Elucidating the molecular targets of bacterial nano-syringes

第一年结束时确认：MIBTP学生在第一年接受一段时间的培训。这包括统计、编程、数据分析、人工智能和迷你研究项目中的必修教学模块。确认项目：阐明细菌纳米注射器的分子目标细菌与其宿主之间的进化“军备竞赛”导致了一系列复杂的策略和机制，这些策略和机制能够操纵宿主细胞，往往导致致命性。近年来，特别是自(低温)电子显微镜领域的“分辨率革命”以来，人们已经清楚地看到，这些机制可以以大分子复合体的形式以及更“常规”的外毒素等形式变得非常复杂。在这些由细菌衍生的大分子复合体中，最优雅和最著名的是各种“分泌系统”，特别是类型3和类型6，它们有效地充当嵌入细菌细胞壁的‘鱼叉’，介导生物材料的细胞间转移。最近，人们发现了起到纳米级“鱼雷”或注射器作用的结构，这些结构由细菌释放到细胞外环境中，能够“远距离”发挥作用。这些所谓的“毒力盒”最初是在沙雷氏菌和梭菌属细菌中发现的，它们是由大约16个基因组成的短基因簇，包含生产、自发自组装和装载(通常是有毒的)货物所需的一切。这些“纳米注射器”在进化上与噬菌体有关，它们的功能非常相似：一旦在细胞外环境中游离，它们就会扩散并‘寻找’目标细胞。盒式磁带中的一个基因产生了一种所谓的尾部纤维蛋白，它类似于噬菌体，负责与相关的细胞表面标记/受体结合。一旦被结合，纳米注射器载体就会收缩，一层蛋白质内鞘被推出来刺穿细胞屏障。随着细胞屏障的穿透，纳米注射器载体中包含的有效载荷被释放到细胞胞浆中。有待阐明的关键特征之一是“纳米注射器”如何与靶细胞相互作用--它们的尾部纤维准确地结合到靶细胞膜上的什么？到目前为止的工作表明，可能的候选者包括细胞表面(糖蛋白)、特定的糖脂成分，或者可能是两者的某种组合。这个合作项目将把华威大学在生产和分析“纳米注射器”尾部蛋白质方面的专业知识与阿斯顿大学的细胞膜专业知识结合起来。特别是，将使用诸如SMALP(苯乙烯、马来酸、脂质颗粒)等聚合物脂颗粒方法来识别与纯化的尾蛋白结合的膜成分。这些脂质颗粒很重要，因为它们将在天然的脂质双层环境中呈现膜蛋白，代表纳米注射器的所有可能的分子靶点。然后，将对与尾部蛋白结合的成分进行更详细的鉴定和研究，以研究相互作用的亲和力和特异性。新的衍生公司Nanosyrinx正在利用这些PVC作为潜在的生物技术和医疗保健应用的输送载体。在理解目标和设计/重定目标这一系统的能力方面取得的进展将立即产生翻译影响。

To be confirmed at end of year 1: MIBTP students undertake a period of training during their first year. This includes compulsory taught modules in statistics, programming, data analysis, AI and mini Research projects.Confirmed project: Elucidating the molecular targets of bacterial nano-syringesThe evolutionary 'arms race' between bacteria and their hosts has led to a wide range of sophisticated strategies and mechanisms which enable manipulation of host cells, often resulting in lethality. In recent years, particularly since the "Resolution Revolution" in the field of (cryo) Electron Microscopy, it has become apparent that these mechanisms can be extremely elaborate in the form of large macromolecule complexes, as well as more 'conventional' exotoxins etc.Among the most elegant and well known of these bacterially-derived macromolecular complexes are the various "Secretion Systems", particularly the Type 3 and Type 6, which effectively function as 'harpoons' embedded in the cell wall of the bacterium, which mediate cell-to-cell transfer of biologic material. More recently, structures which function as nano-scale 'torpedos' or syringes have been discovered, which are released by the bacteria into the extracellular milieu and are capable of acting 'at a distance'. First discovered in Serratia and Photorhabdus species of bacteria, these so-called "Virulence Cassettes" are short gene clusters of approximately 16 genes, which contain everything necessary to produce, spontaneously self-assemble, and load with (typically toxic) cargoes, these remarkable entities. These 'nano-syringes' are evolutionarily related to bacteriophages, and function in a very similar way: Once free in the extracellular environment, they will diffuse and 'seek out' a target cell. One of the genes within the cassette gives rise to a so-called 'tail fibre' protein which, similar to bacteriophage, is responsible for binding to the relevant cell surface marker/receptor. Once bound, the nanosyringe vehicle contracts and an inner sheath of protein is propelled out to puncture the cell barrier. With the cell barrier pierced, the payloads contained within the nanosyringe vehicle are released into the cell cytosol.One of the key features remaining to be elucidated is how the 'nano-syringes' interact with the target cell - what in the target membrane do their tail fibres bind to precisely? Work to date suggests possible candidates include cell surface (glyco)proteins, specific glycolipid components, or potentially some combination of the two. This collaborative project will combine expertise from the University of Warwick in producing and assaying the 'nano-syringe' tail proteins, with membrane expertise at Aston University. In particular polymer lipid particle approaches such as SMALPs (styrene maleic acid lipid particles) will be utilised in order to identify membrane components that bind to the purified tail proteins. These lipid particles are important as they will present membrane proteins within a native lipid bilayer environment, representing all possible molecular targets of the nano-syringes. Components that bind to the tail proteins will then be identified and investigated in more detail to study the affinity and specificity of the interaction.These PVCs are being exploited by the new spinout company Nanosyrinx as potential delivery vehicles for biotechnological and healthcare applications. Advances made in understanding the targeting, and capacity to engineer/retarget this system will have immediate translational impact.