Nanomechanics of bacterial adhesion

 描述(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附着在感染部位，例如粘液流动和在粘膜中咳嗽，或在口腔中咀嚼和刷牙引起的扰动。虽然在细菌中已经描述了几种黏附结构，但对细菌-宿主黏附部位高机械耐受性的分子机制知之甚少。主要原因是缺乏可以探测受力下的粘连连接的经典批量实验，这极大地限制了我们对体内连接的了解，更重要的是，阻止了我们开发针对病原体粘连的药物。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连连接的行为，所有这些都是在生理上相关的机械扰动下进行的。我们将考虑涉及三种革兰氏阳性生物菌毛(菌毛)的各种类型的粘附性相互作用：白喉棒状杆菌(白喉)、无乳链球菌(出生前感染)和口腔放线菌(牙菌斑)。革兰氏阳性细菌是独一无二的，因为他们的菌毛组装成单个连续的多肽，由重复的折叠单位组成，可以生长到几微米长。目前尚不清楚这种大小的单个串联模块蛋白质如何经受住巨大的机械力。拟议的新的单分子分析基于最新的里程碑式技术，这些技术允许将蛋白质可靠地机械连接到表面，并基于我们使用力光谱仪器研究蛋白质力学的丰富经验，包括使用AFM和磁镊子。我们的目标是识别粘连连接的“阿喀琉斯跟腱”，即那些对体内粘连连接的耐久性至关重要的分子元件。我们将测量它们的机械性能，以及它们如何在细菌菌毛中成熟为完全功能的元素。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分析来研究机械稳定的二硫键是如何在革兰氏阳性细菌的菌毛中引入和修饰的。我们还将结合我们的HaloTag共价锚定和磁性镊子，对活着的细菌中完整菌毛的机制进行一天的记录。我们的发现将被用来构建革兰氏阳性菌毛的计算模型，该模型结合了所有已识别的“阿喀琉斯跟腱” 并将它们与延伸多肽的物理学相结合。我们将把布朗动力学应用到我们的模型中，以预测菌毛在咳嗽等生理刺激下的机械行为。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附着在目标组织上的能力来发挥作用。鉴于对当前几类抗生素具有抗药性的细菌的快速生长，开发新的方法来阻止细菌感染是一项对社会具有重要意义的紧迫努力。

 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.