Table 2_EPEC autotransporter adhesin (Eaa): a novel adhesin identified in atypical enteropathogenic Escherichia coli.docx

Enteropathogenic Escherichia coli (EPEC) is a pathogen that causes diarrhea that can be subdivided into typical (tEPEC) and atypical (aEPEC), based on the production of an adhesin termed Bundle-Forming Pilus (BFP) in the former group. aEPEC is one of the main bacterial pathogens isolated from individuals with diarrhea, and some serotypes have been implicated in diarrheal outbreaks in Brazil, such as the O2:H16. A comparative genomic analysis of aEPEC of this serotype led to the identification of a gene encoding a previously uncharacterized autotransporter protein. In the present study, this novel autotransporter protein was characterized and named EPEC Autotransporter Adhesin (Eaa). The Eaa-encoding gene (eaa) is located in a chromosomal prophage region of 17,014 base pairs, organized in 20 open reading frames and inserted downstream to the threonine-tRNA. A recombinant plasmid termed pIC (pBAD/Myc-His A harboring the eaa gene from aEPEC BA92) was transformed in the MS427 host bacteria, and the MS427(pIC) was used in phenotypic assays. Immunogold-labelling transmission electron microscopy, using anti-Eaa antibodies, showed the presence of Eaa in the cell surface of the wild-type BA92 and MS427(pIC) strains. Subsequently, we demonstrated that Eaa mediates bacterial autoaggregation, biofilm formation and binding to several components of the extracellular matrix, including fibrinogen, plasma and cellular fibronectin, type I, III as well as V collagen and laminin. In summary, we demonstrated that Eaa harbors several adherence properties and may contribute to the pathogenicity of some aEPEC isolates by mediating the interaction of this pathogen with biotic and abiotic surfaces.

肠致病性大肠杆菌（Enteropathogenic Escherichia coli, EPEC）是一类可引发腹泻的病原菌，根据是否产生被称为束状菌毛（Bundle-Forming Pilus, BFP）的黏附素，可将其划分为典型肠致病性大肠杆菌（tEPEC）与非典型肠致病性大肠杆菌（aEPEC）两类。其中aEPEC是从腹泻患者体内分离得到的主要细菌性病原菌之一，部分血清型已被证实与巴西地区的腹泻暴发疫情相关，例如O2:H16血清型。针对该血清型aEPEC的比较基因组分析（comparative genomic analysis），成功鉴定出一个编码此前未被表征的自转运蛋白（autotransporter protein）的基因。本研究对该新型自转运蛋白进行了系统表征，并将其命名为EPEC自转运黏附素（EPEC Autotransporter Adhesin, Eaa）。编码Eaa的基因（eaa）位于一段长度为17014碱基对（base pairs）的染色体原噬菌体区域（chromosomal prophage region）内，该区域包含20个开放阅读框（open reading frames），且插入于苏氨酸tRNA（threonine-tRNA）的下游位置。将携带aEPEC BA92株eaa基因的重组质粒（recombinant plasmid）pIC（pBAD/Myc-His A载体）转化至MS427宿主菌（host bacteria）中，获得的重组菌MS427(pIC)被用于后续表型实验（phenotypic assays）。利用抗Eaa抗体（anti-Eaa antibodies）进行的免疫金标记透射电子显微镜（Immunogold-labelling transmission electron microscopy）检测显示，野生型（wild-type）BA92株与重组菌MS427(pIC)的细胞表面均存在Eaa蛋白。后续实验证实，Eaa可介导细菌发生自身聚集（autoaggregation）、形成生物膜（biofilm formation），并能结合细胞外基质（extracellular matrix）的多种组分，包括纤维蛋白原（fibrinogen）、血浆纤连蛋白（plasma fibronectin）、细胞纤连蛋白（cellular fibronectin），以及I型、III型与V型胶原（collagen）和层粘连蛋白（laminin）。综上，本研究证实Eaa具备多种黏附相关特性，其可通过介导该病原菌与生物及非生物表面（biotic and abiotic surfaces）的相互作用，参与部分aEPEC分离株的致病过程（pathogenicity）。