腸出血性大腸桿菌是一種具有致病性的大腸桿菌菌株，對公共衛生造成重大威脅，其中以 O157:H7 血清型最為普遍。腸出血性大腸桿菌感染主要透過受污染的食物與水源傳播，導致嚴重的食源性疾病，臨床症狀涵蓋輕微腹瀉、嚴重腹痛、出血性結腸炎，甚至溶血性尿毒症症候群，後者表現為溶血性貧血、血小板減少及急性腎損傷。鑑於抗生素的使用可能會誘導腸出血性大腸桿菌中的溶原性噬菌體進入裂解期，導致釋放志賀毒素，進一步加重病情，因此在缺乏特效治療的情況下，了解其致病機制並發展降低其毒力的策略就顯得格外重要。訊號抑制因子 X (SixA)為一種磷酸組胺酸磷酸酶，負責去磷酸化特定蛋白。過去實驗室的研究指出，剔除sixA基因可降低腸出血性大腸桿菌 EDL933 菌株的毒性。因此，本研究旨在探討SixA在 EDL933 毒力調控中的作用。本研究首先證實，SixA突變株中數個毒力基因的表現下調，而在 SixA 回補株中則恢復正常表現。由於鐵是細菌生長與代謝所必需的重要微量元素，參與多種生理反應，因此鐵的獲取也被認為是一項毒力因子的判斷，因此我們想要了解SixA是否會影響EDL933獲取鐵的能力進而去影響毒力的發展，因此，我們探討SixA是否影響鐵攝取，然而實驗結果顯示在缺失sixA的條件下，鐵的攝取不會受到影響。在蛋白質純化過程中，我們透過質譜分析鑑定到可能與 SixA 交互作用的候選蛋白 RplB 和 SlyD，隨後以GST下拉試驗與細菌雙雜交系統驗證，但均未顯示有結合現象。接著，我們進行蛋白質晶片篩選，發現三個可能與 SixA 交互作用的蛋白質：YgiF、YafJ 及 YafS。為進一步驗證，我們再次採用細菌雙雜交系統，但雙雜交實驗仍未出現陽性結果，而我們再次進行GST下拉試驗，YgiF 與 YafS 並未顯示與 SixA 有可偵測的交互作用，而 YafJ 則顯示出可能的結合現象。未來，可以直接透過比較WT、∆sixA mutant以及回補株的磷酸化差異，進而了解SixA是如何影響何種蛋白質的磷酸化，從而改變腸出血性大腸桿菌的毒性。

Enterohemorrhagic Escherichia coli (EHEC) is a pathogenic strain of E. coli that poses a significant threat to public health, with the O157:H7 serotype being one of the most prevalent. EHEC infections are transmitted through contaminated food and water, leading to severe foodborne illnesses. These can range from mild diarrhea to severe abdominal pain, hemorrhagic colitis, and even hemolytic uremic syndrome (HUS), which is characterized by hemolytic anemia, thrombocytopenia, and acute kidney injury. Since the use of antibiotics may induce prophages in EHEC to enter the lytic cycle, leading to the release of Shiga toxin and worsening the patient's condition, understanding its pathogenic mechanisms and developing strategies to reduce its virulence are particularly important in the absence of effective treatments. Signal inhibitory factor X (SixA) is a phosphohistidine phosphatase responsible for dephosphorylating target proteins. Previous research by Ms. Pei-Yu Hsu showed that the deletion of the sixA gene reduces the toxicity of the EHEC strain EDL933. Therefore, this study aims to investigate how SixA regulates toxicity in EDL933. We first demonstrated that several virulence genes were downregulated in the sixA mutant strain, and their expression was restored in the SixA-complemented strain. Since iron is an essential micronutrient required for bacterial growth and metabolism and is involved in various physiological processes, iron acquisition is considered a key virulence factor. Therefore, we aim to investigate whether SixA affects the ability of EDL933 to acquire iron, thereby influencing the development of its virulence. We next examined whether SixA affects iron uptake; however, iron uptake was not affected by the absence of sixA. During protein purification, we identified candidate proteins that may interact with SixA—RplB and SlyD—via mass spectrometry (MS) analysis. Subsequently, we performed a protein microarray screening and identified three candidate proteins that may interact with SixA: YgiF, YafJ, and YafS. To further validate these interactions, we employed the bacterial two-hybrid system; however, no positive interactions were detected. We then carried out GST pull-down assays, in which no binding was observed between SixA and either YgiF or YafS, whereas YafJ displayed a possible binding with SixA. In the future, we can directly compare the phosphorylation differences between WT, ∆sixA mutant and complemented strains by mass spectrometer to understand how SixA affects the phosphorylation of certain proteins, thereby changing the toxicity of EHEC.

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