臺灣博碩士論文加值系統

氧化逆境 (oxidative stress) 在細胞層面上扮演許多不同的角色，例如在巨噬細胞 (macrophage) 中進行的殺菌作用 (bactericidal effect) 。然而，活性氧分子 (reactive oxygen species, ROS) 殺菌的機制到目前為止仍不清楚。本篇論文嘗試以過氧化氫 (雙氧水， hydrogen peroxide, H2O2) 代表活性氧分子來探討細菌第二型拓樸異構酶 (Type II topoisomerases, Topo IIs) 在活性氧分子所造成之細菌死亡中所扮演的角色。我們的實驗結果指出過氧化氫可以毒化 (poison) 第二型拓樸異構酶而使之與DNA共價鍵結形成複合體 (cleavable complex) 的形式，這樣的毒化複合體持續存在會導致DNA的斷裂，並進一步造成細菌的死亡。細菌第二型拓樸異構酶在巨噬細胞進行吞噬作用 (phagocytosis) 所造成的殺菌效果中扮演重要角色的推論可由以下結果推得： (1) 過氧化氫可大量活化細菌在遭受DNA損害時的典型SOS反應 (SOS response)； (2)過氧化氫可以導致細菌DNA斷裂形成大分子的DNA片段； (3) 這些過氧化氫所造成的大片段DNA具有蛋白質共價連結其上； (4) 這些過氧化氫造成大片段DNA的斷裂反應是可逆的； (5) 細菌第二型拓樸異構酶的抑制劑coumermycin A1，可有效地擷抗過氧化氫所造成的大片段DNA的形成及降低過氧化氫所引起的SOS反應的發生； (6) 最重要的是，細菌第二型拓樸異構酶的抑制劑被證實可以抑制過氧化氫所造成之細菌毒殺作用 (cytotoxicity) 以及巨噬細胞中所進行的吞噬作用之殺菌的效果。總結以上，本篇論文證明了過氧化氫可以有效地藉由毒化細菌第二型拓樸異構酶來造成DNA損壞，並進一步導致細菌的死亡。這個實驗結果提供了一個在吞噬作用中新的殺菌機制：在氧化逆境導致的殺菌作用，特別是在巨噬細胞進行的吞噬作用中，細菌第二型拓樸異構酶扮演了關鍵的角色。

Oxidative stress plays important roles in many cellular states including bactericidal effect during phagocytosis. However, the bacterial killing mechanism by reactive oxygen species (ROS) remains obscure. Here, we investigated the role of bacterial type II topoisomerases (Topo IIs) in oxidative stress-mediated cell death. Hydrogen peroxide (H2O2) was used to induce oxidative stress in bacteria. The involvement of Topo II-mediated DNA damage in bactericidal activity during phagocytosis is supported by the following observations. (1) Cellular exposure of H2O2 led to activation of SOS response, an observation suggestive of DNA damage in E. coli. (2) H2O2 rapidly induced cleavage of nucleoid DNA into high-molecular-weight (HMW) DNA fragments in E. coli. (3) H2O2-induced HMW DNA fragments were covalently linked with proteins. (4) H2O2-induced HMW DNA fragmentations were highly reversible. (5) Coumermycin A1, a bacterial Topo II catalytic inhibitor, efficiently antagonized the formation of H2O2-induced HMW DNA fragments and the H2O2-induced SOS response. (6) Most importantly, coumermycin A1 attenuated the bacterial cell killing by H2O2 and bactericidal effect during phagocytosis in macrophage. Taken together, we concluded that H2O2 effectively targets bacterial Topo IIs and causes DNA breakage, which then leads to cell death. Our results therefore present the first demonstration that bacterial Topo IIs are the bactericidal target during phagocytosis.

ACKNOWLEDGEMENTS
中文摘要
ABSTRACT
INTRODUCTION ................................................. 1
- DNA topoisomerases
Type I topoisomerases (Topo Is)
Type II topoisomerases (Topo IIs)
- DNA topoisomerase-targeting drugs
Poisons for DNA topoisomerases
Inhibitors for DNA topoisomerases
- Bactericidal effects of phagocytosis
Antimicrobial peptides (AMPs) and proteases
Reactive oxygen species (ROS)
SPECIFIC AIMS ................................................. 11
MATERIALS & METHODS ...................................... 12
- Chemicals
- Bacterial strains and growth condition
- Encapsulation of bacterial cells and drug treatment conditions
- Pulsed-field gel electrophoresis (PFGE)
- Bioluminescent reporter assay
- Cytotoxicity assay
- Efficiency of plating (EoP) assay
- Cell and culture media
- Phagocytosis assay
RESULTS ........................................................ 16
- Treatment with H2O2 induced the activation of SOS response, an observation suggestive of nucleoid DNA damage
- H2O2 rapidly induced excision of nucleoid DNA into 50~100-kb HMW DNA fragments
- H2O2- and norfloxacin-induced HMW DNA fragments were covalently linked with proteins
- Both H2O2- and norfloxacin-induced excision of HMW DNA fragments was highly reversible
- Coumermycin A1, a bacterial Topo II inhibitor, efficiently antagonized the HMW DNA fragmentation induced by H2O2 or norfloxacin
- The activation of SOS response induced by either H2O2 or norfloxacin treatment was through Topo II-mediated DNA damage
- H2O2-induced oxidative response was not compromised by the addition of coumermycin A1
- Bactericidal effects caused by H2O2 or norfloxacin were antagonized by pretreatment with coumermycin A1
- Gyrase, one isoform of two bacterial Topo IIs, played the major role in bactericidal effect caused by H2O2 or norfloxacin
- Coumermycin A1 effectively antagonized the bacterial cell killing by phagocytosis in macrophage
- Gyrase played a role in bacterial cell killing by phagocytosis in macrophage
DISCUSSIONS ................................................... 22
- The underlying mechanisms of H2O2-induced DNA damage
- The differential roles that gyrase and Topo IV play in the bactericidal activity exhibited by oxidative stress and during phagocytosis
- Various chemically reactive compounds are used in the physiological defending system to eliminate invasive bacteria
- A natural gyrase inhibitor, GyrI, might serve as a bacterial virulence factor
- Chronic granulomatous disease (CGD) patients
REFERENCES ................................................... 27
FIGURES ........................................................ 31
- Figure 1 The catalytic cycle of gyrase
- Figure 2 Domain structures and their associated functions of gyrase
- Figure 3 Activation of SOS response by H2O2
- Figure 4 The rapid DNA fragmentation induced by H2O2
- Figure 5 The protein-linked DNA fragments induced by H2O2
- Figure 6 The reversibility of DNA fragmentation induced by H2O2
- Figure 7 Coumermycin A1-antagonizing effect in H2O2-induced
DNA fragmentation
- Figure 8 Coumermycin A1-antagonizing effect in H2O2-induced SOS response
- Figure 9 Coumermycin A1-antagonizing effect in H2O2-induced oxidative damage
- Figure 10 Coumermycin A1-antagonizing effect in H2O2-induced bacterial cell death
- Figure 11 The survival abilities of RFM443 and RFM445 under coumermycin A1 treatment
- Figure 12 The role of gyrase in H2O2-induced bacterial cell death
- Figure 13 Coumermycin A1-antagonizing effect in phagocytosis
- Figure 14 The role of gyrase in phagocytosis
- Figure 15 A schematic illustration of bacterial Topo II-mediated DNA damage in phagocytosis
CURRICULUM VITAE ........................................... 47

Belaaouaj, A. (2002) Neutrophil elastase-mediated killing of bacteria: lessons from targeted mutagenesis. Microbes Infect, 4, 1259-1264.
Belkin, S., Smulski, D.R., Vollmer, A.C., Van Dyk, T.K. and LaRossa, R.A. (1996) Oxidative stress detection with Escherichia coli harboring a katG''::lux fusion. Appl Environ Microbiol, 62, 2252-2256.
Bjornsti, M.A. and Wang, J.C. (1987) Expression of yeast DNA topoisomerase I can complement a conditional-lethal DNA topoisomerase I mutation in Escherichia coli. Proc Natl Acad Sci U S A, 84, 8971-8975.
Bokoch, G.M. (2002) Microbial killing: hold the bleach and pass the salt! Nat Immunol, 3, 340-342.
Brogden, K.A. (2005) Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat Rev Microbiol, 3, 238-250.
Brown, K.L. and Hancock, R.E. (2006) Cationic host defense (antimicrobial) peptides. Curr Opin Immunol, 18, 24-30.
Champoux, J.J. (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem, 70, 369-413.
Chatterji, M. and Nagaraja, V. (2002) GyrI: a counter-defensive strategy against proteinaceous inhibitors of DNA gyrase. EMBO Rep, 3, 261-267.
Chatterji, M., Sengupta, S. and Nagaraja, V. (2003) Chromosomally encoded gyrase inhibitor GyrI protects Escherichia coli against DNA-damaging agents. Arch Microbiol, 180, 339-346.
Cohen, M.S., Britigan, B.E., Hassett, D.J. and Rosen, G.M. (1988) Phagocytes, O2 reduction, and hydroxyl radical. Rev Infect Dis, 10, 1088-1096.
Couturier, M., Bahassi el, M. and Van Melderen, L. (1998) Bacterial death by DNA gyrase poisoning. Trends Microbiol, 6, 269-275.
Czapski, G., Goldstein, S., Andorn, N. and Aronovitch, J. (1992) Radiation-induced generation of chlorine derivatives in N2O-saturated phosphate buffered saline: toxic effects on Escherichia coli cells. Free Radic Biol Med, 12, 353-364.
Drlica, K. and Zhao, X. (1997) DNA gyrase, topoisomerase IV, and the 4-quinolones. Microbiol Mol Biol Rev, 61, 377-392.
Evans, M.D., Dizdaroglu, M. and Cooke, M.S. (2004) Oxidative DNA damage and disease: induction, repair and significance. Mutat Res, 567, 1-61.
Forman, H.J. and Torres, M. (2001) Redox signaling in macrophages. Mol Aspects Med, 22, 189-216.
Forman, H.J. and Torres, M. (2002) Reactive oxygen species and cell signaling: respiratory burst in macrophage signaling. Am J Respir Crit Care Med, 166, S4-8.
Forslund, T. and Sundqvist, T. (1997) Nitric oxide-releasing particles inhibit phagocytosis in human neutrophils. Biochem Biophys Res Commun, 233, 492-495.
Gresham, H.D., McGarr, J.A., Shackelford, P.G. and Brown, E.J. (1988) Studies on the molecular mechanisms of human Fc receptor-mediated phagocytosis. Amplification of ingestion is dependent on the generation of reactive oxygen metabolites and is deficient in polymorphonuclear leukocytes from patients with chronic granulomatous disease. J Clin Invest, 82, 1192-1201.
Hampton, M.B., Kettle, A.J. and Winterbourn, C.C. (1998) Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing. Blood, 92, 3007-3017.
Hawkey, P.M. (2003) Mechanisms of quinolone action and microbial response. J Antimicrob Chemother, 51 Suppl 1, 29-35.
Heddle, J.G., Barnard, F.M., Wentzell, L.M. and Maxwell, A. (2000) The interaction of drugs with DNA gyrase: a model for the molecular basis of quinolone action. Nucleosides Nucleotides Nucleic Acids, 19, 1249-1264.
Hirsch, E.C. and Faucheux, B.A. (1998) Iron metabolism and Parkinson''s disease. Mov Disord, 13 Suppl 1, 39-45.
Hsu, Y.H., Chung, M.W. and Li, T.K. (2006) Distribution of gyrase and topoisomerase IV on bacterial nucleoid: implications for nucleoid organization. Nucleic Acids Res, 34, 3128-3138.
Iha, S., Orita, K., Kanno, T., Utsumi, T., Sato, E.F., Inoue, M. and Utsumi, K. (1996) Oxygen-dependent inhibition of neutrophil respiratory burst by nitric oxide. Free Radic Res, 25, 489-498.
Inoue, M., Nishikawa, M., Sato, E.F., Ah-Mee, P., Kashiba, M., Takehara, Y. and Utsumi, K. (1999) Cross-talk of NO, superoxide and molecular oxygen, a majesty of aerobic life. Free Radic Res, 31, 251-260.
Knight, J.A. (2000) Review: Free radicals, antioxidants, and the immune system. Ann Clin Lab Sci, 30, 145-158.
Lai, E., Birren, B.W., Clark, S.M., Simon, M.I. and Hood, L. (1989) Pulsed field gel electrophoresis. Biotechniques, 7, 34-42.
Larsen, A.K., Escargueil, A.E. and Skladanowski, A. (2003) Catalytic topoisomerase II inhibitors in cancer therapy. Pharmacol Ther, 99, 167-181.
Leppard, J.B. and Champoux, J.J. (2005) Human DNA topoisomerase I: relaxation, roles, and damage control. Chromosoma, 114, 75-85.
Li, T.K., Chen, A.Y., Yu, C., Mao, Y., Wang, H. and Liu, L.F. (1999) Activation of topoisomerase II-mediated excision of chromosomal DNA loops during oxidative stress. Genes Dev, 13, 1553-1560.
Li, T.K. and Liu, L.F. (1998) Modulation of gyrase-mediated DNA cleavage and cell killing by ATP. Antimicrob Agents Chemother, 42, 1022-1027.
Li, T.K. and Liu, L.F. (2001) Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol, 41, 53-77.
Liu, L.F. (1983) DNA topoisomerases--enzymes that catalyse the breaking and rejoining of DNA. CRC Crit Rev Biochem, 15, 1-24.
Mani, N., Gross, C.H., Parsons, J.D., Hanzelka, B., Muh, U., Mullin, S., Liao, Y., Grillot, A.L., Stamos, D., Charifson, P.S. and Grossman, T.H. (2006) In vitro characterization of the antibacterial spectrum of novel bacterial type II topoisomerase inhibitors of the aminobenzimidazole class. Antimicrob Agents Chemother, 50, 1228-1237.
Maxwell, A. (1997) DNA gyrase as a drug target. Trends Microbiol, 5, 102-109.
McCord, J.M. and Fridovich, I. (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J Biol Chem, 244, 6049-6055.
Meresse, P., Dechaux, E., Monneret, C. and Bertounesque, E. (2004) Etoposide: discovery and medicinal chemistry. Curr Med Chem, 11, 2443-2466.
Morais Cabral, J.H., Jackson, A.P., Smith, C.V., Shikotra, N., Maxwell, A. and Liddington, R.C. (1997) Crystal structure of the breakage-reunion domain of DNA gyrase. Nature, 388, 903-906.
Nagaraja, V., Sikder, D. and Jain, P. (2002) DNA topoisomerase I from mycobacteria--a potential drug target. Curr Pharm Des, 8, 1995-2007.
Nakanishi, A., Imajoh-Ohmi, S. and Hanaoka, F. (2002) Characterization of the interaction between DNA gyrase inhibitor and DNA gyrase of Escherichia coli. J Biol Chem, 277, 8949-8954.
Nakanishi, A., Oshida, T., Matsushita, T., Imajoh-Ohmi, S. and Ohnuki, T. (1998) Identification of DNA gyrase inhibitor (GyrI) in Escherichia coli. J Biol Chem, 273, 1933-1938.
Reeves, E.P., Lu, H., Jacobs, H.L., Messina, C.G., Bolsover, S., Gabella, G., Potma, E.O., Warley, A., Roes, J. and Segal, A.W. (2002) Killing activity of neutrophils is mediated through activation of proteases by K+ flux. Nature, 416, 291-297.
Roos, D. and Winterbourn, C.C. (2002) Immunology. Lethal weapons. Science, 296, 669-671.
Segal, A.W. (2005) How neutrophils kill microbes. Annu Rev Immunol, 23, 197-223.
Skinner, A.M. and Turker, M.S. (2005) Oxidative mutagenesis, mismatch repair, and aging. Sci Aging Knowledge Environ, 2005, re3.
Trigueros, S. and Roca, J. (2002) Failure to relax negative supercoiling of DNA is a primary cause of mitotic hyper-recombination in topoisomerase-deficient yeast cells. J Biol Chem, 277, 37207-37211.
Underhill, D.M. and Ozinsky, A. (2002) Phagocytosis of microbes: complexity in action. Annu Rev Immunol, 20, 825-852.
Vollmer, A.C., Belkin, S., Smulski, D.R., Van Dyk, T.K. and LaRossa, R.A. (1997) Detection of DNA damage by use of Escherichia coli carrying recA''::lux, uvrA''::lux, or alkA''::lux reporter plasmids. Appl Environ Microbiol, 63, 2566-2571.
Wang, J.C. (1985) DNA topoisomerases: nature''s solution to the topological ramifications of the double-helix structure of DNA. Harvey Lect, 81, 93-110.
Wang, J.C. (1996) DNA topoisomerases. Annu Rev Biochem, 65, 635-692.
Wang, J.C. (2002) Cellular roles of DNA topoisomerases: a molecular perspective. Nat Rev Mol Cell Biol, 3, 430-440.
Watson, J.D. and Crick, F.H. (1953) Genetical implications of the structure of deoxyribonucleic acid. Nature, 171, 964-967.
Xiao, H., Li, T.K., Yang, J.M. and Liu, L.F. (2003) Acidic pH induces topoisomerase II-mediated DNA damage. Proc Natl Acad Sci U S A, 100, 5205-5210.
Zasloff, M. (2002) Antimicrobial peptides of multicellular organisms. Nature, 415, 389-395.