Exploiting protein import to interrogate energy transduction through the bacterial cell envelope

细菌既是朋友，也是敌人。据估计，我们的身体含有比人类细胞更多的细菌细胞(微生物群)。因此，细菌对健康生活至关重要；例如，肠道细菌对食物的消化是必不可少的。然而，许多细菌都是疾病的病原体。即使是我们体内的宿主，当暴露在不同的利基环境中时，也可能成为机会性病原体，就像在手术中发生的那样。细菌对抗生素耐药性的增加使这种情况变得更加频繁，这增加了这样一种可能性，即在未来20-30年内，由于缺乏有效的抗生素，常规的医院程序甚至分娩将变得危险。本申请集中在一组称为细菌素的蛋白质分子上。这些是自然产生的抗菌剂，细菌在与邻国进行战争以获得更多资源的过程中产生这些抗菌剂。细菌素有可能被重新配置来杀死病原菌，但关于它们的作用模式仍有许多需要了解。这项建议中研究的细菌素针对的是革兰氏阴性细菌，换句话说，即有两层膜的细菌。外膜是一种独特的不对称脂质双层，它排除了许多种类的抗生素，这些抗生素对革兰氏阳性菌有活性，而革兰氏阳性菌缺乏这一额外的膜。因此，外膜是革兰氏阴性菌在抗生素耐药性方面最有问题的原因之一。近年来，我们对细菌素杀死革兰氏阴性菌的潜在作用机制有了很多了解，特别是针对大肠杆菌、铜绿假单胞菌和肺炎克雷伯菌的细菌素。这项最近的工作，其中大部分来自牛津大学，尚未出版，确定了几个关键信息。首先，细菌素通常使用一种称为β-发夹的结构基序，在运输之前停靠在细菌表面。其次，细菌素有可能进口比外膜渗透性过滤器通常允许的质量大得多的质量(1000倍)。这些过滤器是被称为孔蛋白的蛋白质。第三，细菌素的这种特性与它们利用细胞能量的能力有关，细胞能量与细菌的内膜有关。通过利用这种被称为质子动力的能源，细菌素催化它们穿过外膜的运输，甚至携带DNA和有机分子等货物分子。我们将利用这些发现来了解细菌素β-发夹与其孔蛋白受体联系的结构基础。我们还将使用细菌素，我们设计的细菌素比普通细菌素大得多，能够将它们附着在聚苯乙烯珠子上，这样我们就可以实时可视化单个细菌素的进口。通过这样做，我们可以开始询问输入的能量学，换句话说，细菌素利用质子动力穿过内膜，将自己运输到外膜的分子机制。开发这些新的进口检测方法也将告诉我们仍然笼罩在神秘中的能量转导系统本身。最后，通过实现这些目标，我们将为了解外膜对细菌素进入的限制奠定基础，以便我们能够适当地设计这些未来的抗菌剂。

Bacteria are both friend and foe. It is estimated that our bodies contain more bacterial cells (the microbiome) than human cells. Bacteria are therefore vital to healthy living; for example, gut bacteria are essential to the digestion of food. However, many bacteria are causative agents of disease. Even those we host in our bodies can become opportunistic pathogens when exposed to different niches, as happens during surgery. The rise of antibiotic resistance amongst bacterial species has made this occurrence all the more frequent, raising the prospect that in the next 20-30 years routine hospital procedures and even giving birth will become hazardous because of the lack of effective antibiotics. The present application focuses on a group of protein molecules known as bacteriocins. These are naturally-occurring antimicrobials that bacteria produce during the warfare they wage with their neighbours to gain greater access to resources. Bacteriocins have the potential to be reconfigured to kill pathogenic bacteria but much still needs to be understood as to their mode of action. The bacteriocins investigated in this proposal target Gram-negative bacteria, in other words bacteria that have two membranes. The outer membrane is a unique asymmetric lipid bilayer that excludes many classes of antibiotics that are active against Gram-positive bacteria, which lack this additional membrane. Hence, the outer membrane is one of the reasons why Gram-negative bacteria are some of the most problematic in terms of antibiotic resistance. In recent years, we have learnt much about the underlying mechanisms of action of bacteriocins that kill Gram-negative bacteria, especially those that target E. coli, P. aeruginosa and K. pneumoniae. This recent work, much of it from Oxford and unpublished, has identified several critical pieces of information. First, bacteriocins often use a structural motif known as a beta-hairpin to dock onto the surface of the bacterium prior to transport. Second, bacteriocins have the potential to import significantly greater mass (1000x) than is normally permitted by the permeability filters of the outer membrane. These filters are proteins known as porins. Third, this property of bacteriocins is linked to their ability to tap into the energy of the cell, which is associated with the inner membrane of the bacterium. By tapping into this energy source, known as the proton motive force, bacteriocins catalyse their transport across the outer membrane, even carrying cargo molecules such as DNA and organic molecules.We will exploit these discoveries to understand the structural basis for bacteriocin beta-hairpin association with their porin receptors. We will also use the bacteriocins we've engineered to be much larger than normal to be able to attach them to polystyrene beads so that we can visualise the import of single bacteriocins in real time. By doing so, we can begin to interrogate the energetics of import, in other words the molecular mechanisms by which bacteriocins harness the proton motive force across the inner membrane to transport themselves across the outer membrane. Developing these new import assays will also tell us about the energy transduction systems themselves which are still shrouded in mystery. Finally, by achieving these goals we'll be laying the foundations for understanding the constraints the outer membrane places on bacteriocin entry so that we can engineer these future antimicrobials appropriately.