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

在感染开始时，细菌利用长长的毛发状附属物附着在宿主的组织上，这种附属物被称为菌毛。这些菌毛是由百分之几的菌毛蛋白连续连接而成，顶端附着蛋白直接与暴露在靶细胞中的配体相互作用。为了分离细菌和防止感染，宿主用一系列的防御机制来回应——比如咳嗽或打喷嚏——挑战细菌建立的机械链，使单个的pilin蛋白受到能够展开任何已知蛋白质的力。然而，为了克服这些挑战，细菌进化出了独特的化学特征，赋予它们的毛蛋白特殊的机械特性，使它们能够在这些强大的压力下保持牢固的附着。例如，尖端支柱通常含有一种罕见的内部硫酯键，据称这种键使它们能够与宿主细胞建立共价和持久的相互作用。同样，轴柱蛋白具有独特的内部异肽键，为这些蛋白质提供了出色的机械稳定性。由于这些原因，匹林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，特别是考虑到抗生素耐药细菌的威胁日益增加。然而，开发针对毛蛋白力学的药物需要了解这些蛋白质在大机械力下的行为，这是传统的结构或生化技术无法做到的。这就需要采用新的实验方法来测量pilin蛋白的力学性能，为新型抗粘剂的合理设计提供基础依据。在这里，我们提出了一个多尺度的研究计划来揭示尖端和轴柱蛋白的机械化学特性，以开发抗粘附化合物，从而消除它们的机械特性并防止入侵。我们将首先使用磁镊子力谱来探测两种细菌病原体，化脓性葡萄球菌（坏死性筋膜炎）和肺炎葡萄球菌（肺炎）尖端柱中的硫酯的机制，设计阻断这种结合的肽。其次，我们将研究三种含有异肽键- s的细菌的轴柱。变异菌（蛀牙），肺炎链球菌和白喉链球菌（白喉），并设计模拟肽来阻断这些异肽键。最后，我们将在活细菌上测试这些抗粘附策略，评估这些病原体在用阻断肽处理时如何在力下附着。由于牵头实验室和合作伙伴实验室的综合专业知识，开展这项研究将成为可能。牵头实验室将带来其在蛋白质纳米力学和磁镊子力光谱方面的专业知识，提供一种独特的实验方法来测量这些受力的蛋白质；合作伙伴实验室将贡献其生物化学专业知识和处理细菌的经验。总的来说，我们将开发一个创新的研究项目，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针对青霉素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.