Contact-Dependent Signaling in Burkholderia

伯克霍尔德杆菌中的接触依赖性信号传导

• 批准号: 9197358
• 负责人: Peggy A Cotter
• 金额: $ 29.57万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2020-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9197358
• 关键词: Alleles; Animals; Antibiotics; Bacteria; Behavior; Binding; Binding Proteins; Biochemical; Burkholderia; C-terminal; Cell Death; Cells; Cleaved cell; Communities; Competitive Behavior; Complex; Contact Inhibition; Control Locus; Cooperative Behavior; Cytoplasm; DNA; Data; Development; Disease; Environment; Enzymes; Gene Expression; Genes; Genetic; Genetic Epistasis; Gram-Negative Bacteria; Growth; Human; Immunity; Immunoblotting; Immunoprecipitation; In Vitro; Insecta; Life; Mass Spectrum Analysis; Measures; Mediating; Medical Device; Membrane; Microbial Biofilms; Modeling; Molecular; Mutagenesis; Nucleotides; Plant Roots; Production; Proteins; Proteobacteria; RNA; Reaction; Regulon; Role; Second Messenger Systems; Shellfish; Shipping; Ships; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Structure; Surface; System; Transducers; abstracting; base; hookah; killings; member; mutant; novel therapeutics; nuclease; pathogenic bacteria; polypeptide; prevent; reconstitution; research study; second messenger; transcriptome sequencing; transmission process

Abstract: Bacteria typically live in complex sociomicrobiological communities, often as biofilms, on surfaces as diverse as water pipes, ship hulls, plant roots, insects, shellfish, indwelling medical devices, and mucosal surfaces. Biofilm growth in humans can cause or exacerbate disease, and biofilm growth in environmental niches can facilitate transmission of pathogenic bacteria to humans and other animals. Understanding how bacteria recognize, cooperate and compete with their neighbors in diverse environments is critical for developing strategies to control microbiological community composition, to prevent biofilm development, and to eliminate pre-existing biofilms and their consequent diseases. Contact-Dependent Growth Inhibition (CDI) is a phenomenon in which bacteria use the toxic C-terminus of a large exoprotein to kill or inhibit the growth of neighboring bacteria upon cell-cell contact. Production of a small immunity protein protects bacteria against CDI. Using the Gram-negative bacterium Burkholderia thailandensis as a model, we have discovered that in addition to using CDI system proteins to kill their neighbors, bacteria can use these proteins for signal transduction, causing a change in gene expression that leads to the production of cooperative behaviors, such as biofilm formation, when neighboring bacteria are recognized as ‘self’. Here, we propose experiments to determine the molecular mechanisms underlying this Contact-Dependent Signaling (CDS) phenomenon. Our preliminary data support a model in which the C-terminus of the large BcpA exoprotein is delivered into neighboring bacteria where it forms a ‘CDS complex’ that catalyzes a reaction that increases or decreases the concentration of a signal molecule, which may be a small nucleotide second messenger. Experiments described in Aim 1 will define the CDS regulatory network and identify the signal molecule, which will help us identify the enzymatic activity of the CDS complex. Experiments described in Aim 2 will identify the members of the CDS complex. We will reconstitute the CDS complex in vitro using purified proteins and will measure its catalytic activity. We have discovered that a small protein called BcpO is required for CDS. We proposed experiments in Aim 3 to determine if BcpO localizes to the inner leaflet of the outer membrane, as predicted, and if that localization is important for its function. We will also identify proteins with which BcpO interacts so that we can understand how it functions in CDS.

摘要： 细菌通常生活在复杂的社会微生物群落中，通常以生物膜的形式存在于表面 种类繁多，如水管、船体、植物根、昆虫、贝类、留置医疗器械和 粘膜表面。人体内生物膜的生长可导致或加重疾病，而生物膜的生长 在环境中的壁龛可以促进致病菌传播给人类和其他 动物。了解细菌如何识别、合作和与邻居竞争 多样化的环境对于制定控制微生物群落的策略至关重要 成分，以防止生物膜的发展，并消除先前存在的生物膜及其 随之而来的疾病。接触依赖性生长抑制(CDI)是一种现象， 细菌利用一个大的外源蛋白的有毒C末端来杀死或抑制邻近细菌的生长 细菌在细胞间的接触。一种小免疫蛋白的产生可以保护细菌免受CDI的侵袭。 以革兰氏阴性杆菌泰兰伯克霍尔德氏菌为模型，我们发现 除了使用CDI系统蛋白质杀死它们的邻居外，细菌还可以使用这些蛋白质 对于信号转导，导致基因表达的变化，从而导致产生 合作行为，如形成生物膜，当邻近的细菌被识别为 “自我”。在这里，我们建议进行实验以确定其背后的分子机制。 接触依赖信号(CDS)现象。我们的初步数据支持这样一个模型 大的BCPA外蛋白的C末端被输送到邻近的细菌中，在那里它形成了一个 ‘CDS复合体’，催化增加或降低信号浓度的反应 分子，这可能是一个小核苷酸第二信使。目标1中描述的实验 将定义CDS调控网络并识别信号分子，这将有助于我们识别 CDS复合体的酶活性。目标2中描述的实验将确定 CDS情结的成员。我们将使用纯化的蛋白在体外重建CDS复合体 并将测量其催化活性。我们已经发现一种名为BcpO的小蛋白质 CDS需要。我们在目标3中提出了实验，以确定BcpO是否定位于内部 外膜的小叶，如预测的那样，以及这种定位对其功能是否重要。我们 还将识别BcpO与之相互作用的蛋白质，以便我们能够了解它在 CD。