THE OUTER MEMBRANE AS A VIRULENCE PLATFORM

描述（由申请方提供）：革兰氏阴性病原菌的外膜（OM）代表了分泌和呈递表面定位毒力因子的平台。保守的周质分子伴侣SurA渡轮新生OM蛋白从Sec机械的BamA OM组装复合物，其最终纳入OM。我们的原始工作证明了SurA在尿路致病性大肠杆菌（UPEC）引起的尿路感染的发病机制中的重要性。UPEC中surA的破坏导致OM usher FimD的缺陷，细菌细胞通过OM usher FimD组装粘附1型皮利，该粘附1型菌毛赋予UPEC结合和侵袭膀胱上皮的能力。我们还表明，SurA底物在膀胱炎的后侵袭阶段具有额外的作用，其中UPEC在膀胱上皮细胞内迅速繁殖，形成细胞内细菌群落（IBC），并在上皮内建立持久的生态位，与宿主免疫防御隔离，并免受抗生素杀伤。这种上皮内细菌储库可能作为复发性UTI的种子，这是一种常见且困难的临床问题。我们最近的数据已经确定UPEC外膜蛋白A（OmpA）作为SurA依赖性因子所需的细胞内阶段的膀胱炎。具体而言，UPEC ompA突变体正常侵入膀胱上皮，但不能完成IBC成熟并在尿路上皮内持续存在。这种致病性缺陷在Toll样受体4信号传导缺陷的小鼠中基本上被挽救，这表明OmpA调节宿主免疫机制以促进感染。在这个提议中，我们将首先定义OmpA对宿主先天性反应（包括中性粒细胞募集和膀胱上皮脱落）和适应性反应的贡献。我们将随后解决是否OmpA赋予保护对嗜中性粒细胞的抗病原体活性，特别是先天性抗菌肽的作用。最后，我们将使用染色体互补系统来确定我们观察到的致病表型的OmpA的结构特征。我们预计，这些研究将验证SurA底物OmpA作为膀胱炎细胞内阶段的中心决定因素，并为UTI患者复发周期中断策略的制定提供信息。公共卫生相关性：许多细菌表达表面蛋白质（称为毒力因子），这些蛋白质有助于细菌引起人类感染性疾病。该项目旨在确定这些细菌表面蛋白之一OmpA如何与人体免疫系统相互作用，以促进尿路感染的建立。了解OmpA的功能将导致预防和治疗细菌感染的新策略。

DESCRIPTION (provided by applicant): The outer membrane (OM) of Gram-negative pathogenic bacteria represents a platform for the secretion and presentation of surface-localized virulence factors. The conserved periplasmic chaperone SurA ferries nascent OM proteins from the Sec machinery to the BamA OM assembly complex for their final incorporation into the OM. Our original work demonstrated the importance of SurA in the pathogenesis of urinary tract infections caused by uropathogenic Escherichia coli (UPEC). Disruption of surA in UPEC leads to deficiency of the OM usher FimD, through which the bacterial cell assembles the adhesive type 1 pili that confer upon UPEC the capacity for bladder epithelial binding and invasion. We also showed that SurA substrates have an additional role during the post-invasion phase of cystitis, in which UPEC multiply rapidly within bladder epithelial cells to form intracellular bacterial communities (IBCs) and establish a persistent niche within the epithelium, sequestered from host immune defenses and protected from antibiotic killing. This intraepithelial bacterial reservoir may serve as a seed for recurrent UTI, a common and difficult clinical problem. Our recent data have identified the UPEC outer membrane protein A (OmpA) as the SurA-dependent factor required for this intracellular phase of cystitis. Specifically, the UPEC ompA mutant invades bladder epithelium normally, but cannot complete IBC maturation and persist within the uroepithelium. This pathogenic defect is substantially rescued in mice deficient in Toll-like receptor 4 signaling, suggesting that OmpA modulates host immune mechanisms to promote infection. In this proposal, we will first define the contribution of OmpA to host innate responses (including neutrophil recruitment and bladder epithelial exfoliation) and to adaptive responses. We will subsequently resolve whether OmpA confers protection against the antipathogen activity of neutrophils, particularly the action of innate antimicrobial peptides. Finally, we will use a chromosomal complementation system to determine the structural features of OmpA responsible for the pathogenic phenotypes we have observed. We anticipate that these studies will validate the SurA substrate OmpA as a central determinant of the intracellular phase of cystitis and inform the development of strategies for interrupting the cycle of recurrence in patients with UTI. PUBLIC HEALTH RELEVANCE: Many bacteria express surface proteins (called virulence factors) that help the bacteria to cause infectious diseases in humans. This project aims to determine how one of these bacterial surface proteins, called OmpA, interacts with the human immune system to facilitate the establishment of urinary tract infections. Understanding the functions of OmpA will lead to new strategies for preventing and treating bacterial infections.