腸出血性大腸桿菌（Enterohemorrhagic Escherichia coli, EHEC）O157：H7是一種會引起出血性腹瀉和溶血性尿毒綜合徵（HUS）的病原體。感染案例主要發生在美國、加拿大、日本和歐洲的其他工業化國家。因為抗生素治療可能會惡化EHEC感染的情況，因此發現新目標來開發新藥是非常重要的。在先前的研究中發現與EDL933菌株相比，其ΔsdhA突變體的感染提升了秀麗隱桿線蟲的存活率。SdhA是琥珀酸脫氫酶(succinate dehydrogenase)的其中一個次單元，SdhA參與在中樞代謝途徑TCA循環和電子傳遞鏈中。這意味著中斷TCA循環將影響毒力因子的基因表達，除此之外也表明SdhA是藥物開發中可以做為潛在靶標。有趣的是，ΔsdhA突變體中毒力因子LEE基因的表達已被證明會上調。由LEE基因所轉譯的第三型分泌系統（Type three secretion system, T3SS）使EHEC粘附在宿主腸上皮細胞並在宿主腸上形成A/E lesion的重要因子。在本研究中，我們試圖找出哪個主要因素影響ΔsdhA突變體中LEE基因的表達。首先，我們使用啟動子活性測定的方式想要找出哪個因子可能在ΔsdhA突變體中扮演上調LEE基因表達的主要角色。我們發現Lrp可能在ΔsdhA突變體中起上調LEE表達的作用。 Lrp是亮氨酸反應性調節蛋白(Leucine-responsive regulatory protein)，其在腸出血性大腸桿菌中可以藉由感應丁酸的濃度在通過LeuO和Pch來導致LEE基因上調。接著，我們想藉由突變lrp來確認Δlrp突變體中的LEE表達在ΔsdhAΔlrp雙重突變體中是相同的，但是與野生型相比時EHEC的lrp突變下調LEE基因，這結果不論是在Δlrp突變體或是在ΔsdhAΔlrp雙重突變體中都有相同現象。因為EHEC不會產生丁酸，所以必然有其他分子影響Lrp活性。因此我們使用各別提供有2.5mM琥珀酸鹽(succinate)和富馬酸鹽(fumarate)在M9培養基來檢查是否影響ΔsdhA突變體中的lrp和LEE1啟動子活性。我們發現富馬酸鹽會略微影響lrp啟動子的活性，但不影響ΔsdhA菌株中的LEE1啟動子。這表明富馬酸鹽可能不是調節Lrp和LEE1的主力。除此之外，我們還發現在ΔsdhA菌株中的leuO啟動子的活性遠高於WT。總結，我們知道sdhA的缺失會上調LEE基因的表達並改變lrp和leuO的表達，這個改變並不是因為富馬酸鹽或琥珀酸鹽。因此我們認為在ΔsdhA菌株中存在影響Lrp上調的其他因素，然後LEE基因表達的上調與ΔsdhA菌株中的Lrp-LeuO途徑相關。

Enterohemorrhagic Escherichia coli (EHEC) O157: H7 is a pathogen that can cause bloody diarrhea and hemolytic uremic syndrome (HUS). The cases mainly occur in the U.S., Canada, Japan and other industrialized countries in Europe. Because antibiotic treatment may enhance the severity of EHEC infection, therefore, it is important to discover new targets to develop new drugs. In a previous study, the feeding of EHEC ΔsdhA strain increased the survival rate of Caenorhabditis elegans compared with WT strain. SdhA is a subunit of succinate dehydrogenase (SDH) involved in the central metabolism pathway, TCA cycle and electron transport chain. Thus, disruption of TCA cycle will affect virulence factors expression and SdhA is therefore a promising target for drug development. Interestingly, the expression of the locus of enterocyte effacement (LEE) genes in ΔsdhA strain was upregulated in other studies. Type three secretion system (T3SS), encoded by LEE pathogenicity island, is an important factor for EHEC to adhere directly to host intestinal epithelium and to form attaching and effacing lesions (A/E lesions). In this study, we try to clarify which major factor affect LEE genes expression in ΔsdhA strain. First, we used promoter activity assay trying to find a factor might play a major role that upregulates LEE genes expression in ΔsdhA strain. We found that Lrp might play a role to upregulate LEE expression in ΔsdhA strain. Lrp is a Leucine-responsive regulatory protein that senses butyrate and can also induce LEE genes upregulation through LeuO and Pch in EHEC. We mutated lrp to test LEE genes expression in Δlrp strain will be identical in ΔsdhAΔlrp strain. We confirmed that Δlrp strain and ΔΔlrp strain will downregulate expression of LEE genes while comparing with WT. Because EHEC would not produce butyrate but Lrp still has activity. So we wonder which metabolite affect Lrp. Then, we used M9 medium supplied with 2.5 mM succinate and fumarate respectively to check which supplement could affect lrp promoter activity in ΔsdhA strain. We found out that fumarate will slightly affect the activity of lrp promoter but did not affect LEE1 promoter in ΔsdhA strain. This indicates that fumarate is not the major role to regulate Lrp. To further search, we found that activity of leuO promoter is higher than WT. In conclusion, we know that deletion of sdhA will upregulate LEE genes expression and alter lrp and leuO expression. The alteration is not because of fumarate or succinate. So we suggest there must be other molecules affect Lrp upregulate in ΔsdhA strain. Then, upregulation of LEE genes expression is associated with Lrp-LeuO pathway in ΔsdhA strain.

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