Quorum sensing and virulence in Gram positive pathogens: structure, function and inhibition of the agr system

世界卫生组织认为，耐多种抗生素细菌的出现、迅速传播和持续存在是对人类健康的三大全球威胁之一。抗菌素耐药性（AMR）甚至威胁到简单感染的治疗和结果，以及直到最近才被认为是低风险的常见医疗干预措施（外科、牙科、产科）。在这种背景下，新型抗生素的开发远远落后于对新药的迫切需求。这在一定程度上是因为发现安全有效的新抗生素在科学上具有挑战性，而且因为许多大型制药公司退出了开发昂贵的新药，这些新药可能因耐药性而迅速过时。因此，迫切需要新型抗菌药物，这种药物不会屈服于传统的抗生素耐药机制，不会选择新的耐药形式，也不会损害宿主微生物群。这类药物的发现取决于对致病菌的生理学和分子生物学及其定植宿主组织和对抗宿主免疫防御的策略的透彻理解。这些包括多种毒力因素的部署，如酶和毒素，导致宿主组织损伤和疾病。传统抗生素的作用主要是杀死细菌，因此施加了巨大的选择压力，导致耐药菌株的出现。然而，如果我们不是杀死细菌，而是简单地阻止它们发挥它们的毒力因子，感染应该会减弱，出现耐药性的压力也会减少。对细菌毒力因子及其控制系统的研究使我们能够识别细菌分子靶点的“弱点”，从而可以设计出筛选对这些靶点有活性的类药物化合物的方法，并发现新的抗菌药物。群体感应（quorum sensing， QS）是抗毒剂的一个有希望的靶标。虽然细菌是单细胞生物，但它们可以通过细胞间的交流使种群中所有细胞的活动同步。这是通过产生和感知信号分子来实现的，这些信号分子告知感染细菌它们有足够的数量力量来部署它们的毒力因子并发动攻击。QS系统为抗感染药物提供多种分子靶标，包括QS信号分子的生产、输出和响应。在金黄色葡萄球菌（包括MRSA）和艰难梭菌等多重耐药病原菌中，包括许多主要外毒素在内的毒力因子由agr QS系统控制，该系统采用自诱导肽信号（AIP）分子。在这个研究项目中，我们正试图深入了解金黄色葡萄球菌通过两种称为AgrB和1984的跨膜蛋白产生和输出AIP信号分子的方式，因为它们是QS和毒性的关键。我们建议采用微生物学、化学、结构生物学相结合的多学科方法来阐明AIP产生的关键酶的功能和三维结构。我们还计划发现AIP是如何从细菌细胞中输出的，并开发新的药物样分子，以阻止葡萄球菌中AIP的产生。这些药物将在实验室培养基中单独和与常规抗生素联合进行疗效测试，并使用新型感染成像工具进行测试，这些工具将提供有关agr依赖性QS在何时何地开启或关闭的信息。这项工作将主要集中在金黄色葡萄球菌上，但也将测试有希望的化合物对肠球菌、梭状芽胞杆菌和李斯特菌等的作用。其他葡萄球菌种类。

The emergence, rapid spread and persistence of multi-antibiotic resistant bacteria is considered by the WHO as one of the three greatest global threats to human health. Antimicrobial resistance (AMR) threatens the treatment and outcome of even simple infections and common medical interventions (surgery, dentistry, obstetrics) that until recently were considered low-risk. Against this backdrop, the development of new classes of antibiotics has lagged far behind the urgent requirement for new drugs. This is in part because discovering safe and effective new antibiotics is scientifically challenging and because many major pharmaceutical companies withdrew from developing expensive new drugs likely to become rapidly obsolete through resistance. Consequently, novel antibacterial drugs with that do not succumb to conventional antibiotic resistance mechanisms, nor select for new forms of resistance, nor damage the host microflora, are desperately needed. The discovery of such drugs depends on a thorough understanding of the physiology and molecular biology of pathogenic bacteria and their strategies for colonizing host tissues and combatting host immune defences. These include the deployment of multiple virulence factors such as enzymes and toxins that cause host tissue damage and disease. Conventional antibiotics mostly act by killing bacteria and so exert enormous selective pressures leading to the emergence of resistant strains. If however, instead of killing bacteria, we simply prevent them deploying their virulence factors, infection should be attenuated with less pressure for resistance to emerge. Research on bacterial virulence factors and their control systems enables us to identify molecular target 'weak points' in bacteria so that methods for screening drug-like compounds active against such targets can be designed and new antibacterial drugs discovered. One promising target for anti-virulence agents is quorum sensing (QS). Although bacteria are single cell organisms, they can synchronize the activities of all the cells in a population through cell-to-cell communication. This is achieved through the production and sensing of signal molecules that inform the infecting bacteria that they are present in sufficient numerical strength to deploy their virulence factors and mount an attack. QS systems offer multiple molecular targets for anti-infective agents that include the production, export and response to QS signal molecules. In problematic multi-antibiotic resistant pathogens such as Staphylococcus aureus (including MRSA) and Clostridium difficile, virulence factors including many major exotoxins are controlled by the agr QS system that employs autoinducing peptide signal (AIP) molecules. In this research project we are seeking to understand in depth the way in which S. aureus produces and exports AIP signal molecules via two transmembrane proteins called AgrB and 1984 since these are key to QS and hence virulence. We propose to use a multidisciplinary approach combining microbiology with chemistry, structural biology to elucidate the functions and 3D structures of the key enzymes involved in AIP generation. We also plan to discover how AIPs are exported out of the bacterial cell and to develop new drug-like molecules that block AIP production generation in staphylococci. These will be tested for efficacy alone and in combination with conventional antibiotics in laboratory media and by using novel infection imaging tools that will provide information on when and where agr-dependent QS is switched on or off. The work will focus primarily on S. aureus but promising compounds will also be tested against enterococci, clostridia and listeria and other. other staphylococcal species.