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

利用蛋白质输入来询问通过细菌细胞包膜的能量转导

• 批准号: BB/X016366/1
• 负责人: Colin Kleanthous
• 金额: $ 83.45万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FX016366%2F1
• 关键词: Exploiting; protein; import; interrogate; energy

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 This recent work, much of it from Oxford and unpublished, has identified several critical pieces of information.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.

细菌既是朋友又是敌人。据估计，我们的身体含有比人体细胞更多的细菌细胞（微生物组）。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至关重要。然而，许多细菌是疾病的病原体。即使是那些我们体内的宿主，当暴露在不同的环境中时，也会成为机会性病原体，就像手术期间发生的那样。细菌种类中抗生素耐药性的增加使这种情况更加频繁，提高了前景，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都将变得危险。本申请集中于一组称为细菌素的蛋白质分子。这些是细菌在与邻居进行战争以获得更多资源时产生的天然抗菌剂。细菌素有可能被重新配置以杀死病原菌，但仍需要了解它们的作用模式。本提案中研究的细菌素靶向革兰氏阴性细菌，换句话说，具有两层膜的细菌。外膜是一种独特的不对称脂质双层，它排除了许多类对革兰氏阳性菌有活性的抗生素，而革兰氏阳性菌缺乏这种额外的膜。因此，外膜是革兰氏阴性菌在抗生素耐药性方面最成问题的原因之一。近年来，我们对细菌素杀灭革兰氏阴性菌，特别是大肠杆菌的作用机制有了更多的了解。coli、铜绿假单胞菌和克雷伯氏菌。肺炎。这一最新的工作，其中大部分来自牛津大学和未发表，已经确定了几个关键的信息。首先，细菌素通常使用称为β-发夹的结构基序在运输之前停靠在细菌表面。其次，细菌素有可能输入比外膜的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量来源，细菌素催化它们穿过外膜的运输，甚至携带货物分子，如DNA和有机分子。我们将利用这些发现来理解细菌素β-发夹与其孔蛋白受体结合的结构基础。我们还将使用我们设计的比正常大得多的细菌素，以便能够将它们附着在聚苯乙烯珠上，这样我们就可以在真实的时间内可视化单个细菌素的输入。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送穿过外膜的分子机制。开发这些新的进口分析也将告诉我们仍然笼罩在神秘之中的能量转导系统本身。最后，通过实现这些目标，我们将为理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。