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转运粘附素（Acinetobacter trimeric autotransporter adhesin，Ata）是细菌三聚体自转运粘附素家族的新成员，是鲍曼不动杆菌的重要致病因子。我们已经产生了A. baumannii（Δata）的表达，并分析了Ata在细胞外基质成分、内皮细胞和上皮细胞粘附中的作用。我们建立了一种新的人体器官感染模型（人脐带静脉）模仿细菌-宿主相互作用接近人体的生理情况（蠕动血流条件），并进一步确定了使用大蜡螟感染模型的Ata的作用。Ata表达介导对宿主的粘附，并与诱导受感染内皮细胞的促炎表型和诱导细胞凋亡相关。Ata和新发现的纤溶酶原结合和补体抑制蛋白CipA被证明是A.鲍曼不动杆菌，其抑制旁路补体级联途径的激活。结果表明，Ata在体外、离体和体内的致病性中起重要作用，并影响A.鲍曼不动杆菌。在拟议的工作计划中，我们将解决以下问题：（i）鉴定参与A.鲍曼不动杆菌对人内皮细胞的作用。为此，将采用基于siRNA文库的方法来全身性沉默粘附分子表达，然后在体外（内皮细胞）和离体（动态人体器官感染模型）对鉴定的靶基因进行特异性验证。接下来（ii），我们将分析宿主细胞反应，特别强调炎症（例如，白细胞趋化性和迁移的功能测定）;此外，将分析Ata介导的细胞凋亡激活。(iii)通过使用系统转录组分析（“双重RNA-seq”），我们将鉴定宿主细胞和A.鲍曼不动杆菌的感染，以确定Ata如何影响宿主细胞的反应，并确定毒力性状的A。鲍曼不动杆菌（体外、离体和体内）。(iv)Ata促进补体逃逸的分子机制-结合CipA-将通过研究它们与纯化的补体成分和感染患者血清的相互作用来阐明。我们的实验将有助于阐明Ata在触发宿主反应中的作用，有助于理解A.鲍曼不动杆菌感染和三聚体自身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MDR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.