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研究生:蘇勇誠
研究生(外文):Jurng-Chen Su
論文名稱:LPS合成基因rfaG與rfbX對鼠傷寒沙門氏桿菌之ceftriaxone抗藥性的關聯
論文名稱(外文):Correlation between LPS biosynthesis genes rfaG and rfbX and ceftriaxone resistance of Salmonella enterica Serovar Typhimurium
指導教授:胡文熙
指導教授(外文):Wensi S. Hu
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學生物技術研究所
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:2006
畢業學年度:94
語文別:中文
論文頁數:63
中文關鍵詞:脂多糖體膜蛋白抗生素抗藥性頭孢子類抗生素體抗原鼠傷寒沙門氏桿菌
外文關鍵詞:LPSlipopolysacchariderfaGrfbXSalmonella enterica serovar Typhimuriummembrane proteinceftriaxoneantibiotics resistance
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本實驗室經由逐步加重第三代頭孢子素類抗生素ceftriaxone (CRO) 劑量,進而誘導出能抵抗ceftriaxone 至濃度大於256 mg/l 的鼠傷寒沙門氏桿菌菌株並命名為R200 株(Cheng 2003),之後再利用 Tn5 轉位子任意插入基因座內,並篩選對CRO 抗藥性下降至四倍以下的菌株(Li 2003);其中代號5B7 的菌株是轉位子插入rfaG 基因,以熱酚水萃取法經SDS-PAGE證實具有deep rough LPS的表型及減少的LPS 和外膜蛋白總量,抽取外膜蛋白經二維電泳法發現5 spots 蛋白量增加及4 spots 蛋白量減少,這些都可能造成它對CRO 藥物的敏感性;另外代號4H11 的菌株是轉位子插入rfbX 基因,以熱酚水萃取法經SDS-PAGE 證實具有rough LPS 的表型及減少的LPS 和部份外膜蛋白,抽取外膜蛋白經二維電泳法發現6 spots 蛋白量增加及3 spots 蛋白量減少;經細胞膜通透性實驗證明在5B7 株通透性最高,並伴隨抗藥性的降低,而4H11 株雖然通透性最低,可是減弱的efflux pump 效能亦使其抗藥性降低;而代號5G8 的菌株是轉位子插入tolC 基因,TolC 是位於外膜很重要的一個通道蛋白,與多種抗生素的排出有關,tolC 基因的破壞導致對CRO 敏感(MIC < 2.5 mg/l),也代表TolC 在CRO 抗藥中佔有十足重要的角色。STM3031 是本實驗室早前發現跟CRO 抗藥可能很有關的外膜蛋白(Cheng 2003),在5B7及4H11 兩株其表現量都呈現明顯的減少,在本篇論文中,LPS 合成基因的破壞,除了影響外膜上的膜蛋白表現,也可能影響到外膜通透性及內膜上部份transporter 或efflux pump,進而影響到細菌對CRO藥物的抗藥性。
Using in vitro stepwise induction, a new strain named R200 of Salmonella enterica serovar Typhimurium with high resistance to ceftriaxone (MIC over 256 mg/l) was generated (Cheng 2003). Following, we applied transposon mutagenesis on the R200 strain to generate the mutants with a more-than-four-fold reduction to ceftriaxone resistance (Li 2003).
Among these mutants, a strain 5B7 has a transposon inserted in the rfaG gene and results in susceptible to ceftriaxone. This strain was proved to be the deep rough LPS phenotype using combined hot-phenol water extraction of LPS and SDS-PAGE analysis. The results revealed the reduction of LPS and outer membrane proteins. Nine spots in 2-DE image of outer membrane protein profile were found to be altered. The protein levels of five out of nine spots were significantly increased and those of the other four spots were significantly decreased. All these protein spots change in the strain 5B7 might be related to ceftriaxone (CRO) susceptibility. The other mutant 4H11, a transposon insertion in the rfbX gene was also selected for study. It was proved to have a rough LPS phenotype with reduction of LPS and part of outer membrane proteins, but its MIC to CRO is intermediate. It was found that nine spots in 2-DE image of outer membrane protein profile to be altered. The protein levels of six out of nine spots were significantly increased and those of the other three spots were significantly decreased. The permeability assay proved that the highest permeability in strain 5B7 and reduced efflux pump capacity in both strains 4H11 and 5B7 may contribute to reduction of antibiotics resistance. I also chose strain 5G8 which carried a transposon inserted in tolC gene for study. Since TolC protein associated with multiple drug resistance such as SDS and lipophilic ��-lactams antibiotics, it resulted in reduction of CRO resistance (MIC < 2.5 mg/l). In our previous study, we found a putative outer membrane protein STM3031 correlated with CRO resistance (Cheng 2003). The expression level of STM3031 in strains 5B7 and 4H11 were also found to be reduced. We concluded that a gene disruption in LPS biosynthesis could affect outer membrane protein expression, membrane permeability and the transporters or efflux pumps, all of these may contribute to the susceptibility to ceftriaxone.
中文摘要--------------------------------------------------------------1
英文摘要--------------------------------------------------------------3
緒論--------------------------------------------------------------4
材料與方法--------------------------------------------------------------12
結果--------------------------------------------------------------23
討論--------------------------------------------------------------31
圖表--------------------------------------------------------------40
附錄--------------------------------------------------------------55
參考文獻--------------------------------------------------------------58
Austin, E. A.,T James F. Graves, Lawrence A. Hite, et al. (1990) Genetic Analysis of Lipopolysaccharide Core Biosynthesis by Escherichia coli K-12: Insertion Mutagenesis of the rfa Locus. Journal of Bacteriology.
172(9): 5312-5325.
Binghua, J., M. Freudenberg, and C. Galanos. (1989) Characterization of the lipid A component of genuine smooth-form lipopolysaccharide. Eur. J. Biochem. 180:515-518.
Brown, M.H., Paulsen, I.T. and Skurray, R.A. (1999) The multidrug efflux protein NorM is a prototype of a new family of transporters.Molecular Microbiology. 31: 393-395.
Cetinkaya, Y., Falk, P. and Mayhall, C.G. (2000) Vancomycin-resistant enterococci. Clinical Microbiology Reviews 13: 686-707.
Cheng, C. Y. (2003) Outer membrane protein profile in ceftriaxone resistant Salmonella typhimurium by two-dimensional gel electrophoresis analysis. MS thesis. National Yang-Ming University,Taiwan.
Chopra, I. and Roberts, M. (2001) Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and Molecular Biology Reviews. 65: 232-260.
Chung, Y.J. and Saier, M.H. Jr (2001) SMR-type multidrug
resistance pumps. Current Opinion in Drug Discovery and Development.4: 237-245.
Cox, A. D. and S. G. Wilkinson (1991) Ionizing groups in
lipopolysaccharides of Pseudomonas cepacia in relation to antibiotic resistance. Mol Microbiol. 5(3): 641-646.
Daly, M. and S. Fanning. (2000) Characterization and Chromosomal Mapping of Antimicrobial Resistance Genes in Salmonella enterica Serotype Typhimurium. Applied and Environmental Microbiology. 66(11): 4842-4848.
Diedrich, D. L., M. A. Stein, et al. (1990) Associations of Escherichia coli K-12 OmpF trimers with rough and smooth lipopolysaccharides. J Bacteriol. 172(9): 5307-11.
Donlan, R.M. (2000) Role of biofilms in antimicrobial resistance. ASAIO Journal. 46: S47-S52.
Dupont, M., Emmanuelle De, Renaud Chollet, Jacqueline Chevalier,Jean-Marie Pages. (2004) Enterobacter aerogenes OmpX, a cation-selective channel mar- and osmo-regulated. FEBS Letters. 569:27–30.
Giraud, E., A. Cloeckaert, et al. (2000) Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother. 44(5):1223-1228.
Giraud, Hoare, A., M. Bittner, et al. (2006) The outer core
lipopolysaccharide of Salmonella enterica serovar Typhi is required for bacterial entry into epithelial cells. Infect Immun. 74(3): 1555-64.
Hakenbeck, R., Grebe, T., Zahner, D. and Stock, J.B. (1999) ��-Lactamresistance in Streptococcus pneumoniae: penicillin-binding proteins and non-penicillin-binding proteins. Molecular Microbiology 33:673-678.
Hancock, R.E.W. (1997) The bacterial outer membrane as a drug barrier. Trends in Microbiology. 5: 37-42.
Hancock, R.E.W. and Woodruff, W.A. (1988) Roles of porin and
��-lactamase in ��-lactam resistance of Pseudomonas aeruginosa. Reviews of Infectious Disease. 10: 770-775.
Harder, K. J., H. Nikaido, et al. (1981) Mutants of Escherichia coli that are resistant to certain ��-lactam compounds lack the OmpF porin. Antimicrob Agents Chemother. 20(4): 549-52.
Hoare, A., M. Bittner, et al. (2006) The outer core lipopolysaccharide of Salmonella enterica serovar Typhi is required for bacterial entry into epithelial cells. Infect Immun. 74(3): 1555-64.
Hooper, D.C. (2000) Mechanisms of action and resistance of older and newer fluoroquinolones. Clinical Infectious Diseases. 31(Suppl. 2):S24-S28.
Jack, D.L., Yang, N.M. and Saier, M.H. (2001) The drug/metabolite transporter superfamily. European Journal of Biochemistry. 268:3620-3639.
Jo, Y. L., F. Nara, et al. (1986) Purification and characterization of the OmpR protein, a positive regulator involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli. J Biol Chem. 261(32): 15252-15256.
Kort, G. De, A. Bolton, G. Martin, J. Stephen, and J. A. M. Van De Klundert. (1994) Invasion of Rabbit Ileal Tissue by Enterobacter cloacae Varies with the Concentration of OmpX in the Outer Membrane. Infection and Immunity. 62(11): 4722-4726.
Lambert, B. and Jean-Bernard Le Pecq. (1984) Effect of Mutation, Electric Membrane Potential, and Metabolic Inhibitors on the Accessibility of Nucleic Acids to Ethidium Bromide in Escherichia coli Cells. Biochemistry. 1984(23): 166-176.
Li, P. C. (2003) Overexpression of the response regulator BaeR of the two-component system confer ceftriaxone resistance in Salmonella typhimurium. MS thesis. National Yang-Ming University, Taiwan.
Lin Yin-Hung. (2005) Proteomic analysis on the expression of outer membrane proteins of Salmonella enterica serovar Typhimurium putative transporter gene yjeH associated with ceftriaxone-resistance. MS thesis. National Yang-Ming University, Taiwan.
Lyman , M. B., J. P. Steward, and R. J. Roantree. (1976)
Characterization of the Virulence and Antigenic Structure of Salmonella typhimurium Strains with Lipopolysaccharide Core Defects. Infection and Immunity. 13(6): 1539-1542.
Marolda, C. L., J. Vicarioli, et al. (2004) Wzx proteins involved in biosynthesis of O antigen function in association with the first sugar of the O-specific lipopolysaccharide subunit. Microbiology. 150(Pt 12):
4095-105.
Martinez-Martinez, L., M. C. Conejo, A. Pascual, S. Hernandez-Alles et al. (2000) Activities of Imipenem and Cephalosporins against Clonally Related Strains of Escherichia coli Hyperproducing Chromosomal ��-Lactamase and Showing Altered Porin Profiles.
Antimicrobial Agents and Chemotherapy. 44(9): 2534-2536.
Nakashima, K., Horikoshi K, Mizuno T. (1995) Effect of hydrostatic pressure on the synthesis of outer membrane proteins in Escherichia coli. Biosci Biotechnol Biochem. 59(1):130-132.
Nikaido, H. (1994) Prevention of drug access to bacterial targets: permeability barriers and active efflux. Science. 264: 382-388.
Nikaido, H. (1998) Antibiotic resistance caused by Gram-negative multidrug efflux pumps. Clinical Infectious Diseases. 27:(Suppl. 1) S32–S41.
Nikaido, H. Marina Basina, Vy Nguyen and Emiko Y. Rosenberg (1998) Multidrug Efflux Pump AcrAB of Salmonella typhimurium Excretes Only Those ��-Lactam Antibiotics Containing Lipophilic Side Chains. Journal of Bacteriology. 180(7): 4686-4692.
Nishino, K., and Yamaguchi, A. (2002) EvgA of the two-component signal transduction system modulates production of the YhiUV multidrug transporter in Escherichia coli. J. Bacteriol. 184:2319-2323.
Nishino, K., J. Yamada, et al. (2003) Roles of TolC-dependent multidrug transporters of Escherichia coli in resistance to ��-lactams. Antimicrob Agents Chemother. 47(9): 3030-3.
Osborn, M. J., J. E. Gander, et al. (1972) Mechanism of assembly of the outer membrane of Salmonella typhimurium. Isolation and characterization of cytoplasmic and outer membrane. J. Biological Chemistry. 247(12): 3962-3972.
Otto, K. and Malte Hermansson. (2004) Inactivation of ompX Causes Increased Interactions of Type 1 Fimbriated Escherichia coli with Abiotic Surfaces. Journal of Bacteriology. 186(1):226–234.
Pao, S.S., Paulsen, I.T. and Saier, M.H. Jr (1998) Major facilitator superfamily. Microbiology and Molecular Biology Reviews. 62: 1-34.
Poole, K. Kelly Tetro, Qixun Zhao, Shadi Neshat et al. (1996) Expression of the Multidrug Resistance Operon mexA-mexB-oprM in Pseudomonas aeruginosa: mexR Encodes a Regulator of Operon Expression. Antimicrobial Agents and Chemotherapy. 40(9):2021-2028.
Poole, K. (2002) Mechanisms of bacterial biocide and antibiotic resistance. J. Applied Microbiology Symposium Supplement.92:55S-64S.
Randall, L. P., Woodward M. J. (2002) The multiple antibiotic resistance (mar) locus and its significance. Res Vet Sci. 72(2):87-93.
Rocque, W. J., R. T. Coughlin, and E. J. McGroarty. (1987)
Lipopolysaccharide tightly bound to porin monomers from Escherichia coli K-12. J. Bacteriol. 169:4003-4010.
Stoorvogel, J. Mario J. A. W. M. Van Bussel, and Jos A. M. Van De Klundert (1991) Biological Characterization of an Enterobacter cloacae Outer Membrane Protein (OmpX). Journal of Bacteriology. 173(1):161-167.
Su, L. H., Tsu-Lan Wu, Ju-Hsin Chia, et al. (2005) Increasing ceftriaxone resistance in Salmonella isolates from a university hospital in Taiwan. Journal of Antimicrobial Chemotherapy. 55: 846–852.
Thomson, K.S. and Smith, M.E. (2000) Version 2000: the new
��-lactamases of Gram-negative bacteria at the dawn of the new millennium. Microbes and Infection. 2: 1225-1235.
van Veen, H.W. and Konings,W.N. (1998) The ABC family of multidrug transporters in microorganisms. Biochimica Biophysica Acta. 1365: 31-36.
Wang, P., Nirmalan N., Wang Q., Sims P.F. et al. (2004) Genetic and metabolic analysis of folate salvage in the human malaria parasite Plasmodium falciparum. Mol. Biochem. Parasitol. 135: 77-87
Yang, W., Ian F. Moore, Kalinka P. Koteva et al. (2004) TetX Is a Flavin-dependent Monooxygenase Conferring Resistance to Tetracycline Antibiotics. The Journal of Biological Chemistry. 279(50): 52346-52352.
Yethon, J. A., E. Vinogradov, et al. (2000) Mutation of the
lipopolysaccharide core glycosyltransferase encoded by waaG
destabilizes the outer membrane of Escherichia coli by interfering with core phosphorylation. J Bacteriol. 182(19): 5620-5623.
Yethon, J. A., J. S. Gunn, et al. (2000) Salmonella enterica serovar Typhimurium waaP mutants show increased susceptibility to polymyxin and loss of virulence In vivo. Infect Immun. 68(8): 4485-4491.
Zgurskaya, H.I. and Nikaido, H. (2000) Multidrug resistance
mechanisms: drug efflux across two membranes. Molecular
Microbiology. 37: 219-225.
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