出血性大腸桿菌是一種新興的人畜共通病原菌,會藉由糞口途徑感染並寄殖在人 類的腸道,造成出血性下痢、出血性腸炎、溶血性尿毒症等症狀,甚至會死亡。對於 出血性大腸桿菌的感染,目前在使用抗生素的治療上仍存有諸多限制,所以去探討出 血性大腸桿菌的致病機轉是必要的課題。從我們之前的研究發現,出血大腸桿菌會造 成線蟲產生 A/E 病灶,A/E 病灶會造成線蟲腸道微絨毛的抹除。因此我們利用出血性 大腸桿菌來感染帶有紅色螢光微絨毛肌動蛋白(ACT-5)蛋白的轉殖動物,感染過程中 我們發現出血性大腸桿菌會造成線蟲 ACT-5 的異位表現,ACT-5 蛋白會從線蟲腸道 頂端散佈到腸道細胞質中。然而,參與在出血性大腸桿菌誘發線蟲微絨毛肌動蛋白 (ACT-5)異位表現中的宿主因子尚未被釐清。因此為了找尋參與的宿主因子,我們利 用核糖核酸抑制(RNAi)的技術來篩選抑制出血性大腸桿菌感染初期會被提高表達的 宿主基因。從抑制這些高表達宿主基因的結果中發現,細胞週期蛋白 B3(CYB-3)是出 血性大腸桿菌誘發線蟲 ACT-5 蛋白異位表現中必要的宿主因子。在 G2/M 時期,CYB-3 會藉由結合跟活化週期蛋白依賴性激酶 1(CDK-1)來調控細胞週期。而我們的結果中 也發現,CDK-1 也是出血性大腸桿菌誘發線蟲 ACT-5 蛋白異位表現中必要的宿主因 子,然而其他的細胞週期蛋白跟週期蛋白依賴性激酶並沒有參與在出血性大腸桿菌誘 發線蟲 ACT-5 蛋白異位表現中。再者,從 RNAi 抑制 CDK-1 的內生性調控者跟處理 CDK-1 小分子藥物的實驗,都證實 CDK-1 的活性對於出血性大腸桿菌誘發的這個病 理特徵是重要的。接下來我們更發現,CDK-1 磷酸化的受質動力素相關蛋白(DRP-1) 和它的內生性調控者都參與在出血性大腸桿菌誘發線蟲 ACT-5 蛋白異位表現中。在 最後我們也確認 DRP-1 作用在 CYB-3 的下游且參與在這個出血性大腸桿菌誘發的病 理特徵中。總結我們發現 CYB-3/CDK-1/DRP-1 的訊息傳遞在出血性大腸桿菌感染過 程誘發線蟲 ACT-5 蛋白異位表現中扮演重要角色。

Enterohemorrhagic E. coli (EHEC) is an emerging zoonotic pathogen that infects human by colonizing intestine through the oral-fecal route and can cause bloody diarrhea, hemolytic uremic syndrome (HUS), and even death. The use of antibiotics is constrained for the treatment of EHEC infection, therefore understanding the pathogenesis of EHEC is required. Our previous studies indicated that EHEC can cause specific attaching and effacing (A/E) lesions in C. elegans intestine. A/E lesion in C. elegans is characterized by the effacement of intestinal microvilli. Moreover, EHEC induces ectopic expression of the microvillar actin (ACT-5) from the apical surface to the cytosol in intestinal cells of the transgenic animal with mCherry-tagged ACT-5. However, the host factors required for this EHEC-induced ACT-5 ectopic expression in C. elegans is still unknown. We therefore aimed to identify the host factors required for this EHEC-induced pathology. To this end, we designed a genetic suppressor screen to RNAi the host genes that are significantly up-regulated during early EHEC infection. From RNAi of up-regulated host genes, Cyclin B3 (CYB-3) is a host factor required for this EHEC-induced ACT-5 ectopic expression phenotype. CYB-3 regulates cell cycle through binding to and activating the Cyclin-Dependent Kinase 1 (CDK-1) during G2/M phase. Our results indicated that CDK-1 is also required for the EHEC-induced ACT-5 ectopic expression, while the other Cyclins and CDKs are not. Moreover, from RNAi of the CDK-1 endogenous regulators and inhibition of CDK-1 activity by specific small molecule inhibitor all demonstrated that CDK-1 activity is essential for this EHEC-induced pathology. Next, we found that the CDK-1 substrate DRP-1 and the endogenous regulators of DRP-1 are also involved in this EHEC-induced ACT-5 ectopic expression. Finally, we also confirmed that DRP-1 acts downstream to CYB-3 in this EHEC-induced pathology. Taken together, we showed that the CYB-3/CDK-1/DRP-1 signal axis plays an important role in the EHEC-induced ACT-5 ectopic expression.

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