Understanding the pathogenesis of Burkholderia cenocepacia infections using blocking antibodies & novel chemistry scaffolds to untangle AMR complexity

新绿伯克氏菌是一种机会性革兰氏菌，可感染CF患者和接受化疗的免疫功能低下的癌症患者。在CF b中，洋葱软化引起严重的（不可逆的）肺功能下降，导致危及生命的全身感染，称为洋葱软化综合征。cenocepacia本身对多种抗菌剂具有耐药性（隐藏在细胞内），并表达一系列毒力因子。该博士将开发特定的阻断工具，可用于解除细菌致病武器库的不同组成部分。将详细研究的一种策略是一种系统性耐药机制，它可以中和现有抗生素方法的效力。有趣的是，由于其滥交的性质，这种方法在cenocepacia的部署甚至可以鼓励其他专性或非专性病原体的感染。在cenocepacia芽孢杆菌中，暴露于亚致死浓度的抗生素会诱导化学毒性应激，导致细胞外环境中一组称为脂质体蛋白（我们的重点是BcnA）的保守细菌蛋白的表达和分泌，这些蛋白在抗生素到达细胞内的细菌之前拦截并隔离抗生素。细菌感染策略的这一方面可以通过维生素E等天然化合物部分逆转，维生素E通过似乎是抗氧化和/或通过阻断脂质过氧化作用的机制抑制细菌脂钙素/抗生素结合，从而增加抗菌药物的敏感性（两者都是维生素E的MoAs）。在这里，我们提出了一个更详细和双管齐下的策略来进一步询问和操纵BcnA系统。我们将开发抗BcnA抗体（mab）作为阻止BcnA清除抗生素的工具，同时我们希望在体内（小鼠）这些mab能促进小鼠免疫系统清除和中和BcnA分子。这些单克隆抗体可能还有第三个令人兴奋的用途，即作为诊断探针识别尿液中的BcnA分子，作为感染的早期和特异性指标。同时，我们将使用一种新的超级抗氧化剂（称为Proxison）来确定其保护作用。虽然具有类似MoA的有效分子（500倍Vit E），但目前这是一种生物利用度较差的分子。该项目的一个关键部分将是利用化学来设计和合成更多的生物可利用版本（前药物和生物转化化学），这些版本保留了这种令人印象深刻的效力，但在体外和体内都有提高的效用。本博士的最终目标是将这两种策略的使用结合起来，并确定它们对cenocepacia B.生物学的影响。该项目将以苏格兰生物制剂设施为基础，该设施在重组抗体生产方面享有国际声誉，是蛋白质工程和生物制剂药物发现（包括噬菌体展示）方面的卓越培训中心。博士还需要接受药物化学和现有支架操作方面的培训，以提高生物利用度（化学系和医学科学研究所）。最后，该项目将涉及微生物学培训，包括使用体内感染模型。

Burkholderia cenocepacia is an opportunistic Gram -ve bacterium which can infect CF patients and immuno-compromised cancer patients undergoing chemotherapy. In CF B. cenocepacia causes a severe (and irreversible) decline in lung function resulting in a life-threatening systemic infection known as cepacia syndrome. B. cenocepacia is intrinsically resistant to multiple classes of antimicrobial agents (hiding away inside cells) and expresses an array of virulence factors. This PhD will develop specific blocking tools that can be used to disarm different components of the bacteria's pathogenic arsenal. One strategy that will be studied in detail is a systemic resistance mechanism that neutralises the potency of existing antibiotic approaches. Interestingly, because of its promiscuous nature, deployment of this approach by B. cenocepacia can even encourage infection from other obligate or non-obligate pathogens. In B. cenocepacia, exposure to sublethal concentrations of antibiotics induces chemotoxic stress which results in the expression and secretion, into the extracellular environment, of a group of conserved bacterial proteins called lipocalins (our focus BcnA) which intercept and sequester antibiotics before these drugs can reach the bacteria inside cells. This aspect of the bacteria's infection strategy can be partially reversed by natural compounds such as Vitamin E which increases antimicrobial sensitivity by inhibiting bacterial lipocalin/antibiotic binding through mechanisms that appear to be both antioxidant and/or through the blocking of lipid peroxidation (both recognised MoAs of Vit E). Here we propose a more detailed and two-pronged strategy to further interrogate and hopefully manipulate the BcnA system. We will develop anti-BcnA antibodies (mAbs) as tools to stop BcnA mopping up antibiotics, whilst at the same time we hope in vivo (mouse) these same mAbs will encourage the mouse immune system to mop-up and neutralise the BcnA molecules. These mAbs may have a third exciting use as diagnostic probes to identify BcnA molecules in urine as an early and specific indicator of infection. In parallel we will use a new super-antioxidant (called Proxison) to determine its protective effects. Whilst a potent molecule (500x Vit E) with a similar MoA, this currently is a molecule with poor bioavailability. One key part of this project will be to use chemistry to design and synthesise more bioavailable versions (pro-drug and biotransformation chemistries) that retain this impressive potency but have improved utility in vitro and hopefully in vivo. The final aims of this PhD would be to combine the use of these two strategies and determine their effects on B. cenocepacia biology. This project will be based in the Scottish Biologics Facility which has developed an international reputation for recombinant antibody generation and is a centre of training excellence in protein engineering and biologics drug discovery, including phage display. The PhD will also require training in medicinal chemistry and the manipulation of the existing scaffold to improve bioavailability (Chemistry Dept and Institute of Medical Sciences). Finally the project will involve microbiological training including the use of in vivo infection models.