臺灣博碩士論文加值系統

人類結核病主要起因為結核分枝桿菌感染，在人類史上相當早即有感染案例，又稱為「白色瘟疫」，由於其感染後潛伏期長和抗藥性等因素，結核病的診斷及治療開發上仍有相當大的研究空間。而海洋分枝桿菌和結核分枝桿菌親緣關係相近且在第七型分泌系統具有高度保留性，因此許多關於結核分枝桿菌和結核病之研究皆以安全性較高和生長速度較快的海洋分枝桿菌作為模式生物。
在我們的研究中，即先以海洋分枝桿菌探討第七型分泌系統中的ESX-1對NLRP3發炎小體活化之影響，選擇實驗室先前建構之五株ESX-1前段基因突變株分別感染人類巨噬細胞後以酵素免疫分析法測量THP-1細胞受感染後第一型介白素β分泌差異，發現受∆espG感染之細胞其第一型介白素β分泌顯著下降，且細胞破裂情形亦有顯著下降的情形；此外在西方墨點法分析之結果中，亦發現受∆espG感染之細胞上清液可看到第一型胱天冬酶活化態表現下降；而將細胞預處理NLRP3抑制劑再以海洋分枝桿菌感染，從細胞上清液中第一型介白素β的分泌差異可間接證明海洋分枝桿菌誘發之第一型介白素β分泌為NLRP3發炎小體活化所造成，並可推測espG基因和NLRP3發炎小體活化途徑相關。
隨後我們建構結核分枝桿菌espG1缺失突變株及espG1回補菌株組，以相同實驗條件感染THP-1細胞，觀察細胞之第一型介白素β和第一型胱天冬酶蛋白表現差異。雖然espG1缺失確實影響兩者活化態之分泌量下降，回補株卻無法補回基因完整功能。
鑑於先前有文獻指出結核分枝桿菌之ESAT-6蛋白可影響巨噬細胞NLRP3發炎小體活化，於是我們以西方墨點法分析海洋分枝桿菌espG基因和結核分枝桿菌espG1基因所造成之第一型介白素β分泌差異是否和ESAT-6蛋白表現相關。根據實驗結果發現，∆espG和野生株相比，ESX-1主要致病蛋白ESAT-6和CFP-10在細菌上清液中表現皆下降，在細菌裂解液中則表現上升，推測espG可影響ESAT-6和CFP-10分泌；而結核分枝桿菌中亦可發現此趨勢，因此我們推測結核分枝桿菌espG1基因同樣可影響ESAT-6和CFP-10分泌。

Human tuberculosis, also known as "the white plague", mainly results from Mycobacterium tuberculosis infection which was found in early human history. Due to the long incubation period and drug resistance of M. tuberculosis, the researches of diagnosis and treatment in tuberculosis still remain a considerable development space. Generally, many studies on M. tuberculosis and tuberculosis use Mycobacterium marinum with higher safety and faster growth rate as a model in laboratory works. M. marinum shows high conservation with M. tuberculosis in genetic relationship and the type seven secretion system, which is critical for virulence.
In our study, first we used five constructed M. marinum ESX-1 gene deletion mutants: ∆espE, ∆espF, ∆espG, ∆espH and ∆eccA1 to investigate the role of the type seven secretion system ESX-1 in NLRP3 inflammasome activation. After infecting THP-1 differentiated macrophages with these deletion mutants respectively, we found ∆espG induced less IL-1β in cell supernatant through enzyme-linked immunosorbent assay (ELISA). Our data from Western blot also showed ∆espG reduced activated caspase-1 secretion in infected macrophages. Additionally, THP-1 cells pretreated with NLRP3 inhibitor before infected with wildtype M. marinum showed the difference in secretion of IL-1β, while ∆espG-infected cells did not show this pattern. The data could indirectly demonstrate that M. marinum induces IL-1β through activating NLRP3 inflammasomes, and espG may involve in NLRP3 inflammasome activation.
Subsequently, we constructed the M. tuberculosis espG1 deletion mutant and the espG1 complemented strains, infected THP-1 cells with M. tuberculosis, and analyzed the protein expression level of IL-1β and caspase-1 in cell lysate and supernatant. Although M. tuberculosis espG1 deletion mutant reduced the secretion of IL-1β significantly, the complemented strain did not repair the function of espG1 completely.
In view of the previous studies that ESAT-6 in M. tuberculosis is involved in activation of NLRP3 inflammasomes, we used Western blot to analyze whether the difference in IL-1β secretion is related to the expression of ESAT-6 in M. marinum espG and M. tuberculosis espG1. According to the results, the ESX-1 major virulent proteins, ESAT-6 and CFP-10, showed substantial decrease in both M. marinum ∆espG and M. tuberculosis ∆espG1::pMN437 supernatants respectively, but were increased in bacterial lysates. Thus, we suggested that M. marinum espG gene and M. tuberculosis espG1 gene are involved in secretion of ESAT-6 and CFP-10.

目錄
口試委員會審定書 I
誌謝 II
中文摘要 III
Abstract IV
第一章 緒論 1
1.1 海洋分枝桿菌 (Mycobacterium marinum) 1
1.2 結核分枝桿菌 (Mycobacterium tuberculosis) 2
1.3 第七型分泌系統 (Type VII secretion system) 3
1.4 NLRP3發炎小體 (NLRP3 Inflammasome) 4
1.5 研究目標 (Aim) 6
第二章 材料與方法 7
2.1 菌種 (Bacterial strains) 7
2.2 細胞株 (Cell line) 8
2.3 巨噬細胞感染 (Macrophage infection) 8
2.4 酵素免疫分析法 (Enzyme-linked immunosorbent assay；ELISA) 9
2.5 乳酸脫氫酶細胞毒性測試 (LDH Cytotoxicity Assay) 9
2.6 西方墨點法 (Western blot) 9
2.6.1感染海洋分枝桿菌之細胞裂解液樣本製備 9
2.6.2感染海洋分枝桿菌之細胞上清液樣本製備 10
2.6.3感染結核分枝桿菌之細胞裂解樣本液製備 10
2.6.4感染結核分枝桿菌之細胞上清樣本製備 10
2.6.5海洋分枝桿菌及結核分枝桿菌裂解液和上清液樣本製備 11
2.6.6蛋白樣本分離及抗體偵測 11
2.7 結晶紫測定 (Crystal violet Assay) 12
2.8 即時定量聚合酶連鎖反應 (Real-time PCR) 12
2.9 統計分析 (Statistical analysis) 13
第三章 結果 13
3.1 確認菌株 13
3.2 測試海洋分枝桿菌引發細胞第一型介白素分泌之適當感染劑量 13
3.3 分析海洋分枝桿菌缺失突變株引發細胞第一型介白素差異量 14
3.4 分析海洋分枝桿菌感染後細胞相對破裂比例 14
3.5 分析海洋分枝桿菌espG缺失突變株感染後細胞內外之第一型胱天冬酶及 15
第一型介白素蛋白表現 15
3.6 抑制NLRP3發炎小體以分析受海洋分枝桿菌espG缺失突變株感染後之 16
第一型介白素活化途徑 16
3.7 觀察海洋分枝桿菌espG缺失突變株之第七型分泌系統ESX-1主要致病蛋白表現 16
3.8 結核分枝桿菌espG1基因回補菌株組建構並測量espG1相對基因表現 17
3.9 分析結核分枝桿菌espG1缺失突變株及回補菌株組引發之細胞第一型介白素差異量 17
3.10 以西方墨點法分析受結核分枝桿菌espG1回補菌株組感染後細胞內外第一型胱天冬酶 及第一型介白素蛋白表現 18
3.11 觀察結核分枝桿菌espG1回補菌株組之第七型分泌系統ESX-1主要致病蛋白表現 18
3.12 以感染後細胞之第一型介白素分泌量差異篩選海洋分枝桿菌跳躍子突變株 19
第四章 討論 19
第五章 結語 22
第六章 參考文獻 40

圖表目錄
圖一、實驗菌株和質體之引子相對位置和目標基因位置示意圖 23
圖二、實驗所用引子之位置示意圖 24
圖三、以聚合酶連鎖反應確認海洋分枝桿菌缺失突變株 25
圖四、分析以海洋分枝桿菌不同菌量感染THP-1細胞後第一型介白素β分泌量 26
圖五、分析THP-1細胞受海洋分枝桿菌缺失突變株感染後第一型介白素β分泌量 差異及細胞破裂比例 27
圖六、分析感染後胞內外分泌的第一型胱天冬酶及第一型介白素β蛋白表現量 28
圖七、分析THP-1細胞預處理NLRP3抑制劑後受海洋分枝桿菌感染之第一型介白素β分泌量差異 29
圖八、以西方墨點法分析海洋分枝桿菌espG缺失突變株第七型分泌系統ESX-1之主要致病蛋白表現 30
圖九、以聚合酶連鎖反應確認結核分枝桿菌espG1基因回補菌株組 31
圖十、以即時定量聚合酶連鎖反應分析結核分枝桿菌espG1回補菌株組ESX-1基因群mRNA相對表現量 32
圖十一、分析THP-1細胞受結核分枝桿菌espG１缺失突變株及基因回補菌株組 33
圖十二、以西方墨點法分析結核分枝桿菌espG1基因回補菌株組感染後胞內外 34
圖十三、以西方墨點法分析結核分枝桿菌espG1基因回補菌株組於第七型分泌系統ESX-1之主要致病蛋白表現 35
圖十四、以受感染細胞第一型介白素β分泌量差異篩選海洋分枝桿菌致病力相關之跳躍子突變株組 36

1.Riera, J., et al., Septic arthritis caused by Mycobacterium marinum. Arch Orthop Trauma Surg, 2016. 136(1): p. 131-4.
2.Hashish, E., et al., Mycobacterium marinum infection in fish and man: epidemiology, pathophysiology and management; a review. Vet Q, 2018. 38(1): p. 35-46.
3.Johnson, M.G. and J.E. Stout, Twenty-eight cases of Mycobacterium marinum infection: retrospective case series and literature review. Infection, 2015. 43(6): p. 655-62.
4.M. A. Bhatty, D.P.J.T.a.S.T.C., Mycobacterium marinum hand infection- case reports and review of literature. 2000.
5.Rombouts, Y., et al., Structural analysis of an unusual bioactive N-acylated lipo-oligosaccharide LOS-IV in Mycobacterium marinum. J Am Chem Soc, 2010. 132(45): p. 16073-84.
6.Jason A. Jarzembowski, M., PhD; Michael B. Young, MD, Nontuberculous Mycobacterial Infections. Arch Pathol Lab Med, 2008.
7.Lai, L.Y., et al., Role of the Mycobacterium marinum ESX-1 Secretion System in Sliding Motility and Biofilm Formation. Front Microbiol, 2018. 9: p. 1160.
8.Stinear, T.P., et al., Insights from the complete genome sequence of Mycobacterium marinum on the evolution of Mycobacterium tuberculosis. Genome Research, 2008. 18(5): p. 729-741.
9.Tobin, D.M. and L. Ramakrishnan, Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis. Cell Microbiol, 2008. 10(5): p. 1027-39.
10.Kugelberg, E., Immune evasion: Mycobacteria hide from TLRs. Nat Rev Immunol, 2014. 14(2): p. 62-3.
11.Ramakrishnan, L., Revisiting the role of the granuloma in tuberculosis. Nat Rev Immunol, 2012. 12(5): p. 352-66.
12.Aubry, A., et al., Mycobacterium marinum. Microbiol Spectr, 2017. 5(2).
13.WHO guidelines on tuberculosis infection prevention and control. 2019.
14.Basaraba, R.J. and R.L. Hunter, Pathology of Tuberculosis: How the Pathology of Human Tuberculosis Informs and Directs Animal Models. Microbiology Spectrum, 2017. 5(3).
15.Delogu, G., M. Sali, and G. Fadda, The Biology of Mycobacterium Tuberculosis Infection. Mediterranean Journal of Hematology and Infectious Diseases, 2013. 5(1).
16.Lenaerts, A., C.E.B. III, and V.e. Dartois, Heterogeneity in tuberculosispathology, microenvironmentsand therapeutic responses. Immunological Reviews, 2015.
17.Gambhir, S., et al., Imaging in extrapulmonary tuberculosis. Int J Infect Dis, 2017. 56: p. 237-247.
18.Gordon, S.V. and T. Parish, Microbe Profile: Mycobacterium tuberculosis: Humanity''s deadly microbial foe. Microbiology, 2018. 164(4): p. 437-439.
19.Huang, L., E.V. Nazarova, and D.G. Russell, Mycobacterium tuberculosis: Bacterial Fitness within the Host Macrophage. Microbiol Spectr, 2019. 7(2).
20.Ates, L.S., E.N.G. Houben, and W. Bitter, Type VII Secretion: A Highly Versatile Secretion System, in Virulence Mechanisms of Bacterial Pathogens, Fifth Edition. 2016. p. 357-384.
21.Green, E.R. and J. Mecsas, Bacterial Secretion Systems: An Overview, in Virulence Mechanisms of Bacterial Pathogens, Fifth Edition. 2016. p. 215-239.
22.Bottai, D., M.I. Groschel, and R. Brosch, Type VII Secretion Systems in Gram-Positive Bacteria. Curr Top Microbiol Immunol, 2017. 404: p. 235-265.
23.Stanley, S.A., et al., Acute infection and macrophage subversion by Mycobacterium tuberculosis require a specialized secretion system. Proceedings of the National Academy of Sciences, 2003. 100(22): p. 13001-13006.
24.Gey Van Pittius, N.C., et al., The ESAT-6 gene cluster of Mycobacterium tuberculosis and other high G+C Gram-positive bacteria. Genome Biol, 2001. 2(10): p. Research0044.
25.van der Woude, A.D., J. Luirink, and W. Bitter, Getting Across the Cell Envelope: Mycobacterial Protein Secretion, in Pathogenesis of Mycobacterium tuberculosis and its Interaction with the Host Organism. 2012. p. 109-134.
26.Groschel, M.I., et al., ESX secretion systems: mycobacterial evolution to counter host immunity. Nat Rev Microbiol, 2016. 14(11): p. 677-691.
27.Dumas, E., et al., Mycobacterial Pan-Genome Analysis Suggests Important Role of Plasmids in the Radiation of Type VII Secretion Systems. Genome Biol Evol, 2016. 8(2): p. 387-402.
28.Bosserman, R.E. and P.A. Champion, Esx Systems and the Mycobacterial Cell Envelope: What''s the Connection? J Bacteriol, 2017. 199(17).
29.Abdallah, A.M., et al., Type VII secretion--mycobacteria show the way. Nat Rev Microbiol, 2007. 5(11): p. 883-91.
30.Wong, K.W., The Role of ESX-1 in Mycobacterium tuberculosis Pathogenesis. Microbiol Spectr, 2017. 5(3).
31.Tiwari, S., et al., Infect and Inject: How Mycobacterium tuberculosis Exploits Its Major Virulence-Associated Type VII Secretion System, ESX-1. Microbiol Spectr, 2019. 7(3).
32.Latz, E., T.S. Xiao, and A. Stutz, Activation and regulation of the inflammasomes. Nat Rev Immunol, 2013. 13(6): p. 397-411.
33.von Moltke, J., et al., Recognition of Bacteria by Inflammasomes. Annual Review of Immunology, 2013. 31(1): p. 73-106.
34.Guo, H., J.B. Callaway, and J.P. Ting, Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat Med, 2015. 21(7): p. 677-87.
35.Swanson, K.V., M. Deng, and J.P. Ting, The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol, 2019.
36.Hamilton, C. and P.K. Anand, Right place, right time: localisation and assembly of the NLRP3 inflammasome. F1000Res, 2019. 8.
37.Franchi, L., R. Munoz-Planillo, and G. Nunez, Sensing and reacting to microbes through the inflammasomes. Nat Immunol, 2012. 13(4): p. 325-32.
38.Ablasser, A. and A. Dorhoi, Inflammasome Activation and Function During Infection with Mycobacterium Tuberculosis. Curr Top Microbiol Immunol, 2016. 397: p. 183-97.
39.Mishra, B.B., et al., Mycobacterium tuberculosis protein ESAT-6 is a potent activator of the NLRP3/ASC inflammasome. Cell Microbiol, 2010. 12(8): p. 1046-63.
40.Wong, K.-W. and W.R. Jacobs Jr, Critical role for NLRP3 in necrotic death triggered by Mycobacterium tuberculosis. Cellular Microbiology, 2011. 13(9): p. 1371-1384.
41.Chakraborty, P. and A. Kumar, The extracellular matrix of mycobacterial biofilms: could we shorten the treatment of mycobacterial infections? Microb Cell, 2019. 6(2): p. 105-122.
42.Chen, Y.Y., et al., Mycobacterium marinum mmar_2318 and mmar_2319 are Responsible for Lipooligosaccharide Biosynthesis and Virulence Toward Dictyostelium. Front Microbiol, 2015. 6: p. 1458.
43.Amaral, E.P., et al., Lysosomal Cathepsin Release Is Required for NLRP3-Inflammasome Activation by Mycobacterium tuberculosis in Infected Macrophages. Front Immunol, 2018. 9: p. 1427.
44.Shah, S., et al., A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria. Infection and Immunity, 2015. 83(11): p. 4349-4361.
45.Brodsky, I.E. and D. Monack, NLR-mediated control of inflammasome assembly in the host response against bacterial pathogens. Seminars in Immunology, 2009. 21(4): p. 199-207.
46.Gurung, P. and T.D. Kanneganti, Novel roles for caspase-8 in IL-1beta and inflammasome regulation. Am J Pathol, 2015. 185(1): p. 17-25.
47.Monteleone, M., J.L. Stow, and K. Schroder, Mechanisms of unconventional secretion of IL-1 family cytokines. Cytokine, 2015. 74(2): p. 213-8.
48.Sagulenko, V., et al., AIM2 and NLRP3 inflammasomes activate both apoptotic and pyroptotic death pathways via ASC. Cell Death Differ, 2013. 20(9): p. 1149-60.
49.Gregersen, I., et al., Interleukin 27 is increased in carotid atherosclerosis and promotes NLRP3 inflammasome activation. PLoS One, 2017. 12(11): p. e0188387.
50.Qu, Y., et al., Nonclassical IL-1 Secretion Stimulated by P2X7 Receptors Is Dependent on Inflammasome Activation and Correlated with Exosome Release in Murine Macrophages. The Journal of Immunology, 2007. 179(3): p. 1913-1925.
51.Shamaa, O.R., et al., Monocyte Caspase-1 Is Released in a Stable, Active High Molecular Weight Complex Distinct from the Unstable Cell Lysate-Activated Caspase-1. PLoS One, 2015. 10(11): p. e0142203.
52.Bottai, D., et al., ESAT-6 Secretion-Independent Impact of ESX-1 Genes espF and espG1 on Virulence of Mycobacterium tuberculosis. The Journal of Infectious Diseases, 2011. 203(8): p. 1155-1164.
53.Ekiert, D.C. and J.S. Cox, Structure of a PE-PPE-EspG complex from Mycobacterium tuberculosis reveals molecular specificity of ESX protein secretion. Proceedings of the National Academy of Sciences, 2014. 111(41): p. 14758-14763.
54.Houben, E.N., K.V. Korotkov, and W. Bitter, Take five - Type VII secretion systems of Mycobacteria. Biochim Biophys Acta, 2014. 1843(8): p. 1707-16.
55.Daleke, M.H., et al., Specific chaperones for the type VII protein secretion pathway. J Biol Chem, 2012. 287(38): p. 31939-47.
56.Elena Stylianou, a.R.H.-K., a Julia Beglov,a Naomi Bull,a Nawamin Pinpathomrat,a Gwendolyn M. Swarbrick,b and b. Deborah A. Lewinsohn, c David M. Lewinsohn,c,d Helen McShane, Identification and Evaluation of Novel Protective Antigens for the Development of a Candidate Tuberculosis Subunit Vaccine. 2018.
57.McEvoy, C.R., et al., Comparative analysis of Mycobacterium tuberculosis pe and ppe genes reveals high sequence variation and an apparent absence of selective constraints. PLoS One, 2012. 7(4): p. e30593.