Analysis of Acinetobacter trimeric adhesin (Ata) in multi-drug resistant clinical isolates and its contribution to the pathogenic potential of Acinetobacter baumannii

外膜蛋白不动杆菌三聚体自体转运蛋白黏附素(ATA)是细菌三聚体自体转运蛋白家族的新成员，是鲍曼不动杆菌重要的致病因子。我们已经产生了Ata缺失的鲍曼不动杆菌(Δata)突变体，并分别分析了Ata在黏附细胞外基质成分和内皮细胞和上皮细胞中的作用。我们建立了一个新的人体器官感染模型(人脐静脉)，模拟了细菌与宿主之间的相互作用，接近人类的生理状况(蠕动血流条件)，并进一步利用梅隆格列氏菌感染模型确定了ATA的作用。ATA的表达介导了与宿主的黏附，并与感染内皮细胞的致炎表型的诱导和细胞凋亡的诱导有关。ATA和新发现的纤溶酶原结合和补体抑制蛋白CIPA被证明是鲍曼不动杆菌补体抵抗的两个关键决定因素，它们抑制了补体替代级联途径的激活。我们的数据表明，Ata在体外、体外和体内的致病性中起着至关重要的作用，并影响鲍曼不动杆菌感染的结局。在拟议的工作计划中，我们将解决以下问题：(I)参与鲍曼不动杆菌与人内皮细胞ATA依赖的黏附的宿主细胞受体的鉴定。为此，将采用基于siRNA文库的方法系统地沉默黏附分子的表达，然后在体外(内皮细胞)和体外(动态人体器官感染模型)对识别的靶基因进行特异性验证。下一步(Ii)，我们将分析宿主细胞的反应，特别强调炎症(例如，白细胞趋化和迁移的功能分析)；此外，还将分析ATA介导的细胞凋亡激活。(Iii)通过系统的转录组分析(“双重RNA-seq”)，我们将确定宿主细胞和鲍曼不动杆菌中的信号通路，以确定ATA如何影响感染后的宿主细胞反应，并鉴定鲍曼不动杆菌的毒力特性(体外、体外和体内)。(Iv)ATA促进补体逃逸的分子机制--与CIPA结合--将通过研究它们与纯化的补体成分和感染患者的血清的相互作用来阐明。我们的实验将阐明ATA在触发宿主反应中的作用，有助于了解鲍曼不动杆菌感染的病理生物学以及三聚体自体转运蛋白粘附素的作用。这可能有助于开发新的治疗概念来对抗多药耐药细菌的感染。

The outer membrane protein Acinetobacter trimeric autotransporter adhesin (Ata) is a new member of the family of bacterial trimeric autotransporter adhesins and represents an important pathogenicity factor of Acinetobacter baumannii. We have generated Ata-deficient mutants of A. baumannii (Δata) and analysed the role of Ata in adherence to extracellular matrix components and endothelial and epithelial cells, respectively. We established a novel human organ infection model (human umbilical cord veins) mimicking the bacteria-host interactions close to the physiological situation in humans (peristaltic blood flow conditions) and, furthermore, determined the role of Ata using the Galleria mellonella infection model. Ata expression mediates adhesion to the host and correlates with the induction of a proinflammatory phenotype of infected endothelial cells and the induction of apoptosis. Ata and the newly discovered plasminogen-binding and complement-inhibitory protein CipA proved to be two key determinants of complement resistance of A. baumannii which inhibit activation of the alternative complement cascade pathway. Our data suggest that Ata plays a crucial role in the pathogenicity in vitro, ex vivo and in vivo and influences the outcome of infections with A. baumannii. In the proposed work programme, we will address the following questions: (i) Identification of host cell receptors involved in Ata-dependent adhesion of A. baumannii to human endothelial cells. For this purpose, a siRNA library-based approach to systemically silence adhesion-molecule expression will be employed, followed by specific validation of identified target genes in vitro (endothelial cells) and ex vivo (dynamic human organ infection model). Next (ii), we will analyse the host cell response with a special emphasis on inflammation (e.g., functional assays of leukocyte chemotaxis and transmigration); moreover, Ata-mediated activation of apoptosis will be analysed. (iii) By using systematic transcriptome analysis ("dual RNA-seq") we will identify signalling pathways in host cells and in A. baumannii to determine how Ata affects the host cell response upon infection and to identify virulence traits of A. baumannii (in vitro, ex vivo and in vivo). (iv) Molecular mechanisms by which Ata facilitates complement evasion -in combination with CipA- will be elucidated by investigating their interactions with purified complement components and sera of infected patients. Our experiments will clarify the role of Ata in triggering the host response, help to understand the pathobiology underlying A. baumannii infections and the role of trimeric autotransporter adhesins in general. This might aid to develop novel therapeutic concepts to combat infections with MDR bacteria.