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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 王錦堂(Jin-Town Wang) | |
dc.contributor.author | Li-Yin Lai | en |
dc.contributor.author | 賴俐吟 | zh_TW |
dc.date.accessioned | 2021-06-17T07:30:29Z | - |
dc.date.available | 2021-02-23 | |
dc.date.copyright | 2021-02-23 | |
dc.date.issued | 2020 | |
dc.date.submitted | 2020-12-20 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/73360 | - |
dc.description.abstract | 海洋分枝桿菌和結核分枝桿菌在親緣關係上屬於極為密切的菌株,會造成變溫動物感染並引起系統性類結核病,也會造成人類皮膚的感染,大部分分枝桿菌屬的細菌其滑行能力與生物膜形成及毒力相關,在我們的研究中,我們使用海洋分枝桿菌NTUH-M6885 的2,304 株跳躍子突變株篩選了海洋分枝桿菌中滑動能力相關的基因,並鑑定了具有降低滑動運動性的5 株跳躍子突變株,其中3 株突變株分別被跳躍子擊中屬於第七型分泌系統的ESX-1 系統的相關基因,包含espE(MMAR_5439)、espF(MMAR_5440)和eccA1(MMAR_5443)。接著,我們利用即時聚合酶鏈反應分析espE、espF 和eccA 所座落的操縱組範圍包含MMAR_5438 至MMAR_5450,同時我們也建構espE、espF、espG(MMAR_5441)和espH(MMAR_5442)缺失突變株進行分析,由結果顯示espE、espF、espG 和espH 缺失突變株的滑動能力和生物膜形成能力有顯著降低,而基因互補菌株能夠恢復其滑動和生物膜形成的能力,這些結果表明ESX-1 系統在海洋分枝桿菌NTUH-M6885 的滑動能力和生物膜形成中扮演重要的角色。 結核病由結核分枝桿菌所引發的傳染性呼吸系統疾病,不僅名列全球十大死因之一,也是國內法定傳染病中每年確診數及死亡數最多的疾病,結核病成功的治療需要長期使用多種抗生素,而利福平是治療結核病的一線藥物,抗利福平結核分枝桿菌的發展將使治療更加困難。本研究針對台灣抗利福平臨床分離菌株作為研究對象,其中四株利福平抗藥性菌株其RpoB 分別帶有G158R V170F、V168A V170F、V170F S188P 及S431G Q432insQ 的胺基酸序列改變,而在過去研究中RpoB G158R、RpoB V168A、RpoB S188P 及RpoB Q432insQ 皆未被證實與抗藥性有關,為了確認上述基因突變與利福平抗性產生的關聯,將攜帶這些突變的rpoB 基因的質體(pMN437)轉型到結核分枝桿菌H37Rv 參考菌株中,其實驗結果顯示G158R、V168A 和S188P 不影響利福平的最小抑制濃度(MIC),然而,攜帶RpoB Q432insQ 的H37Rv 其利福平的MIC 增加,為了進一步確認利福平抗性與Q432insQ 之間的相關性,我們使用攜帶插入片段的rpoB 片段(編碼RpoB Q432insQ)的自殺式質體(pGOAL19)利用同源重組置換H37Rv 的rpoB 片段,所有表達RpoB Q432insQ 的突變株均對利福平具有抗性(MIC > 1 mg/L),這些結果表示RpoB Q432insQ 在結核分枝桿菌中引起利福平抗性。 | zh_TW |
dc.description.abstract | Mycobacterium marinum is a close relative of M. tuberculosis that can cause systemic tuberculosis-like infections in ectotherms and skin infections in humans. The sliding motility in Mycobacterium spp. correlates with biofilm formation and virulence in most species. In this study, we examined sliding-associated genes in M. marinum. We used a sliding motility assay to screen 2,304 transposon mutants of M. marinum NTUH-M6885 and identified 5 transposon mutants with decreased sliding motility. Transposons interrupted on the Type VII secretion system ESX-1-related genes, espE (MMAR_5439), espF (MMAR_5440) and eccA1 (MMAR_5443), were present in 3 mutants. We performed reverse-transcription polymerase chain reaction to verify genes from MMAR_5438 to MMAR_5450, which were found to belong to a single transcriptional unit. Deletion mutants of espE, espF, espG (MMAR_5441), and espH (MMAR_5442) displayed significant attenuation regarding sliding motility and biofilm formation. Complementation restored sliding motility and biofilm formation. Thus, the M. marinum ESX-1 secretion system mediates sliding motility and is crucial for biofilm formation. Tuberculosis (TB) is an infectious respiratory disease caused by Mycobacterium tuberculosis. It is not only one of the top 10 causes of death worldwide but also the highest incidence and mortality rate among all communicable diseases in Taiwan. Treating TB is challenging; successful treatment requires a long course of multiple antibiotics. Rifampicin (RIF) is a first-line drug for treating TB, and the development of RIF-resistant M. tuberculosis makes treatment even more difficult. In this study, we focus on clinically isolated RIF resistant M. tuberculosis in Taiwan. Four isolates had high-level RIF resistance and unique mutations encoding RpoB G158R, RpoB V168A, RpoB S188P, and RpoB Q432insQ, respectively. To evaluate their correlation with RIF resistance, plasmids (pMN437) carrying rpoB genes encoding these mutant proteins were transfected into the H37Rv reference strain. The plasmid complementation of RpoB indicated that G158R, V168A, and S188P did not affect the minimum inhibitory concentration (MIC) of RIF. However, the MIC of RIF was increased in H37Rv carrying RpoB Q432insQ. To confirm the correlation between RIF resistance and Q432insQ, we cloned an rpoB fragment carrying the insertion (encoding RpoB Q432insQ) into H37Rv by homologous recombination using a suicide vector (pGOAL19). All replacement mutants expressing RpoB Q432insQ were resistant to RIF (MIC > 1 mg/L). These results indicate that RpoB Q432insQ causes RIF resistance in M. tuberculosis. | en |
dc.description.provenance | Made available in DSpace on 2021-06-17T07:30:29Z (GMT). No. of bitstreams: 1 U0001-0912202015014900.pdf: 4796266 bytes, checksum: 5178267ed233f98e5067853214e5d99f (MD5) Previous issue date: 2020 | en |
dc.description.tableofcontents | 口試委員會審定書 i 誌謝 ii 中文摘要 iii Abstract v 目 錄 viii 表 目 錄 xiv 圖 目 錄 xv 第一章 前言 1 1-1 海洋分枝桿菌(Mycobacterium marinum) 1 1-2 滑動能力(Sliding ability) 2 1-3 生物膜形成能力(Biofilm formation ability) 3 1-4 第七型分泌系統(Type VII secretion system;T7SS) 4 1-5 結核分枝桿菌(Mycobacterium tuberculosis) 5 1-6 結核病(Tuberculosis;TB) 6 1-7 結核病的診斷(Tuberculosis diagnosis) 8 1-8 結核病之治療(Tuberculosis treatment) 9 1-9 抗利福平結核病(Rifampicin-resistant tuberculosis;RR-TB) 10 第二章 材料與方法 12 2-1 菌種(Bacteria strains) 12 2-2 建立跳躍子突變基因庫(Transposon mutant library) 12 2-3 DNA 萃取(DNA extraction) 13 2-4 半隨機聚合酶鏈反應(Semi-random polymerase chain reaction;semi-random PCR) 14 2-6 快速擴增基因5’ 端(5’-rapid amplification of cDNA ends;RACE) 16 2-7 即時定量聚合酶鏈反應(Q-RT-PCR) 16 2-8 建構缺失突變株(Deletion mutants) 17 2-9 建構互補菌株(Complementation strains) 17 2-10 測量生長曲線(Growth curve) 18 2-11 測量生物膜形成(Biofilm formation) 18 2-12 共軛焦顯微鏡觀察生物膜形成(Confocal laser scanning microscopy;CLSM) 19 2-13 抗酸染色(Acid fast stain) 20 2-14 二維薄層層析(Two-dimensional thin-layer chromatography;2D-TLC) 20 2-15 細菌對溶劑之黏附(Bacterial adhesion to solvents;BATS) 21 2-16 海洋分枝桿菌表面電荷(surface chage) 21 2-17 西方墨點法(Western blotting) 22 2-18 阿爾瑪藍微量盤法(Microplate alamar blue assay;MABA) 24 2-19 瓊脂稀釋法(Agar dilution method) 25 2-20 錯配擴增突變測定聚合酶鏈反應(Mismatch amplification mutation assay polymerase chain reaction;MAMA-PCR) 25 2-21 建構質體轉型突變株(Plasmid transformation) 25 2-22 單股去氧核醣核酸重組(Single-stranded DNA recombineering) 26 2-23 利用pGOAL19 建構點突變株(Site-directed mutagenesis) 26 2-24 引子訂購及定序服務(Primer ordering and sequencing services) 27 2-25 蛋白結構電腦分析(Protein structure determination) 27 2-26 統計分析(Statistical analysis) 28 第三章 結果 29 3-1 利用滑行能力篩選跳躍子突變株基因庫(Screening an M. marinum transposon library for sliding motility) 29 3-2 分析跳躍子破壞的基因及插入位點(Analyzation of the insertion sites on the destroyed genes) 30 3-3 測量跳躍子突變株的生長速度(Detection of growth rates in transposon mutants) 30 3-4 找ESX-1 基因簇中與滑動基因相關的操縱組及其5’ 端的轉錄起始點(Transcriptional units and 5’ start site of sliding related gene operon in the ESX-1 gene cluster) 31 3-5 建構海洋分枝桿菌espE、espF、espG、espH 和eccA1 的基因缺失突變株(Construction of unmarked deletion mutants of espE, espF, espG and espH and eccA1 in M. marinum) 32 3-6 測量基因缺失突變株的滑動能力(Measurements of the sliding motility of unmarked deletion mutants) 34 3-7 建構ESX-1 相關的基因回補菌株(Construction of ESX-1 related gene complemented strains) 35 3-8 測量基因回補株的滑動能力(Measurements of the sliding motility of complemented strains) 35 3-9 測量突變株的生物膜形成能力(Measurements of the biofilm formation in mutants) 36 3-10 海洋分枝桿菌突變株的型態變化(The morphological changes in M. marinum mutants) 37 3-11 測量海洋分枝桿菌表面疏水性、表面電性及LOS 生合成(Measurements of the surface charge, hydrophobicity and LOS biosynthesis of M. marinum) 37 3-12 測量22-B5 的espE、espF、espG、espH 和eccA1 的基因表現量(Quantification of mRNA levels of espE, espF, espG, espH, and eccA1 in strain 22-B5) 38 3-13 △espE、△espF、△espG 和△espH 的EsxB 分泌量(EsxB Secretion in △espE, △espF, △espG and △espH mutants) 39 3-14 建構結核分枝桿菌基因缺失突變株(Construction of deletion mutants in M. tuberculosis) 39 3-15 espG1 和espH1 不影響結核分枝桿菌的滑動能力(The espG1 and espH1 genes do not affect sliding motility of M. tuberculosis) 41 3-16 espG1 和espH1 不影響結核分枝桿菌的生物膜形成(The espG1 and espH1 genes do not affect the biofilm formation of M. tuberculosis) 41 3-17 阿爾瑪藍微量盤法與瓊脂稀釋法進行藥敏試驗之比較(Drug susceptibility test by MABA compared with agar dilution assay) 42 3-18 篩選點突變株的檢驗方法(The process for distinguishing the point mutations) 43 3-19 建立建構結核分枝桿菌跳躍子突變菌株庫的實驗條件(Establishing the experimental conditions for generation of M. tuberculosis transposon mutant library) 44 3-20 定序檢測臨床抗藥菌株的突變位點(Detection of mutation sites in clinical drug-resistant strains by sequencing) 45 3-21 利用質體轉型建構RpoB 突變株(Construction of RpoB mutant strains by plasmid transformation) 47 3-22 阿爾瑪藍微量盤法測量RpoB 突變株的RIF 最小抑制濃度(Measurement of the MIC of RIF in RpoB mutants by MABA) 48 3-23 建構點突變株(Construction of site-directed mutagenesis strains) 49 3-24 RpoB Q432insQ 可能影響RIF 和RpoB 的結合力(RpoB Q432insQ decreases the binding ability between RpoB and RIF) 51 第四章 討論 53 第五章 結論 61 參考文獻 62 | |
dc.language.iso | zh-TW | |
dc.title | i. 海洋分枝桿菌ESX-1 分泌系統參與滑動運動和生物膜形成 ii. 結核分枝桿菌RpoB Q432insQ 造成利福平抗藥性 | zh_TW |
dc.title | i. Mycobacterium marinum ESX-1 secretion system involved in sliding motility and biofilm formation ii. A glutamine insertion at codon 432 of RpoB confers rifampicin resistance in Mycobacterium tuberculosis | en |
dc.type | Thesis | |
dc.date.schoolyear | 109-1 | |
dc.description.degree | 博士 | |
dc.contributor.oralexamcommittee | 董馨蓮(Shin-Lian Doong),林妙霞(Miao-Hsia Lin),陳宜君(Yee-Chun Chen),賴信志(Hsin-Chih Lai) | |
dc.subject.keyword | 海洋分枝桿菌,結核分枝桿菌,滑動能力,生物膜形成,第七型分泌系統,利福平抗藥性結核病,rpoB, | zh_TW |
dc.subject.keyword | Mycobacterium marinum,Mycobacterium tuberculosis,Type VII secretion system,Sliding ability,Biofilm formation,Rifampicin resistant TB,rpoB, | en |
dc.relation.page | 136 | |
dc.identifier.doi | 10.6342/NTU202004408 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2020-12-21 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 微生物學研究所 | zh_TW |
顯示於系所單位: | 微生物學科所 |
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