Elucidating the molecular targets of bacterial nano-syringes

在第一年结束时确认：MIBTP学生在第一年接受一段时间的培训。这包括统计学、编程、数据分析、人工智能和微型研究项目的必修教学模块。已确认的项目：阐明细菌纳米生物学的分子靶点细菌与其宿主之间的进化“军备竞赛”导致了广泛的复杂策略和机制，这些策略和机制能够操纵宿主细胞，通常导致致命性。近年来，特别是自从（冷冻）电子显微镜领域的“分辨率革命”以来，已经变得明显的是，这些机制可以以大分子复合物以及更“常规”外毒素等的形式极其复杂。在这些细菌衍生的大分子复合物中，最优雅和众所周知的是各种“分泌系统”，特别是3型和6型，其有效地起到嵌入细菌细胞壁中的“鱼叉”的作用，其介导生物材料的细胞间转移。最近，已经发现了用作纳米级“鱼雷”或注射器的结构，这些结构由细菌释放到细胞外环境中，并且能够“在一定距离”起作用。首先在沙雷氏菌属和光杆状菌属的细菌中发现，这些所谓的“毒力Cascadium”是大约16个基因的短基因簇，其中包含产生、自发自组装和装载（通常是有毒的）货物所需的一切，这些非凡的实体。这些“纳米注射器”在进化上与噬菌体有关，并且以非常相似的方式发挥作用：一旦在细胞外环境中自由，它们将扩散并“寻找”靶细胞。盒中的一个基因产生所谓的“尾纤维”蛋白，其类似于噬菌体，负责结合相关的细胞表面标记/受体。一旦结合，纳米注射器载体收缩，蛋白质的内鞘被推出以刺穿细胞屏障。随着细胞屏障被刺穿，纳米注射器载体中的有效载荷被释放到细胞胞质溶胶中。其中一个尚待阐明的关键特征是“纳米注射器”如何与靶细胞相互作用--它们的尾纤维精确地结合在靶细胞膜的什么地方？迄今为止的工作表明，可能的候选者包括细胞表面（糖）蛋白，特定的糖脂成分，或潜在的两者的某种组合。这个合作项目将联合收割机的专业知识，从沃里克大学在生产和分析的'纳米注射器'尾部蛋白，与膜专业知识在阿斯顿大学。特别地，将使用聚合物脂质颗粒方法如SMALP（苯乙烯马来酸脂质颗粒）以鉴定结合纯化的尾蛋白的膜组分。这些脂质颗粒很重要，因为它们将在天然脂质双层环境中呈递膜蛋白，代表纳米注射器的所有可能的分子靶标。随后将对与尾蛋白结合的组分进行鉴定和更详细的研究，以研究相互作用的亲和力和特异性。这些PVC正被新分拆公司Nanosyrinx开发，作为生物技术和医疗保健应用的潜在递送载体。在了解目标方面取得的进展，以及设计/重新定位这一系统的能力将产生直接的转化影响。

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.