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

革兰氏阳性细菌粘附素的机械化学——合理设计抗侵入策略

• 批准号: EP/Y001125/1
• 负责人: Rafael Tapia-Rojo
• 金额: $ 20.14万
• 依托单位: King's College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FY001125%2F1
• 关键词: Mechanochemistry; gram; positive; bacterial; adhesins

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.

感染开始时，细菌利用长发状的附属物（称为菌毛）附着在宿主的组织上。这些菌毛是由数百个菌毛蛋白顺序串联而成，顶端粘附素蛋白与靶细胞中暴露的配体直接相互作用。为了分离细菌并防止感染，宿主通过一系列防御机制（例如咳嗽或打喷嚏）做出反应，这些机制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，细菌进化出了独特的化学特性，赋予它们的菌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，尖端菌毛蛋白通常含有罕见的内部硫酯键，据称使它们能够与宿主细胞建立共价且持久的相互作用。同样，轴菌毛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，菌毛蛋白被认为是毒力因子，并已成为开发新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，开发针对菌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，而这无法通过经典的结构或生化技术来完成。这就需要实施新的实验方法来测量菌毛蛋白的机械特性，为新型抗粘附化合物的合理设计提供基础依据。 在这里，我们提出了一项多尺度研究计划，以揭示尖端和轴菌毛蛋白的机械化学特性，以开发可以消除其机械特性并防止入侵的抗粘附化合物。我们将首先使用磁镊力谱来探测两种细菌病原体——化脓性链球菌（坏死性筋膜炎）和肺炎链球菌（肺炎）尖端菌毛中硫酯的机制，设计阻断这种键的肽。其次，我们将研究三种含有异肽键-S的细菌的菌毛轴。变形链球菌（蛀牙）、肺炎链球菌和白喉杆菌（白喉），并设计阻断这些异肽键的模拟肽。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作用下粘附。得益于牵头实验室和合作伙伴实验室的综合专业知识，这项研究的开展才得以实现。牵头实验室将利用其在蛋白质纳米力学和磁镊力谱方面的专业知识，提供独特的实验方法来测量受力下的这些蛋白质；合作伙伴实验室将贡献其生物化学专业知识和细菌研究经验。总体而言，我们将开发一项创新研究计划，该计划将极大地促进我们对细菌粘附的基本理解，进一步为开发针对菌毛纳米力学的新一代抗菌药物奠定基础。