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研究生:邱祖廷
研究生(外文):Tsu-Ting Chiu
論文名稱:Stenotrophomonas maltophilia smeU1-V-W-U2-X Operon於氧化壓力反應中之角色
論文名稱(外文):Roles of smeU1-V-W-U2-X Operon in Oxidative stress response of Stenotrophomonas maltophilia
指導教授:楊翠青
指導教授(外文):Tsuey-Ching Yang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術暨檢驗學系
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:48
中文關鍵詞:Stenotrophomonas maltophiliasmeU1-V-W-U2-X Operon氧化壓力
外文關鍵詞:Stenotrophomonas maltophiliasmeU1-V-W-U2-X OperonOxidative stress response
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Stenotrophomonas maltophilia 為臨床上常見的院內感染的伺機性病原菌,具有多重抗藥性,而輸出幫浦為其重要機制之一。除了排出抗生素受質外,許多研究指出輸出幫浦也具有其他非抗藥之生理功能,如:緩解細菌所面臨之氧化壓力。S. maltophilia K279a目前已知有八套 RND-type 輸出幫浦,分別為 SmeABC、SmeDEF、SmeGH、SmeIJK、SmeMN、SmeOP、SmeVWX及SmeYZ。其中,SmeVWX 輸出幫浦和其他 RND efflux pumps 不同,其由五個基因分別為(smeU1VWU2X)組成一個 operon。RND-type 輸出幫浦組成之膜間蛋白、內膜蛋白及外膜蛋白分別由 smeV、smeW 及 smeX 轉譯。smeU1 及 smeU2 所轉譯的蛋白為short-chain dehydrogenases/reductases (SDRs)可催化NAD(P)(H)-dependent氧化還原反應。調控基因smeRv反向位於 smeU1VWU2X operon 上游,可正調控smeU1VWU2X operon之表現。先前研究已知,在不外加任何刺激下smeU1VWU2X operon 為不表現,其大量表現時造成 tetracycline、quinolones 及chloramphenicol 抗藥性。本研究主要探討 S. maltophilia 中 SmeVWX 輸出幫浦與緩解氧化壓力之間的關係。研究結果顯示:(i)在氧化壓力 (H2O2及MD) 刺激下,會促使 smeU1VWU2X operon 表現上升,而抗生素受質並不會使其表現上升。(ii)當 smeU1VWU2X operon 大量表現時,能緩解 MD 氧化壓力,但smeU1VWU2X operon對於緩解H2O2氧化壓力貢獻不明顯。(iii)SmeU2及SmeVWX個別存在時即有緩解MD氧化壓力的功能,兩者共同存在時,緩解MD氧化壓力具有加成性。(iv) H2O2 促使smeU1VWU2X operon表現上升受到smeRv之正調控,但不受oxyR 及soxR調控。(v) MD 促使 smeU1VWU2X operon表現上升完全受 soxR 正調控及部分smeRv正調控,而不受 oxyR 所調控。
Stenotrophomonas maltophilia is a common nosocomial pathogens characterized by resistance to a variety of antimicrobial agents. Efflux pumps overexpression is a major mechanism contributing to antibiotics resistance. In addition to antibiotics resistance, efflux pumps also have other physiological roles in the environmental adaption, for example, alleviation of oxidative stress. S. maltophilia K279a harbors eight putative RND efflux pumps, including SmeABC, SmeDEF, SmeGH, SmeIJK, SmeMN, SmeOP, SmeVWX, and SmeYZ. Of them, SmeVWX efflux pump proteins, distinct from other RND efflux pumps, are encoded by a five-gene operon, smeU1VWU2X. Proteins encoded by smeV, smeW, and smeX are the membrane fusion protein, inner membrane protein, and outer membrane protein, respectively. The proteins encoded by smeU1 and smeU2 are short-chain dehydrogenases/reductases (SDRs), which involved in catalyzing NAD(P)(H)-dependent oxidation/reduction reactions. Regulator gene smeRv is divergently located upstream from the smeU1VWU2X operon and plays a positive regulatory role in smeU1VWU2X expression. It have been known that smeU1VWU2X operon is intrinsically unexpressed and overexpression of SmeVWX pump contributes to resistance toward tetracyclines, quinolones, and chloramphenicol. This study aimed to characterize the relation between smeU1VWU2X operon and oxidative stresses alleviation. The result demonstrated that (i) SmeU1VWU2X operon was upregulated by the challenge of H2O2 and MD, but not the antibiotics extruded by SmeVWX pump. (ii) Overexpression of smeU1VWU2X operon participated in alleviation of MD-mediated oxidative stresses, but not H2O2-mediated oxidative stresses. (iii) SmeU2 and SmeVWX pump individually were involved in oxidative stress alleviation, and this function was enhanced when SmeU2 and SmeVWX pump were simultaneously present. (iv) H2O2-mediated smeU1VWU2X operon upregulation was positively regulated by smeRv, but less related to oxyR and soxR. (v) MD-mediated smeU1VWU2X operon upregulation totally depended on soxR, partially relied on smeRv, and was little affected by oxyR.
目錄
中文摘要... i
Abstract ... ii
目錄... iv
圖目錄... vii
表目錄... viii
第一章緒論 ... 1
第一節 研究背景 ... 1
1.1.1 Stenotrophomonas maltophilia ... 1
1.1.2 輸出幫浦(efflux pumps)之介紹 ... 1
1.1.3 S. maltophilia之 RND-type 輸出幫浦 ... 2
1.1.4 S. maltophilia之SmeVWX輸出幫浦 ... 3
1.1.5 RND-type輸出幫浦之生理功能... 4
1.1.6 氧化壓力(oxidative stress) ... 5
第二節 研究目的 ... 5
第二章 材料與方法 ... 7
第一節 研究材料 ... 7
2.1.1 質體與菌株 ... 7
2.1.2 培養基 ... 7
2.1.3 PCR 引子 ... 7
2.1.4 實驗抗生素及藥品 ... 7
2.1.5 試劑與緩衝溶劑 .... 8
2.1.6 儀器設備 ... 8
第二節 實驗方法 ... 9
2.2.1 菌種的培養與保存 ... 9
2.2.2聚合酶連鎖反應 (polymerase chain reaction, PCR) ... 9
2.2.3洋菜膠體電泳分析 (agarose gel electrophoresis) ... 9
2.2.4菌落快速檢驗聚合酶連鎖反應 (Colony PCR) ... 10
2.2.5勝任細胞 (competent cell) 之製備 ... 10
2.2.6 轉型作用 (transformation) ... 10
2.2.7接合作用 (conjugation) ... 11
2.2.8. C23O (catechol 2,3-dioxygenase) 活性測試 ... 11
2.2.9即時定量聚合酶連鎖反應 (qRT-PCR)... 11
2.2.10 H2O2感受性試驗 ...12
2.2.11 menadione耐受性試驗 ... 12
第三章 研究結果 ... 13
第一節 氧化壓力(H2O2及menadione)會促使 smeU1VWU2X operon 表現上升 ... 13
第二節 探討 smeRv-smeU1VWU2X cluster 在細菌緩解氧化壓力所扮演的角色 ... 14
3.2.1 smeRv-smeU1VWU2X cluster不參與H2O2壓力之緩解 ... 14
3.2.2 smeRv-smeU1VWU2X cluster參與menadione壓力之緩解 ... 14
第三節 探討soxR、oxyR及smeRv在H2O2促使smeU1VWU2X operon表現所扮演之調控角色 ... 16
第四節 探討soxR、oxyR 及 smeRv 在 menadione 促使 smeU1VWU2X operon表現所扮演之調控角色 ... 17
3.4.1 soxR和smeRv對於MD-mediated smeU1VWU2X upexpression為正調控 ...17
3.4.2 soxR正調控smeRv之轉錄表現 ...17
第四章 結論 ... 18
第五章 討論 ... 19
參考文獻... 41
附錄... 46

圖目錄
Fig. 1. KJVWX23之示意圖...23
Fig. 2. KJVWX23在抗生素壓力及氧化壓力下表現的C23O活性...24
Fig. 3. KJ在H2O2氧化壓力下smeW、smeX及smeRv之RNA表現量...25
Fig. 4. KJ、KJ09C及KJ09CΔ5之H2O2氧化壓力耐受性試驗...26
Fig. 5. KJ09C與其所衍生之smeRv-smeU1VWU2X cluster相關突變菌株之menadione耐受性試驗...27
Fig. 6. KJ與其所衍生之smeRv-smeU1VWU2X cluster相關突變菌株之menadione耐受性試驗...28
Fig. 7.KJ09C(pRK415)、KJ09C△5(pRK415)、KJ09C△5(pSmeU2)、KJ09C△U2(pRK415)及KJ09C△U2(pSmeX)之menadione耐受性試驗...29
Fig. 8. KJ、KJ△SoxR、KJ△OxyR及KJ△Rv在H2O2氧化壓力下smeW及smeX之RNA表現量...30
Fig. 9. soxR、oxyR及smeRv對於MD-mediated smeU1VWU2X operon upexpression之調控...31
Fig. 10. SoxR在有無外加氧化壓力下對於smeRv 轉錄之調控...32
Fig. 11. H2O2促使smeU1VWU2X operon表現之示意圖...33
Fig. 12. MD促使smeU1VWU2X operon表現及緩解之示意圖...34
表目錄
Table 1. KJ在H2O2氧化壓力下smeRv-smeU1VWU2X cluster之transcriptome分析...35
Table 2. 本次研究所使用之菌株、質體與引子...36
Table 3. 本研究中所使用的抗生素及藥品...39
Table 4. KJ、KJ09C及KJ09C△5之MIC...40
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