出血性大腸桿菌血清型O157:H7的感染能夠在人類造成出血性腹瀉及溶血性尿毒症，並且導致腸壁損害 (attaching and effacing lesion, A/E lesion)，此為出現在腸道的特殊病灶，其特色為出血性大腸桿菌會抹除腸道表面之微絨毛並且造成腸道表面形成凸起結構 (pedestal-like structure)，再透過此結構緊密黏附在腸道上。根據先前的研究發現細菌的黏附是由LEE致病基因群 (the pathogenicity island locus of enterocyte effacement) 調控，然而微絨毛抹除的致病機制目前仍然有待釐清。因此我們藉由全基因體篩選，試圖找出出血性大腸桿菌誘發線蟲腸道微絨毛異位 (ACT-5 mislocalization) 的毒理因子。首先，我們透過基因轉殖在微絨毛肌動蛋白 (microvillar actin protein, ACT-5) 表現紅色螢光的秀麗隱桿線蟲 (Caenorhabditis elegans)，並在感染出血性大腸桿菌後觀察其紅色螢光表現，我們將螢光從腸道表現轉移至細胞質中同時異常聚集的現象定義為腸道微絨毛異位表現。從17802個跳躍子突變菌株中篩選出48個降低線蟲腸道微絨毛異位表現的突變菌株，其中脂多醣生合成相關基因佔了大多數，然而，我們選擇了一功能未知基因，名為z1205，當作目標基因。為了證實此基因與微絨毛抹除有關，我們透過基因缺失突變與基因回補實驗，發現此基因的缺失確實能降低出血性大腸桿菌抹除線蟲腸道微絨毛的能力，而回補此基因後，細菌的抹除能力又能得到回復。在蛋白功能研究方面，我們利用西方墨點法偵測細菌的Z1205蛋白表現，卻無法在野生型的細菌中偵測到蛋白，因此，我們推測此基因要在細菌感染宿主的過程中才會表現而設計了報導基因偵測實驗 (reporter assay)，由此發現z1205啟動子需要在感染線蟲的過程中才會活化，進而推論z1205基因要在感染過程中才會表現。概括上述，z1205基因是出血性大腸桿菌造成宿主腸道微絨毛異位表現的毒理因子，且此基因需要在感染過程中才會表現。

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) infection leads to hemorrhagic diarrhea and hemolytic uremic syndrome in human. EHEC can induce a characteristic pathology called attaching and effacing (A/E) lesion, which is that EHEC effaces host intestinal microvilli and attaches to intestinal epithelium through a pedestal-like structure. Previous study found that bacterial attachment is mediated by the pathogenicity island locus of enterocyte effacement (LEE); however, the mechanism of microvillar effacement remains unclear. Here, we conducted a genome-wide transposon library screening to identify the bacterial factors involved in EHEC-induced microvillar actin rearrangement in C. elegans. We used a specific transgenic C. elegans with the expression of the microvillar actin protein, ACT-5, labeled with mCherry and examined the florescent expression pattern upon EHEC infection. We defined microvillar actin rearrangement is that the ACT-5 re-localizes from apical side to cytosol, as ACT-5 mislocalization. After screening 17,802 transposon mutants, we identified 48 hits that showed reduced ACT-5 mislocalization in C. elegans. Among them, LPS biosynthesis related genes were the majority; however, one gene with unknown function named z1205 attracted our attention. To reconfirm our results, we generated an z1205 isogenic deletion mutant and found that C. elegans exhibited reduced ACT-5 mislocalization while feeding this z1205 mutant. We also performed a gene complementation test and found that C. elegans exhibited compatible ACT-5 mislocalization ratio to the EHEC wild type. To investigate the protein function of Z1205, we generated an antibody specifically against Z1205. However, we failed to detect Z1205 protein expression in wild-type EHEC strain but not in the complementation strain. Therefore, we hypothesized that the Z1205 protein might express during EHEC infection in vivo. We established a reporter assay and found z1205 promoter was activated when EHEC infected C. elegans. Overall, our result suggested that the z1205 gene is one of the virulence determinants of EHEC effacement and the z1205 gene might express during EHEC infection in vivo.

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