Nanomechanics of bacterial adhesion

细菌粘附的纳米力学

• 批准号: 9145721
• 负责人: Julio M Fernandez
• 金额: $ 35.75万
• 依托单位: COLUMBIA UNIV NEW YORK MORNINGSIDE
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9145721
• 关键词: Actinomyces Infections; Actinomycetales Infections; Adherence; Adherens Junction; Adhesions; Adhesives; Antibiotics; Bacteria; Bacterial Adhesins; Bacterial Adhesion; Bacterial Infections; Bacterial Pili; Behavior; Benchmarking; Beryllium; Biochemistry; Biogenesis; Biological Assay; Cell Adhesion; Cells; Computer Simulation; Corynebacterium diphtheriae; Coughing; Dental Plaque; Development; Drug Targeting; Engineering; Environment; Extracellular Protein; Fimbriae Proteins; Fingerprint; Free Energy; Funding; Goals; Gram-Positive Bacteria; Grant; Health; In Vitro; Indium; Infection; Intervention; Kinetics; Laboratories; Lead; Length; Life; Ligands; Magnetism; Mastication; Measures; Mechanics; Memory; Methods; Modeling; Molecular; Mucous Membrane; Mucous body substance; Oral cavity; Organism; Pharmacology; Physics; Physiological; Pilum; Polyproteins; Property; Proteins; Proxy; Resolution; Role; Shock; Site; Sneezing; Societies; Source; Spectrum Analysis; Stimulus; Streptococcus Group B; Structure; Surface; Techniques; Technology; Time; Tissues; Vaccines; Work; antimicrobial; bacterial resistance; base; disulfide bond; experience; fimbria; in vivo; instrumentation; mechanical behavior; mouse model; nanomechanics; nanometer; novel; novel strategies; pathogenic bacteria; physical model; polypeptide; prevent; rapid growth; research study; resilience; response; single molecule; success

 DESCRIPTION (provided by applicant): Bacteria have evolved to remain attached to infection sites even in the presence of strong mechanical perturbations, such as those induced by mucus flow and coughing in the mucosa, or chewing and brushing in the mouth. Although several adhesive structures have been described in bacteria, very little is known about the molecular mechanisms responsible for the high mechanical endurance of bacteria-host adhesion sites. The main reason is the absence of classical bulk experiments that can probe adhesive junctions under force, greatly limiting our understanding of junctions in vivo and, more importantly, preventing us from developing drugs that target adhesion of pathogenic bacteria. Here, we propose to develop novel single-molecule techniques based on robust mechanical fingerprints that will unambiguously probe the behavior of adhesive junctions that lead to infection, all under physiologically relevant mechanical perturbations. We will consider various types of adhesive interactions involving the pili (fimbriae) of three gram-positive organisms: Corynebacterium diphtheriae (diphtheria), Streptococcus agalactiae (pre-natal infections), and Actinomyces oris (dental plaques). Gram positive bacteria are unique because their pili are assembled as a single continuous polypeptide of repeating folded units that can grow up to several micrometers in length. It is unknown how a single tandem modular protein of that size can withstand large mechanical forces. The proposed new single-molecule assays are based on recent milestone technologies that allow reliable mechanical tethering of proteins to surfaces and on our extensive experience studying the mechanics of proteins using force-spectroscopy instrumentation, both with AFM and magnetic tweezers. Our aim is to identify the "Achilles heels" of adhesive junctions, i.e. those molecular elements that are essential to the endurance of the junction in vivo. We will measure their mechanical properties and how they mature into fully functional elements in bacterial pili. For instance, we will use our recently developed singl-molecule oxidative folding and mechanical memory assays to examine how mechanically stable disulfide bonds are introduced and modified in pilins of Gram-positive bacteria. We will also combine our HaloTag covalent anchor with Magnetic tweezers to make daylong recordings of the mechanics of intact pili in living bacteria. Our findings will be used to construct a computational model for gram-positive pili that incorporates all of the "Achilles heels" identified in this proposal and combines them with the physics of an extending polypeptide. We will use Brownian Dynamics applied to our model to predict the mechanical behavior of pili in response to physiological shocks such as coughing. Our model will serve as a quantitative platform for the identification of a novel class of antibiotics and vaccines that work by blocking the ability of bacteria to adhere to their target tissues. Given the rapid growth of bacteria that are resistant t the current classes of antibiotics, developing novel approaches for blocking bacterial infections is an urgent endeavor of great importance to society.

 描述（由申请方提供）：细菌已经进化为即使在存在强烈机械扰动的情况下也能保持附着在感染部位，例如由粘膜中的粘液流动和咳嗽或口腔中的咀嚼和刷牙引起的扰动。尽管已经描述了细菌中的几种粘附结构，但对细菌-宿主粘附位点的高机械耐久性的分子机制知之甚少。主要原因是缺乏经典的批量实验，可以探测力下的粘附连接，极大地限制了我们对体内连接的理解，更重要的是，阻止我们开发针对致病菌粘附的药物。在这里，我们建议开发新的单分子技术的基础上强大的机械指纹，将明确探测的行为，导致感染的粘合剂连接，所有生理相关的机械扰动。我们将考虑涉及三种革兰氏阳性菌的皮利（菌毛）的各种类型的粘附相互作用：白喉棒状杆菌（白喉）、无乳链球菌（产前感染）和口腔放线菌（牙菌斑）。革兰氏阳性细菌是独特的，因为它们的皮利被组装成重复折叠单元的单个连续多肽，其长度可长至几微米。目前还不清楚这种大小的单个串联模块蛋白如何承受大的机械力。提出的新的单分子测定是基于最近的里程碑式的技术，允许可靠的蛋白质的机械拴系到表面和我们的广泛经验，研究蛋白质的力学使用力光谱仪器，无论是与原子力显微镜和磁镊子。我们的目的是确定“阿喀琉斯之踵”的粘合剂连接，即那些分子元素，是必不可少的耐久性的连接在体内。我们将测量它们的机械特性以及它们如何在细菌皮利中成熟为完全功能的元件。例如，我们将使用我们最近开发的单分子氧化折叠和机械记忆测定来研究如何在革兰氏阳性菌的菌毛中引入和修饰机械稳定的二硫键。我们还将联合收割机结合我们的HaloTag共价锚与磁性镊子，使一整天的记录力学完整的皮利在活细菌。我们的研究结果将被用来构建一个计算模型的革兰氏阳性皮利，包括所有的“阿基里斯脚跟”确定 并将它们与延伸多肽的物理学相结合。我们将使用布朗动力学应用到我们的模型来预测皮利的机械行为，以响应生理冲击，如咳嗽。我们的模型将作为一个定量平台，用于识别一类新型抗生素和疫苗，这些抗生素和疫苗通过阻断细菌粘附到其靶组织的能力来发挥作用。鉴于对当前抗生素类别具有抗性的细菌的快速增长，开发用于阻断细菌感染的新方法是对社会非常重要的紧迫奋进。