Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/97557
標題: 鮑氏不動桿菌醣蛋白A1S_3744之功能探討
Functional characterization of the Acinetobacter baumannii glycoprotein A1S_3744
作者: 陳品君
Pin-Chun Chen
關鍵字: 鮑氏不動桿菌;醣蛋白;蛋白酶;Acinetobacter baumannii;glycoprotein;protease
引用: 莊斯凱 (2015) 鮑氏不動桿菌抗原NcsP與TonB-R之特性分析,國立中興大學分子生物學研究所碩士論文。 楊宗翰 (2015) 鮑氏不動桿菌醣蛋白A1S_3744特性分析以及免疫載體蛋白質P64KN對保護性抗原至免疫力的影響,國立中興大學分子生物學研究所碩士論文。 Aizenman E, Engelberg-Kulka H and Glaser G. (1996) An Escherichia coli chromosomal 'addiction module' regulated by 3',5'-bispyrophosphate: a model for programmed bacterial cell death. Proc Natl Acad Sci U S A. 93: 6059-63. Asally M, Kittisopikul M, Rué P, Du Y, Hu Z, Çağatay T, Robinson AB, Lu H, Garcia-Ojalvo J and Süel GM. (2012) Localized cell death focuses mechanical forces during 3D patterning in a biofilm. Proc Natl Acad Sci U S A. 109: 18891-6. Alexeyev MF and Shokolenko IN. (1995) Mini-Tn10 transposon derivatives for insertion mutagenesis and gene delivery into the chromosome of gram-negative bacteria. Gene. 160: 59-62. Adams-Haduch JM, Paterson DL, Sidjabat HE, Pasculle AW, Potoski BA, Muto CA, Harrison LH and Doi Y. (2008) Genetic basis of multidrug resistance in Acinetobacter baumannii clinical isolates at a tertiary medical center in Pennsylvania. Antimicrob Agents Chemother. 52: 3837-43. Beijerinck M. (1911) Pigmenten als oxydatieproducten gevormd door bacterien. Vers Konin Akad Wet Ams. 19: 1092–1103. Bouvet PJ and Grimont PA. (1986) Taxonomy of the genus Acinetobacter with the recognition of Acinetobacter baumannii sp. nov, Acinetobacter haemolyticus sp. nov, Acinetobacter johnsonii sp. nov and Acinetobacter junii sp. nov and emended description of Acinetobacter calcoaceticus and Acinetobacter lwoffii. Int J Syst Bacteriol. 36: 228–40. Boyer HW and Roulland-dussoix D. (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 41: 459-72. Bou G and Martínez-Beltrán J. (2000) Cloning, nucleotide sequencing, and analysis of the gene encoding an AmpC beta-lactamase in Acinetobacter baumannii. Antimicrob Agents Chemother. 44: 428-32. Bugg TD and Brandish PE. (1994) From peptidoglycan to glycoproteins: common features of lipid-linked oligosaccharide biosynthesis. FEMS Microbiol Lett. 119: 255-62. Bullitt E and Makowski L. (1995) Structural polymorphism of bacterial adhesion pili. Nature. 373: 164-7. Barrett AJ and Rawlings ND. (2001) Evolutionary lines of cysteine peptidases. Biol Chem. 382: 727-33. Becker C, Shutov AD, Nong VH, Senyuk VI, Jung R, Horstmann C, Fischer J, Nielsen NC and Müntz K. (1995) Purification, cDNA cloning and characterization of proteinase B, an asparagine-specific endopeptidase from germinating vetch (Vicia sativa L.) seeds. Eur J Biochem. 228: 456-62. Canchaya C, Proux C, Fournous G, Bruttin A and Brüssow H. (2003) Prophage genomics. Microbiol Mol Biol Rev. 67: 238-76. Castric P. (1995) pilO, a gene required for glycosylation of Pseudomonas aeruginosa 1244 pilin. Microbiology. 141: 1247-54. Danial NN and Korsmeyer SJ. (2004) Cell death: critical control points. Cell. 116: 205-19. Dwyer DJ, Kohanski MA, Hayete B and Collins JJ. (2007) Gyrase inhibitors induce an oxidative damage cellular death pathway in Escherichia coli. Mol Syst Biol. 3: 91. Dwyer DJ, Camacho DM, Kohanski MA, Callura JM and Collins JJ. (2012) Antibiotic-induced bacterial cell death exhibits physiological and biochemical hallmarks of apoptosis. Mol Cell. 46: 561-72. Dell A, Galadari A, Sastre F and Hitchen P. (2010) Similarities and differences in the glycosylation mechanisms in prokaryotes and eukaryotes. Int J Microbiol. 2010: 148178. Donlan RM. (2002) Biofilms: microbial life on surfaces. Emerg Infect Dis. 8: 881-90. Fournier PE and Richet H. (2006) The epidemiology and control of Acinetobacter baumannii in health care facilities. Clin Infect Dis. 42: 692-9. Faridmoayer A, Fentabil MA, Mills DC, Klassen JS and Feldman MF. (2007) Functional characterization of bacterial oligosaccharyltransferases involved in O-linked protein glycosylation. J Bacteriol. 189: 8088-98. Flemming HC, Neu TR and Wozniak DJ. (2007) The EPS matrix: the 'house of biofilm cells'. J Bacteriol. 189: 7945-7. Fux CA, Costerton JW, Stewart PS and Stoodley P. (2005) Survival strategies of infectious biofilms. Trends Microbiol. 13: 34-40. Feldman MF. (2013) A common pathway for O-linked protein-glycosylation and synthesis of capsule in Acinetobacter baumannii. Mol Microbiol. 89: 816-30. Grodzicker T, Arditti RR and Eisen H. (1972) Establishment of repression by lambdoid phage in catabolite activator protein and adenylate cyclase mutants of Escherichia coli. Proc Natl Acad Sci U S A. 69: 366-70. Giannouli M, Di Popolo A, Durante-Mangoni E, Bernardo M, Cuccurullo S, Amato G, Tripodi MF, Triassi M, Utili R and Zarrilli R. (2012) Molecular epidemiology and mechanisms of rifampicin resistance in Acinetobacter baumannii isolates from Italy. Int J Antimicrob Agents. 39: 58-63. Guerry P. (2007) Campylobacter flagella: not just for motility. Trends Microbiol. 15: 456-61. Grudkowska M and Zagdańska B. (2004) Multifunctional role of plant cysteine proteinases. Acta Biochim Pol. 51: 609-24. Hochman A. (1997) Programmed cell death in prokaryotes. Crit Rev Microbiol. 23: 207-14. Hazan R, Sat B and Engelberg-Kulka H. (2004) Escherichia coli mazEF-mediated cell death is triggered by various stressful conditions. J Bacteriol. 186: 3663-9. Hsueh PR, Teng LJ, Chen CY, Chen WH, Yu CJ, Ho SW and Luh KT. (2002) Pandrug-resistant Acinetobacter baumannii causing nosocomial infections in a university hospital, Taiwan. Emerg Infect Dis. 8: 827-32. Héritier C, Poirel L and Nordmann P. (2006) Cephalosporinase over-expression resulting from insertion of ISAba1 in Acinetobacter baumannii. Clin Microbiol Infect. 12: 123-30. Hentzer M and Givskov M. (2003) Pharmacological inhibition of quorum sensing for the treatment of chronic bacterial infections. J Clin Invest. 112: 1300-7. Hug I and Feldman MF. (2011) Analogies and homologies in lipopolysaccharide and glycoprotein biosynthesis in bacteria. Glycobiology. 21: 138-51. Hall-Stoodley L and Stoodley P. (2009) Evolving concepts in biofilm infections. Cell Microbiol. 11: 1034-43. Harshey RM. (2003) Bacterial motility on a surface: many ways to a common goal. Annu Rev Microbiol. 57: 249-73. Hara-Nishimura I, Inoue K and Nishimura M. (1991) A unique vacuolar processing enzyme responsible for conversion of several proprotein precursors into the mature forms. FEBS Lett. 294: 89-93. Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E, Stratilo C, Reiz B, Cordwell SJ, Whittal R, Schild S and Feldman MF. (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog. 8: e1002758. Kerr JF, Wyllie AH and Currie AR. (1972) Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer. 26: 239-57. Koza A, Hallett PD, Moon CD and Spiers AJ. (2009) Characterization of a novel air-liquid interface biofilm of Pseudomonas fluorescens SBW25. Microbiology. 155: 1397-406. Lees-Miller RG, Iwashkiw JA, Scott NE, Seper A, Vinogradov E, Schild S and Montefour K, Frieden J, Hurst S, Helmich C, Headley D and Martin M. (2008) Acinetobacter baumannii: an emerging multidrug-resistant pathogen in critical care. Crit Care Nurse. 28:15–25. Marchand I, Damier-Piolle L, Courvalin P and Lambert T. (2004) Expression of the RND-type efflux pump AdeABC in Acinetobacter baumanniiis regulated by the AdeRS two-component system. Antimicrob Agents Chemother. 48: 3298 –3304. Mescher MF and Strominger JL. (1976) Purification and characterization of a prokaryotic glycoprotein from the cell envelope of Halobacterium salinarium. J Biol Chem. 251: 2005-14. Martí S, Rodríguez-Baño J, Catel-Ferreira M, Jouenne T, Vila J, Seifert H and Dé E. (2011) Biofilm formation at the solid-liquid and air-liquid interfaces by Acinetobacter species. BMC Res Notes. 4: 5. Mussi MA, Gaddy JA, Cabruja M, Arivett BA, Viale AM, Rasia R, Actis LA. (2010) The opportunistic human pathogen Acinetobacter baumannii senses and responds to light. J Bacteriol. 192: 6336-45. Nedelcu AM, Driscoll WW, Durand PM, Herron MD and Rashidi A. (2011) On the paradigm of altruistic suicide in the unicellular world. Evolution. 65: 3-20. Nothaft H and Szymanski CM. (2010) Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol. 8: 765-78. O'Toole GA and Kolter R. (1998) Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development. Mol Microbiol. 30: 295-304. Pandey DP and Gerdes K. (2005) Toxin-antitoxin loci are highly abundant in free-living but lost from host-associated prokaryotes. Nucleic Acids Res. 33: 966-76. Peleg AY, Seifert H and Paterson DL. (2008) Acinetobacter baumannii: emergence of a successful pathogen. Clin Microbiol Rev. 21: 538-82. Reddien PW and Horvitz HR. (2004) The engulfment process of programmed cell death in caenorhabditis elegans. Annu Rev Cell Dev Biol. 20: 193-221. Ren Y and Savill J. (1998) Apoptosis: the importance of being eaten. Cell Death Differ. 5: 563-8. Richmond GE, Evans LP, Anderson MJ, Wand ME, Bonney LC, Ivens A, Chua KL, Webber MA, Sutton JM, Peterson ML and Piddock LJ. (2016) The Acinetobacter baumannii Two-Component System AdeRS Regulates Genes Required for Multidrug Efflux, Biofilm Formation, and Virulence in a Strain-Specific Manner. MBio. 7: e00430-16. Rasmussen TB and Givskov M. (2006) Quorum-sensing inhibitors as anti-pathogenic drugs. Int J Med Microbiol. 296: 149-61. Rodríguez-Baño J, Martí S, Soto S, Fernández-Cuenca F, Cisneros JM, Pachón J, Pascual A, Martínez-Martínez L, McQueary C, Actis LA, Vila J; Spanish Group for the Study of Nosocomial Infections (GEIH). (2008) Biofilm formation in Acinetobacter baumannii: associated features and clinical implications. Clin Microbiol Infect.14: 276-8. Seifert H, Dijkshoorn L, Gerner-Smidt P, Pelzer N, Tjernberg I and Vaneechoutte M. (1997) Distribution of Acinetobacter species on human skin: comparison of phenotypic and genotypic identification methods. J Clin Microbiol. 35: 2819–25. Silhavy TJ, Kahne D and Walker S. (2010) The bacterial cell envelope. Cold Spring Harb Perspect Biol. 2: a000414. Simon R, Priefer U and Pühler A. (1983) A broad host rangemobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. J Bacteriol. 1: 784–79. Smedley JG III, Jewell E, Roguskie J, Horzempa J, Syboldt A, Stolz DB and Castric P. (2005) Influence of pilin glycosylation on Pseudomonas aeruginosa 1244 pilus function. Infect Immun. 73: 7922-31. Scott MP, Jung R, Muntz K and Nielsen NC. (1992) A protease responsible for post-translational cleavage of a conserved Asn-Gly linkage in glycinin, the major seed storage protein of soybean. Proc Natl Acad Sci U S A. 89: 658-62. Skiebe E, de Berardinis V, Morczinek P, Kerrinnes T, Faber F, Lepka D, Hammer B, Zimmermann O, Ziesing S, Wichelhaus TA, Hunfeld KP, Borgmann S, Gröbner S, Higgins PG, Seifert H, Busse HJ, Witte W, Pfeifer Y, Wilharm G. (2012) Surface-associated motility, a common trait of clinical isolates of Acinetobacter baumannii, depends on 1,3-diaminopropane. Int J Med Microbiol. 302: 117-28. Tanouchi Y, Pai A, Buchler NE and You L. (2012) Programming stress-induced altruistic death in engineered bacteria. Mol Syst Biol. 8: 626. Tanouchi Y, Lee AJ, Meredith H and You L. (2013) Programmed cell death in bacteria and implications for antibiotic therapy. Trends Microbiol. 21: 265-70. Turton JF, Ward ME, Woodford N, Kaufmann ME, Pike R, Livermore DM and Pitt TL. (2006) The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol Lett. 258: 72-7. Tomaras AP, Dorsey CW, Edelmann RE and Actis LA. (2003) Attachment to and biofilm formation on abiotic surfaces by Acinetobacter baumannii: involvement of a novel chaperone-usher pili assembly system. Microbiology. 149: 3473-84. Vik A, Aas FE, Anonsen JH, Bilsborough S, Schneider A, Egge-Jacobsen W and Koomey M. (2009) Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae. Proc Natl Acad Sci U S A. 106: 4447-52. Wyllie AH, Kerr JF and Currie AR. (1980) Cell death: the significance of apoptosis. Int Rev Cytol. 68: 251-306. West SA, Diggle SP, Buckling A, Gardner A and Griffin AS. (2007) The social lives of microbes. Annu Rev Ecol Evol S. 38: 53–77. Williams JJ and Hergenrother PJ. (2012) Artificial activation of toxin-antitoxin systems as an antibacterial strategy. Trends Microbiol. 20: 291-8. Wendt C, Dietze B, Dietz E and Rüden H. (1997) Survival of Acinetobacter baumannii on dry surfaces. J Clin Microbiol. 35: 1394-7. Yamaguchi Y and Inouye M. (2011) Regulation of growth and death in Escherichia coli by toxin-antitoxin systems. Nat Rev Microbiol. 9: 779-90. Zhang X, McDaniel AD, Wolf LE, Keusch GT, Waldor MK and Acheson DW. (2000) Quinolone antibiotics induce Shiga toxin-encoding bacteriophages, toxin production, and death in mice. J Infect Dis. 181: 664-70. Zhou J, Thompson DK, Xu Y, Tiedje JM. (2004) Microbial Functional Genomics. 1st ed., 225-229. New Jersey: John Wiley & Sons.
摘要: 
鮑氏不動桿菌 (Acinetobacter baumannii, AB菌) 屬於革蘭氏陰性菌,為伺機性病原菌,感染者會引發腦膜炎、肺炎、菌血症等,甚至導致死亡。AB菌能適應較為嚴苛的環境,進而成為嚴重的院內感染病原菌,另外多重抗藥性 (multiple drug resistance, MDR) AB菌迅速發展,造成醫學治療上的困難,因此除了尋找新型抗生素外,探討對細菌生理和/或病理功能至關重要之基因,藉以發展新型抗菌治療也更顯為重要。目前已證實AB菌在O-glycosylation缺陷的情況下,其毒力會衰減,因此本研究以探討AB菌之醣蛋白A1S_3744 (以下稱為GP2) 功能為目標。為了達成此目標,我構築了以下的AB菌株 : GP2缺陷之菌株AB(ΔGP2)、互補株AB(ΔGP2+) 及mock組菌株AB(ΔGP2-IIc)。在所有分析的表型性狀,包含複製、貼附、毒性、生物膜與pellicle生成,及菌體存活率,其中pellicle生成及穩定生長期 (stationary phase) 之菌體存活率在野生型與GP2缺陷之菌株AB(ΔGP2) 以及AB(ΔGP2-IIc) 間具顯著性差異。然而,這些性狀並無在互補株AB(ΔGP2+) 中恢復。利用西方墨點法分析之下,結果顯示AB(ΔGP2+) 之GP2蛋白質表現量約為野生型之3倍,因此互補株AB(ΔGP2+) 無法恢復這些性狀可能是因為GP2蛋白質過量表現所造成,這也代表適量表現GP2蛋白質對其功能的重要性。除此之外,以西方墨點法分析結果揭示GP2蛋白質之分子量於穩定生長期後逐漸從~34 kDa下降至~30-32 kDa,呈現加工之現象 ; 藉由隨機突變之方式,我也鑑定出參與GP2蛋白質加工之蛋白酶。進一步,我發現缺乏此蛋白酶之菌株其OD600及菌體存活率皆較野生型低,且培養至12小時後觀察到菌體裂解之情形,因此推論此蛋白酶可能在菌體分裂中扮演關鍵的角色。最後,在過量表現GP2蛋白質之AB菌株中發現菌體聚集之現象,且此現象在缺乏上述蛋白酶的情況下更為嚴重。綜合上述之結果,本研究顯示適量表現GP2蛋白質對AB菌之菌體存活率及細胞膜結構是至關重要的。

Acinetobacter baumannii (AB) belongs to Gram-negative and is an opportunistic pathogen. Infected patients may cause meningitis, pneumonia, bacteremia and even death. AB can adapt to severe environments, thus become a serious nosocomial infection pathogen. Furthermore, multiple drug resistance AB develops rapidly, causing medical treatment ineffectively. Therefore, in addition to searching for new antibiotics, it is important to explore the genes that are crucial for bacterial physiological and/or pathological functions that may lead to development of new antibacterial therapies. It has been demonstrated that AB deficient in O-glycosylation is less virulent. This study aimed to investigate the function of A1S_3744 (GP2), one of the identified glycoproteins in AB. To achieve this goal, I constructed the following AB strains: a GP2 deficient strain AB(ΔGP2), a complementary strain AB(ΔGP2+), and a mock strain AB(ΔGP2-IIc). Among the examined phenotypic traits including replication, adhesion, cytotoxicity, biofilm and pellicle formation, and viability, significant differences in pellicle formation and viability during the stationary phase were observed between the wild type and GP2 deficient strain AB(ΔGP2) as well as AB(ΔGP2-IIc). However, these traits were not recovered in the complementary strain AB(ΔGP2+). Western blot analysis showed that the level of GP2 protein in the AB(ΔGP2+) is ~3 fold of that in the wild type. The failure to recover the pellicle formation and viable count may due to the overexpressing of GP2 protein in AB(ΔGP2+) suggesting that an appropriate amount of GP2 protein is important for its function. In addition, Western blot analysis revealed that the apparent molecular weight of GP2 protein gradually decreased from ~34 kDa to ~30-32 kDa during the stationary phase, an indication of processing. By random mutagenesis, I identified a protease that is involved in GP2 protein processing. Next, I found the bacteria growth measured by OD600 and viable count were significantly reduced and bacteriolysis was observed in 12-h culture and thereafter inferring that this protease may also play a role in cell division. Finally, bacteria aggregation was observed in AB strains overexpressing GP2 protein, and this phenomenon was more severe in the absence of the above protease. Together, this study demonstrates that appropriately expression of GP2 protein is critical in AB viability and membrane structure.
URI: http://hdl.handle.net/11455/97557
Rights: 同意授權瀏覽/列印電子全文服務,2021-08-30起公開。
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