THE OUTER MEMBRANE AS A VIRULENCE PLATFORM

描述(申请人提供)：革兰氏阴性致病菌的外膜(OM)代表了一个分泌和呈现表面局部毒力因子的平台。保守的周质伴侣Sura将新生OM蛋白从SEC机制运送到BAMA OM组装复合体，最终将它们整合到OM中。我们的原创工作证明了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引导者FimD的缺失，细菌细胞通过该FimD组装黏附的1型菌毛，赋予UPEC结合和侵袭膀胱上皮的能力。我们还表明，在膀胱炎的侵袭后阶段，SurA底物还有一个额外的作用，在这个阶段，UPEC在膀胱上皮细胞内快速繁殖，形成细胞内细菌群落(IBCs)，并在上皮内建立一个持久的生态位，与宿主的免疫防御隔离，免受抗生素的杀伤。这种上皮内细菌储存库可能是复发尿路感染的种子，尿路感染是临床上常见和困难的问题。我们最近的数据已经确定UPEC外膜蛋白A(OmpA)是这一细胞内期膀胱炎所需的SurA依赖因子。具体地说，UPEC OmpA突变体正常侵入膀胱上皮，但不能完成IBC成熟并持续存在于尿路上皮内。在缺乏Toll样受体4信号的小鼠中，这种致病缺陷基本上得到了挽救，这表明OmpA调节宿主免疫机制以促进感染。在这项提案中，我们将首先定义OmpA对宿主先天反应(包括中性粒细胞招募和膀胱上皮脱落)和适应性反应的贡献。我们随后将解决OmpA是否对中性粒细胞的抗病原体活性，特别是天然抗菌肽的作用提供保护。最后，我们将使用染色体互补系统来确定OmpA的结构特征，该结构特征与我们观察到的致病表型有关。我们期望这些研究将证实SurA底物OmpA作为膀胱炎细胞内期的核心决定因素，并为制定阻断尿路感染患者复发周期的策略提供信息。与公共卫生相关：许多细菌表达表面蛋白(称为毒力因子)，帮助细菌引起人类传染病。该项目旨在确定其中一种名为OmpA的细菌表面蛋白如何与人类免疫系统相互作用，以促进尿路感染的建立。了解OmpA的功能将为预防和治疗细菌感染提供新的策略。

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.