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標題: | 探討銅離子於尿道致病性奇異變形桿菌中表面移行與抗氧化壓力所扮演的角色及其銅解毒系統 The effects of copper on swarming motility, oxidative stress tolerance and the copper detoxification system in uropathogenic Proteus mirabilis |
作者: | Wei-Syuan Huang 黃偉軒 |
指導教授: | 廖淑貞(Shwu-Jen Liaw) |
關鍵字: | 銅,奇異變形桿菌,表面移行,抗氧化壓力,銅解毒系統,金屬壓力,細胞分化, copper,proteus,mirabilis,swarming,oxidative stress,detoxification,metal stress,cell differentiation, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 銅,為生命必須微量元素,但累績過多卻也對細胞造成氧化傷害。因此,細菌演化出銅解毒系統嚴格調控體內銅含量。革蘭氏陰性的Proteus mirabilis為臨床常見的尿道致病菌,常伺機感染長期植入尿導管的病人。研究發現人體泌尿道感染時,尿銅濃度顯著增加作為抵禦病菌的武器。因此,本文的目的欲探討銅對於P. mirabilis毒力因子的影響。我們發現在不影響生長的1 mM銅濃度下可抑制P. mirabilis的表面移行能力,泳動、溶血素、尿素酶與細胞分化也受到抑制,但提升生物膜生成能力。此外,低濃度的銅則提升抗氧化能力,但不影響藥物感受性。 我們利用transcriptome搭配蛋白質直系同源群(COGs)分析,發現參與細胞分化的基因中cueO受銅induce最多,為了探討CueO對於P. mirabilis表面移行是否扮演角色,我們先過度表現cueO發現移行能力與細胞分化受抑制,之後構築cueO突變株,分析其與野生株在表現型的差異。結果顯示cueO突變株的表面移行與細胞分化提升,泳動、鞭毛生成、生物膜生成、尿素酶活性與尿結石生成能力與野生株並無差異,而抗氧化能力則顯著下降。此結果顯示CueO不參與在銅抑制鞭毛表現的路徑,但是參與P. mirabilis細胞分化。已有研究顯示UPEC與Pseudomonas aeruginosa中CueO與Fe3+ permease共同參與Fe2+的攝取調控。UPEC cueO缺失造成Fe2+累積且UPEC與Salmonella cueO缺失皆導致對H2O2感受性提升。我們推論cueO突變株的抗氧化能力下降可能肇因於細菌體內Fe2+累積,也暗示P. mirabilis cueO在鐵攝取可能扮演角色。 已有許多研究顯示,當細菌遇到外界銅壓力時,CueR結合Cu+活化並提升cueO及copA的表現,CopA為P-type ATPase將Cu+ pump out periplasm,再由CueO將Cu+氧化為毒性較低的Cu2+。為了探討P. mirabilis是否也具有這樣的調控路徑,我們用real-time PCR證明1 mM銅環境下copA與cueO基因表現增加,兩者的promoter活性在有銅環境下也顯著提升。最後,利用實驗室前人建構的cueR跳躍子突變株證明銅透過CueR的參與正調控copA與cueO。 總結,當外界出現銅壓力,P. mirabilis透過CueR提升cue銅解毒系統cueO與copA基因表現進而緩解銅毒性,cueO增加抑制細胞分化,銅壓力則可能透過其他路徑抑制flhDC表現導致表面移行能力下降。CueO在P. mirabilis除了緩解銅毒性也對抗氧化能力扮演角色,不排除在鐵攝取調控也扮演角色。 Copper, an essential trace element for all lives but toxic when intracellular accumulation, is also an anti-microbial agent used by the human immune system. Researches show that cooper effects bacterial virulence, thus, bacteria evolve copper detoxification system to maintain intracellular homeostasis. Proteus mirabilis with swarming characteristic often causes catheter-associated urinary tract infection, however, the effects of copper on P. mirabilis remains unclear. First, we found that 1 mM of copper decreased the swarming motility but not effected the growth, and reduced swimming motility, haemolysin, urease activity but enhance biofilm formation under this condition. To investigate the effect of copper on swarming motility, flagellin level and cell differentiation were examined. We found that flagellin levels significantly reduced and shorter swarmer cells under copper stress. Using transcriptomic analysis and the clusters of orthologous groups of proteins database, we found that cueO, encoding a periplasmic multicopper oxidase, increased 29-fold in cell division category. We further investigate the role of cueO in cell differentiation and found that overexpression cueO decreased swarming motility and swarmer cell length. Then, we constructed the cueO mutant and found that enhanced swarming motility and cell differentiation compared to wild-type. Loss of cueO did not affect swimming motility, flagella levels, biofilm forming ability, urease activity and urine stone-forming ability, suggesting that cueO may play a role in cell differentiation in P. mirabilis. Researches showed that CueO took part in ferrous iron acquisition with ferric iron permease in UPEC and Pseudomonas aeruginosa, loss of cueO caused ferrous iron accumulation and increased H2O2 sensitivity in UPEC and Salmonella, respectively. Our data indicated that lower tolerance of oxidative stress in the cueO mutant may due to ferrous iron accumulation. When encountered environmental copper stress, CueR sensed copper and upregulated copA and cueO for detoxification in many bacteria. A putative cue detoxification system encoded by cueO, cueR, and copA, was found in P. mirabilis and showed high protein similarities to E. coli. We confirmed that the copper-induced expression of cueO and copA through CueR regulation. In summary, CueR activated cue detoxification system through upregulating cueO and copA under copper stress in P. mirabilis. Upregulation of cueO affected cell differentiation, and copper stress, instead, reduced flhDC expression via unknown regulators. Both contributed to reducing swarming motility under copper stress in P. mirabilis. Also, cueO may play a role in ferrous iron uptake and alleviation of oxidative stress in P. mirabilis. To the best of our knowledge, this is the first report investigating the effects of copper on virulence factors and the role of cueO in P. mirabilis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70838 |
DOI: | 10.6342/NTU202004136 |
全文授權: | 有償授權 |
顯示於系所單位: | 醫學檢驗暨生物技術學系 |
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