Nanomechanics of bacterial adhesion

 描述（由申请人提供）：细菌已经进化到即使在存在强烈的机械扰动的情况下也能保持附着在感染部位，例如由粘液流动和粘膜咳嗽或口腔中咀嚼和刷牙引起的机械扰动。尽管已经描述了细菌中的几种粘附结构，但对于细菌-宿主粘附位点高机械耐久性的分子机制知之甚少。主要原因是缺乏可以探测受力作用下的粘附连接的经典批量实验，极大地限制了我们对体内连接的理解，更重要的是，阻止了我们开发针对病原菌粘附的药物。在这里，我们建议开发基于强大的机械指纹的新型单分子技术，该技术将明确地探测导致感染的粘合连接的行为，所有这些都在生理相关的机械扰动下进行。我们将考虑涉及三种革兰氏阳性生物菌毛（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）和口放线菌（牙菌斑）。革兰氏阳性细菌是独特的，因为它们的菌毛被组装成重复折叠单元的单个连续多肽，长度可以长到几微米。目前尚不清楚这种大小的单个串联模块蛋白如何承受巨大的机械力。拟议的新单分子测定基于最新的里程碑技术，这些技术允许将蛋白质可靠地机械束缚到表面，以及我们使用力谱仪器（包括 AFM 和磁镊）研究蛋白质力学的丰富经验。我们的目标是确定粘合连接的“阿喀琉斯之踵”，即对体内连接持久性至关重要的那些分子元素。我们将测量它们的机械特性以及它们如何成熟为细菌菌毛中的全功能元件。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检查如何在革兰氏阳性细菌的菌毛蛋白中引入和修饰机械稳定的二硫键。我们还将 HaloTag 共价锚与磁性镊子结合起来，对活细菌中完整菌毛的力学进行全天记录。我们的研究结果将用于构建革兰氏阳性菌毛的计算模型，其中包含所有已识别的“致命弱点” 在此提议中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于我们的模型来预测菌毛响应咳嗽等生理冲击的机械行为。我们的模型将作为鉴定一类新型抗生素和疫苗的定量平台，这些抗生素和疫苗通过阻止细菌粘附其目标组织的能力来发挥作用。鉴于对现有抗生素类别具有抗药性的细菌迅速增长，开发阻止细菌感染的新方法是对社会非常重要的紧迫努力。

 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.