物膜形成等行为。这项工作研究了参与处理称为 autoinducer-2 的种间信号分子的蛋白质的结构和功能。了解这种信号分子的处理是利用这种通讯来控制细菌行为的重要一步。