Antibiotic-sparing strategies targeting outer membrane ushers in Gram-negative bacterial pathogens

项目摘要/摘要： 抗菌素耐药性的上升突显出迫切需要制定新的有效战略来对抗 抗生素耐药感染。无处不在的，革兰氏阴性细菌病原体聚集胞外纤维， 称为伴侣-引导者途径(CUP)菌毛，对病原体通过以下途径引起感染的能力至关重要 识别和定居不同的寄主组织和栖息地。因此，针对集合体的治疗学 这些纤维具有潜在的潜力，有望成为治疗多药耐药的急需的替代品。 耐药的革兰氏阴性病原体。这些病原体中包括那些被指定为“紧急威胁”的病原体。 耐碳青霉烯类不动杆菌和耐碳青霉烯类肠杆菌科(CRE)，以及 威胁“耐药弯曲杆菌产超广谱β-内酰胺酶(ESBL) 肠杆菌科、耐多药铜绿假单胞菌、耐药沙门氏菌、志贺氏菌和 百日咳杆菌。在每个杯状菌毛系统中，一个指定的周质伴侣和一个外膜 (OM)引入蛋白质共同作用，将数千个结构亚基组装成每个最终菌毛结构。 大多数杯状菌毛还含有粘附素，这种粘附素可以识别宿主组织中的受体。我们已经做出了 在理解菌毛组装的极其复杂的机制方面取得了相当大的进展。建房 关于我们在杯状菌毛生物发生和Rational开发方面的丰富经验和专业知识 针对杯状菌毛的治疗，该提案寻求开发针对OM的新的节省抗生素的治疗方法 引入多学科方法，包括细菌学、化学生物学、药物化学、 结构生物学和免疫学。基于它们的结构特征和动态性质 多结构域引流蛋白，我们将通过以下途径合理开发小分子引流抑制剂和开孔剂 捕获特定构象状态(目标1)。Usher抑制剂将解除细菌毒力因子，而 开孔剂将增加现有抗生素对细菌外膜的渗透性。此外，我们 将开发出可灭活引座者的单抗，从而防止菌毛的生物发生和感染(目标 2)。虽然我们的前两个目标将集中在两个研究最多的菌毛系统(类型1和P菌毛)，但目标3 将扩大我们对不动杆菌、弯曲杆菌、铜绿假单胞菌、沙门氏菌、志贺氏菌和B。 百日咳。总的来说，我们计划针对多重耐药的革兰氏阴性菌开发合理的治疗方法 细菌病原体。这些发展，连同我们的 多学科U19计划，将协同工作，通过以下方式作为有效的抗生素节约疗法 阻塞引座器和粘附素功能。此外，在这项提案中开发的引座式开孔器将 增加OM通透性，进一步减轻革兰氏阴性病原体的抗生素耐药性，并使我们 重新调整现有药物的用途，以加强目前的抗生素武器库。因此，这些领域的成功发展 在抗击抗生素耐药性方面，方向将具有潜在的变革性。

PROJECT SUMMARY/ ABSTRACT: The rise of antibacterial resistance highlights the urgent need to develop new effective strategies to combat antibiotic-resistant infections. Ubiquitously, Gram-negative bacterial pathogens assemble extracellular fibers, termed chaperone-usher pathway (CUP) pili, that are critical for the pathogen's ability to cause infections by recognizing and colonizing different host tissues and habitats. Thus, therapeutics targeting the assembly of these fibers hold promise in their potential to result in much needed alternatives for the treatment of multidrug- resistant Gram-negative pathogens. Among these pathogens are those designated as “Urgent Threats” carbapenem-resistant Acinetobacter and carbapenem-resistant Enterobacteriaceae (CRE), as well as “Serious Threats” drug-resistant Campylobacter, extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae, multidrug-resistant Pseudomonas aeruginosa, drug-resistant Salmonella, Shigella, and Bordetella pertussis. In each CUP pilus system, a designated periplasmic chaperone and an outer-membrane (OM) usher protein work together to assemble thousands of structural subunits into each final pilus structure. Most CUP pili are also tipped by adhesins that specifically recognize receptors in host tissues. We have made considerable progress towards understanding the remarkably complex mechanisms of pilus assembly. Building on our extensive experience and expertise in CUP pilus biogenesis and in the development of rational therapies targeting CUP pili, this proposal seeks to develop novel antibiotic-sparing therapies targeting the OM ushers using multidisciplinary approaches including bacteriology, chemical biology, medicinal chemistry, structural biology and immunology. Based on the structural characterizations and the dynamic nature of these multi-domain usher proteins, we will rationally develop small molecule usher inhibitors and pore openers by trapping specific conformational states (Aim 1). Usher inhibitors will disarm bacterial virulence factors, whereas pore openers will increase permeability of existing antibiotics into bacterial outer membranes. In addition, we will develop monoclonal antibodies that inactivate usher, thus preventing pilus biogenesis and infection (Aim 2). While our first two aims will concentrate on two of the most studied pilus systems (type 1 and P pili), Aim 3 will expand our studies of ushers in Acinetobacter, Campylobacter, P. aeruginosa, Salmonella, Shigella, and B. pertussis. Collectively, we plan to develop rational therapies against multiple antibiotic-resistant Gram-negative bacterial pathogens. These developments, together with other novel strategies proposed in our multidisciplinary U19 program, will work synergistically to act as efficient antibiotic-sparing therapeutics by blocking usher and adhesin functions. Moreover, the usher pore openers developed in this proposal will increase OM permeability, further alleviating antibiotic resistance in Gram-negative pathogens and allowing us to repurpose existing drugs to enhance the current antibiotic arsenal. Thus, successful developments in these directions will be potentially transformative in combating antibiotic resistance.