Mechanochemistry of gram-positive bacterial adhesins - towards the rational design of anti-invasive strategies

在感染开始时，细菌利用被称为皮利的长毛状附属物附着在宿主的组织上。这些皮利是由百分之一的菌毛蛋白连续连接而成的，其被直接与靶细胞中暴露的配体相互作用的尖端粘附素蛋白封端。为了分离细菌和防止感染，宿主会用一系列防御机制做出反应，比如咳嗽或打喷嚏，这些机制会挑战细菌建立的机械系链，使单个菌毛蛋白受到会展开任何已知蛋白质的力。然而，为了克服这些挑战，细菌已经进化出独特的化学特性，赋予它们的菌毛蛋白特殊的机械性能，使它们能够在这些强大的压力下保持牢固的附着。例如，尖端菌毛蛋白通常含有罕见的内部硫酯键，据称该硫酯键使它们能够与宿主细胞建立共价且持久的相互作用。类似地，轴菌毛蛋白质配备有独特的内部异肽键，其为这些蛋白质提供出色的机械稳定性。由于这些原因，菌毛蛋白是公认的毒力因子，并已成为开发新的抗菌药物的诱人目标，特别是鉴于越来越多的耐药细菌的威胁。然而，开发靶向菌毛蛋白机制的药物需要了解这些蛋白质在大机械力下的行为，这是经典的结构或生化技术无法完成的。这需要实施新的实验方法来测量毛蛋白的机械性能，为合理设计新型抗粘连化合物提供基础依据。 在这里，我们提出了一个多尺度的研究计划，以解开尖端和轴菌毛蛋白的机械化学性质，开发抗粘附化合物，可以消除其机械性能，防止入侵。我们将首先使用磁镊力谱来探测两种细菌病原体S。化脓性（坏死性筋膜炎）和S. pneumoniae（肺炎），设计阻断这种键的肽。其次，我们将研究三种含异肽键的细菌的毛杆蛋白-S。mutans（dental cavities）、S. pneumoniae和C. diphteria（diphteria）-和设计模拟肽阻断这些异肽键。最后，我们将在活细菌上测试这些抗粘附策略，评估当用封闭肽处理时，这些病原体如何在力的作用下粘附。由于牵头实验室和合作实验室的综合专业知识，开展这项研究是可能的。牵头实验室将带来其在蛋白质纳米力学和磁镊力谱方面的专业知识，提供一种独特的实验方法来测量这些蛋白质的力量;合作实验室将贡献其生物化学专业知识和细菌工作经验。总的来说，我们将开发一个创新的研究计划，这将大大有助于我们对细菌粘附的基本理解，进一步为开发新一代靶向菌毛蛋白纳米力学的抗菌药物提供基础。

At the onset of an infection, bacteria attach to the host's tissue using long hair-like appendages, dubbed pili. These pili are built by the sequential concatenation of hundredths of pilin proteins, capped by a tip adhesin protein that directly interacts with ligands exposed in the target cell. Aiming to detach bacteria and prevent infection, the host responds with a battery of defense mechanisms-such as coughing or sneezing- that challenge the mechanical tether established by the bacterium, subjecting the individual pilin proteins to forces that would unfold any known protein. However, to overcome these challenges, bacteria have evolved unique chemical traits that confer their pilin proteins with exceptional mechanical properties allowing them to remain firmly attached despite these formidable stresses. For example, the tip pilins often contain a rare internal thioester bond that allegedly enables them to establish a covalent and long-lasting interaction with the host cells. Similarly, shaft pilin proteins are equipped with unique internal isopeptide bonds that provide these proteins with outstanding mechanical stability. For these reasons, pilin proteins are recognized virulence factors and have become an enticing target for developing new antibacterial drugs, particularly in light of the increasing threat of antibiotic-resistant bacteria. However, developing drugs that target pilin mechanics requires understanding how these proteins behave under large mechanical forces, which cannot be done with classic structural or biochemical techniques. This requires implementing new experimental methods to measure the mechanical properties of pilin proteins, providing the fundamental basis for the rational design of new antiadhesive compounds. Here, we propose a multiscale research program to unravel the mechanochemical properties of tip and shaft pilin proteins to develop anti-adhesive compounds that could obliterate their mechanical properties and prevent invasion. We will first use magnetic tweezers force spectroscopy to probe the mechanics of the thioester in the tip pilin of two bacterial pathogens, S. pyogenes (necrotizing fasciitis) and S. pneumoniae (pneumonia), designing peptides that block this bond. Second, we will study the shaft pilins of three bacteria containing isopeptide bonds-S. mutans (dental cavities), S. pneumoniae, and C. diphteria (diphteria)-and design mimicking peptides that block these isopeptide bonds. Finally, we will test these anti-adhesive strategies on living bacteria, evaluating how these pathogens attach under force when treated with the blocking peptides. Conducting this research will be possible thanks to the combined expertise of the lead and partner laboratories. The lead laboratory will bring its expertise in protein nanomechanics and magnetic tweezers force spectroscopy, providing a unique experimental approach to measure these proteins under force; the partner laboratory will contribute with its biochemistry expertise and experience working with bacteria. Overall, we will develop an innovative research program that will significantly contribute to our fundamental understanding of bacterial adhesion, further providing the ground basis for developing a novel generation of anti-bacterial drugs targeting pilin nanomechanics.