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

盲肠伯克霍尔德氏菌是一种机会革兰氏菌，可感染CF患者和接受化疗的免疫低下的癌症患者。在慢性阻塞性肺疾病中，盲肠闭塞会导致严重的(且不可逆转的)肺功能下降，导致一种危及生命的全身感染，称为洋葱综合征。管圆线虫对多种抗菌剂(隐藏在细胞内)具有内在抗药性，并表达一系列毒力因子。这位博士将开发特定的阻断工具，可用于解除细菌致病军火库的不同成分的武装。将详细研究的一种策略是一种系统性耐药机制，它中和现有抗生素方法的效力。有趣的是，由于其混杂的性质，双毛虫采用这种方法甚至会鼓励来自其他专性或非专性病原体的感染。在盲肠弯曲杆菌中，暴露在亚致死浓度的抗生素下会诱导化学毒性应激，导致一组保守的细菌蛋白Lipocalins(我们的焦点BCNA)在这些药物到达细胞内的细菌之前拦截和隔离抗生素，并分泌到细胞外环境中。细菌感染策略的这一方面可以被维生素E等天然化合物部分逆转，维生素E通过似乎既是抗氧化剂又是通过阻断脂质过氧化的机制(两者都是维生素E的MOAS)抑制细菌Lipocalin/抗生素的结合，从而增加抗菌素的敏感性。在这里，我们提出了一个更详细和双管齐下的战略，以进一步审问并有望操纵BCNA系统。我们将开发抗BCNA抗体(MAbbs)，作为阻止BCNA清除抗生素的工具，同时我们希望在体内(小鼠)这些相同的mAbs将鼓励小鼠免疫系统清除和中和BCNA分子。这些单抗可能还有第三个令人兴奋的用途，作为诊断探针，将尿中的BCNA分子识别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(名为Proxison)来确定其保护作用。虽然这是一种具有类似MOA的有效分子(500倍维生素E)，但目前这是一种生物利用度较低的分子。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，但在体外和有望在体内具有更好的实用性。这一博士学位的最终目标将是将这两种策略的使用结合起来，并确定它们对双毛虫生物学的影响。该项目将设在苏格兰生物制品设施，该设施在重组抗体生成方面享有盛誉，是蛋白质工程和生物药物发现(包括噬菌体展示)方面的卓越培训中心。博士学位还将要求进行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.