鼠傷寒沙門氏菌fimU基因的表現

Expression of fimU in Salmonella enterica serovar Typhimurium

吳幸蓉(Hsing-Jung Wu)

指導教授 ： 葉光勝

國立臺灣大學/獸醫專業學院/獸醫學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201903197

摘要

沙門氏菌是常見的食源性病原菌，也是造成全球食物中毒以及人畜共通感染爆發的重要致病原。沙門氏菌的線毛可以通過黏附蛋白與上皮細胞的寡甘露醣甘鏈之醣蛋白結合，吸附在細胞上。細菌合成線毛的調控複雜且涉及許多基因，鼠傷寒沙門氏菌具有13種不同的線毛基因組，而第一型線毛由fim基因所轉譯，包含了6個結構基因fimA、fimI、fimC、fimD、fimH和fimF以及5個調控基因fimZ、fimY、stm0551、fimW和fimU。其中fimU基因產物tRNAArg (UCU)為可以識別稀有精氨酸密碼子AGA或AGG的tRNA。鼠傷寒沙門氏菌的fimU與大腸桿菌argU具有相似性。在一項研究中顯示AGA / AGG可以停止轉譯並使核醣體增加以至於蛋白質折疊。先前的研究顯示，tRNAArg (UCU) 藉由辨識FimY中的稀有精氨酸密碼子來調控第一型線毛的表現。本研究對fim的基因序列進行分析，顯示出FimI、FimD、FimZ、Stm0551和FimW也具有可被tRNAArg (UCU)識別的精氨酸密碼子。雖然fimU可以藉著辨識稀有密碼子而做轉譯上的調控，但不知fimU受何種因素所調控。由反轉錄聚合酵素鏈鎖反應的分析顯示，當鼠傷寒沙門氏菌在靜置的培養液或固態培養基上生長時，fimU的表達是相同的。同樣的現象也在鼠傷寒沙門氏菌中的fimZ、fimY、fimW、phoQ和crp基因突變菌株上發現。研究也仿照了細菌隨著食物到達腸胃，會經歷了不同的pH值變換、細菌到達人體的溫度差，以及高溫條件，發現fimU表現皆一致。但不同的是在pH值轉變測試下，只有在pH 3.5的酸性環境，能誘導第一型線毛表現。而在溫度轉變測試下，唯獨在高溫條件下，不表現第一型線毛。由以上測試得知，fimU基因可能為鼠傷寒沙門氏菌內的管家基因，任何環境條件都會持續表現。為了觀測fimU基因與第一型線毛的直接關係，在鼠傷寒沙門氏菌中轉形一個帶有fimU的質體，可以加速誘導第一型線毛表現，酵母菌的凝集片段也比野生型的沙門氏菌來的大。目前測試的環境因子以及調控蛋白不足以影響fimU的表現，但細菌體內有額外的fimU存在的確會促進第一型線毛的表現。fimU基因仍有許多特性值得後續研究。

關鍵字

沙門氏菌 ； 第一型線毛 ； fimU ； tRNAArg (UCU) ； 稀有精氨酸密碼 子

並列摘要

Salmonella is a commonly seen causative agent of food poisoning and food-borne zoonotic infections outbreaks worldwide. Fimbriae of Salmonella can bind to the oligomannosidic glycoproteins on the epithelial cells through the adhesion protein and adhere to the cells. Regulation of fimbrial biosynthesis in bacteria is complicate and involves a number of genes. There are 13 different fimbrial gene clusters in S. Typhimurium. Expression of the type 1 fimbriae in S. Typhimurium is encoded by the fim gene cluster, which is composed of six structural genes fimA, fimI, fimC, fimD, fimH and fimF, and five regulatory genes fimZ, fimY, stm0551, fimW, and fimU. The regulatory gene fimU encodes a tRNAArg (UCU) that recognizes the rarely used arginine codon AGA or AGG. The fimU of S. Typhimurium is related to the Escherichia coli argU. In one study, it was shown that AGA /AGG can stop the translation and increase the ribosome for protein folding. Previous studies had revealed that tRNAArg (UCU) controls type 1 fimbrial expression by recognizing the rarely used arginine codon within FimY. Our sequence analysis revealed that FimI, FimD, FimZ, Stm0551, and FimW also possessed such arginine codons that can be recognized by tRNAArg (UCU). Although fimU can regulate the translation of the protein by recognizing the rarely used arginine codons, it is not known what factors may regulate the expression of fimU. Reverse-transcription polymerase-chain reaction indicated that the expression of fimU was identical when S. Typhimurium was grown in either static broth or on the agar plate. This same fimU expression pattern in both culture conditions was also observed when the fimZ, fimY, fimW, phoQ, and crp mutant strains were tested. Our study also simulated the environmental cues that S. Typhimurium may encounter when passing through the gastrointestinal tract with food. These conditions included the pH changes, temperature shift, and high temperature. The expression of fimU is consistent under such conditions. Nevertheless, The acidic condition pH 3.5 induced the expression of type 1 fimbriae. Under the temperature conversion test, only high temperature inhibited type 1 fimbrial expression. From the above tests, it is possible that fimU gene is a housekeeping gene in S. Typhimurium and fimU is constitutively expressed express under different conditions. Transforming a recombinant plasmid possessing the fimU gene into S. Typhimurium accelerated the production of type 1 fimbriae, yielding larger agglutination fragments than the parental strain did. From the above results, the current environmental factors and regulatory proteins tested did not influence the expression of fimU. it is known that environmental factors cannot affect the expression of fimU. However, the presence of extra fimU plasmid did activate the expression of type 1 fimbriae. The fimU gene does possess some characteristics to be further explored.

並列關鍵字

Salmonella ； type 1 fimbriae ； fimU ； tRNAArg (UCU) ； rarely used arginine codon

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