臺灣博碩士論文加值系統

陰道鞭毛蟲 (T. vaginalis) 是一種厭氧性的單細胞原生寄生蟲，主要寄生於人類的泌尿生殖道，引起陰道鞭毛蟲症 (trichomoniasis)，是最常見的非病毒的性接觸傳染疾病。流行病學研究指出感染陰道鞭毛蟲會使病人受到人類後天免疫缺乏病毒(HIV-1)感染的機會增加1.5倍。鐵藉由影響陰道鞭毛蟲的生理特性，例如：黏附因子合成、表面抗原的表達以及許多過氧化氫體(hydrogenosome)中的酵素活性，對陰道鞭毛蟲的生長與存活來說，是不可缺少的微量元素。我們利用蛋白質體學研究方法，比較在補充有12 �嵱及70 �n�嵱檸檬酸鐵(ammonium iron citrate)、12 �嵱血紅素(haemoglobin)或12 �嵱高鐵血紅素(hemin)等不同來源鐵的無鐵YI-S培養液(yeast-extract iron serum medium)中，陰道鞭毛蟲(T. vaginalis strain 30236 (JH31A, USA))的基因表達差異，並將有明顯差異的蛋白質利用胜肽質量指紋譜鑑定(peptide mass fingerprint)方法鍵定出其身份。其中，紅素氧還蛋白(rubrerythrin, TVAG_064490)是一種非血晶質(haem)鍵結的蛋白質，是一種過氧化氫酶，在檸檬酸鐵環境下有高量表現，且表現量隨著鐵濃度降低而降低，在血紅素環境下幾乎不表現，而在高鐵血紅素環境下有高量表現。RT-PCR結果顯示在mRNA表現上也有相同的情形。而在Tritrichomonas foetus和Trichomonas vaginalis strain 30001 (C-1: NIH)，紅素氧還蛋白對鐵的反應也相同。與此同時，同樣具有去除氧化壓力能力的超氧岐化酶(superoxide dismutase)，硫氧還蛋白過氧化酶(thioredoxin peroxidase)，硫氧還蛋白(thioredoxin)及硫氧還蛋白還原酶(thioredoxin reductase)在以檸檬酸鐵或血紅素為鐵來源的培養情況下，分別有不同的基因亞型(isoform)被表現。

Trichomonas vaginalis is an anaerobic parasitic protozoan, which causes trichomoniasis, a sexually transmitted disease. T. vaginalis may play a critical and under-recognized role in amplifying HIV transmission. Epidemiological study indicates that infection with T. vaginalis increases a 1.5-fold risk of HIV-1 acquisition. Iron is an essential element for the growth and survival of T. vaginalis by regulating a variety of trichomonal properties such as adhesin synthesis, gene expression of surface immunogens and several hydrogenosomal enzyme activities of the T. vaginalis. Subproteomics analysis, we compared the gene expression of T. vaginalis strain ATCC 30236 (JH31A, USA) cultivated in the iron free yeast-extract iron serum (YIS) medium supplied with 12 �嵱 (low) or 70�n�嵱 (high) ammonium iron citrate, haemoglobin and hemin which was used for mimicking the habitant environment of T. vaginalis. A 21kDa, pI 8.16 peptide spot could be recognized 15.4-fold expression under high iron supplement than haemoglobin supplement. It was identified with peptide mass fingerprint method as rubrerythrin (RBR, TVAG_064490), a non-haem iron binding protein which was recently identified in T. vaginalis. The RT-PCR results showed RBR TVAG_064490 mRNA was regulated by iron. Not only in different strains of T. vaginalis but also in Tritrichomonas foetus and Trichomonas vaginalis strain 30001 (C-1: NIH), RBR was expressed and induced under iron supplement. At the same time, specific expression pattern of components of oxygen scavenge system such as superoxide dismutase, thioredoxin peroxidase, thioredoxin and thioredoxin reductase isoforms were induced under specific iron source condition.

Index 1
List of tables 2
List of figures 3
Abstract 5
Introduction 7
The aim of the study 13
Materials and methods 14
Results 22
Discussion 30
References 33
Tables 39
Figures 47

Akhmanova,A., Voncken,F., van Alen,T., van Hoek,A., Boxma,B., Vogels,G., Veenhuis,M., and Hackstein,J.H. (1998) A hydrogenosome with a genome Nature. 396: 527-528.
Alderete,J.F., Provenzano,D., and Lehker,M.W. (1995) Iron mediates Trichomonas vaginalis resistance to complement lysis Microb.Pathog. 19: 93-103.
Alderete,J.F., Nguyen,J., Mundodi,V., and Lehker,M.W. (2004) Heme-iron increases levels of AP65-mediated adherence by Trichomonas vaginalis Microb.Pathog. 36: 263-271.
B.M.Honigberg, V.M.King (1964) Structure of Trichomonas vaginalis Donne The Journal of Parasitology 50: 345-364.
Bui,E.T., Johnson,P.J. (1996) Identification and characterization of [Fe]-hydrogenases in the hydrogenosome of Trichomonas vaginalis Mol.Biochem.Parasitol. 76: 305-310.
Carlton,J.M., Hirt,R.P., Silva,J.C., Delcher,A.L., Schatz,M., Zhao,Q., Wortman,J.R., Bidwell,S.L., Alsmark,U.C., Besteiro,S., Sicheritz-Ponten,T., Noel,C.J., Dacks,J.B., Foster,P.G., Simillion,C., Van de,P.Y., Miranda-Saavedra,D., Barton,G.J., Westrop,G.D., Muller,S., Dessi,D., Fiori,P.L., Ren,Q., Paulsen,I., Zhang,H., Bastida-Corcuera,F.D., Simoes-Barbosa,A., Brown,M.T., Hayes,R.D., Mukherjee,M., Okumura,C.Y., Schneider,R., Smith,A.J., Vanacova,S., Villalvazo,M., Haas,B.J., Pertea,M., Feldblyum,T.V., Utterback,T.R., Shu,C.L., Osoegawa,K., de Jong,P.J., Hrdy,I., Horvathova,L., Zubacova,Z., Dolezal,P., Malik,S.B., Logsdon,J.M., Jr., Henze,K., Gupta,A., Wang,C.C., Dunne,R.L., Upcroft,J.A., Upcroft,P., White,O., Salzberg,S.L., Tang,P., Chiu,C.H., Lee,Y.S., Embley,T.M., Coombs,G.H., Mottram,J.C., Tachezy,J., Fraser-Liggett,C.M., and Johnson,P.J. (2007) Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis Science. 315: 207-212.
Cohen,M.S., Hoffman,I.F., Royce,R.A., Kazembe,P., Dyer,J.R., Daly,C.C., Zimba,D., Vernazza,P.L., Maida,M., Fiscus,S.A., and Eron,J.J., Jr. (1997) Reduction of concentration of HIV-1 in semen after treatment of urethritis: implications for prevention of sexual transmission of HIV-1. AIDSCAP Malawi Research Group Lancet. 349: 1868-1873.
Coombs,G.H., Westrop,G.D., Suchan,P., Puzova,G., Hirt,R.P., Embley,T.M., Mottram,J.C., and Muller,S. (2004) The amitochondriate eukaryote Trichomonas vaginalis contains a divergent thioredoxin-linked peroxiredoxin antioxidant system J.Biol.Chem. 279: 5249-5256.
Crouch,M.L., Benchimol,M., and Alderete,J.F. (2001) Binding of fibronectin by Trichomonas vaginalis is influenced by iron and calcium Microb.Pathog. 31: 131-144.
De Jesus,J.B., Cuervo,P., Junqueira,M., Britto,C., Silva-Filho,F.C., Soares,M.J., Cupolillo,E., Fernandes,O., and Domont,G.B. (2007) A further proteomic study on the effect of iron in the human pathogen Trichomonas vaginalis Proteomics. 7: 1961-1972.
Demes,P., Gombosova,A., Valent,M., Fabusova,H., and Janoska,A. (1988a) Fewer Trichomonas vaginalis organisms in vaginas of infected women during menstruation Genitourin.Med. 64: 22-24.
Demes,P., Gombosova,A., Valent,M., Janoska,A., Fabusova,H., and Petrenko,M. (1988b) Differential susceptibility of fresh Trichomonas vaginalis isolates to complement in menstrual blood and cervical mucus Genitourin.Med. 64: 176-179.
Diamond,L.S., Clark,C.G., and Cunnick,C.C. (1995) YI-S, a casein-free medium for axenic cultivation of Entamoeba histolytica, related Entamoeba, Giardia intestinalis and Trichomonas vaginalis J.Eukaryot.Microbiol. 42: 277-278.
Ellis,J.E., Yarlett,N., Cole,D., Humphreys,M.J., and Lloyd,D. (1994) Antioxidant defences in the microaerophilic protozoan Trichomonas vaginalis: comparison of metronidazole-resistant and sensitive strains Microbiology. 140: 2489-2494.
Embley,T.M., Martin,W. (2006) Eukaryotic evolution, changes and challenges Nature. 440: 623-630.
Everse,J., Hsia,N. (1997) The toxicities of native and modified hemoglobins Free Radic.Biol.Med. 22: 1075-1099.
Garcia,A.F., Chang,T.H., Benchimol,M., Klumpp,D.J., Lehker,M.W., and Alderete,J.F. (2003) Iron and contact with host cells induce expression of adhesins on surface of Trichomonas vaginalis Mol.Microbiol. 47: 1207-1224.
Gorrell,T.E. (1985) Effect of culture medium iron content on the biochemical composition and metabolism of Trichomonas vaginalis J.Bacteriol. 161: 1228-1230.
Hall,T.A. (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program. Nucl.Acids.Symp. 41: 95-98.
Heath,J.P. (1981) Behaviour and pathogenicity of Trichomonas vaginalis in epithelial cell cultures: a study by light and scanning electron microscopy Br.J.Vener.Dis. 57: 106-117.
Hentze,M.W., Kuhn,L.C. (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress Proc.Natl.Acad.Sci.U.S.A. 93: 8175-8182.
Jin,S., Kurtz,D.M., Jr., Liu,Z.J., Rose,J., and Wang,B.C. (2002) X-ray crystal structures of reduced rubrerythrin and its azide adduct: a structure-based mechanism for a non-heme diiron peroxidase J.Am.Chem.Soc. 124: 9845-9855.
Kim,Y.S., Song,H.O., Choi,I.H., Park,S.J., and Ryu,J.S. (2006) Hydrogenosomal activity of Trichomonas vaginalis cultivated under different iron conditions Korean J.Parasitol. 44: 373-378.
Kumar,S., Bandyopadhyay,U. (2005) Free heme toxicity and its detoxification systems in human Toxicol.Lett. 157: 175-188.
LeGall,J., Prickril,B.C., Moura,I., Xavier,A.V., Moura,J.J., and Huynh,B.H. (1988) Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster Biochemistry. 27: 1636-1642.
Lehker,M.W., Chang,T.H., Dailey,D.C., and Alderete,J.F. (1990) Specific erythrocyte binding is an additional nutrient acquisition system for Trichomonas vaginalis J.Exp.Med. 171: 2165-2170.
Lehker,M.W., Arroyo,R., and Alderete,J.F. (1991) The regulation by iron of the synthesis of adhesins and cytoadherence levels in the protozoan Trichomonas vaginalis J.Exp.Med. 174: 311-318.
Lehker,M.W., Alderete,J.F. (1992) Iron regulates growth of Trichomonas vaginalis and the expression of immunogenic trichomonad proteins Mol.Microbiol. 6: 123-132.
Leipuviene,R., Theil,E.C. (2007) The family of iron responsive RNA structures regulated by changes in cellular iron and oxygen Cell Mol.Life Sci. 64: 2945-2955.
Lill,R., Muhlenhoff,U. (2005) Iron-sulfur-protein biogenesis in eukaryotes Trends Biochem.Sci. 30: 133-141.
Lindmark,D.G., Muller,M. (1973) Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism J.Biol.Chem. 248: 7724-7728.
Lindmark,D.G., Muller,M. (1974) Superoxide dismutase in the anaerobic flagellates, Tritrichomonas foetus and Monocercomonas sp J.Biol.Chem. 249: 4634-4637.
Linstead,D.J., Bradley,S. (1988) The purification and properties of two soluble reduced nicotinamide: acceptor oxidoreductases from Trichomonas vaginalis Mol.Biochem.Parasitol. 27: 125-133.
Marczak,R., Gorrell,T.E., and Muller,M. (1983) Hydrogenosomal ferredoxin of the anaerobic protozoon, Tritrichomonas foetus J.Biol.Chem. 258: 12427-12433.
McClelland,R.S., Sangare,L., Hassan,W.M., Lavreys,L., Mandaliya,K., Kiarie,J., Ndinya-Achola,J., Jaoko,W., and Baeten,J.M. (2007) Infection with Trichomonas vaginalis increases the risk of HIV-1 acquisition J.Infect.Dis. 195: 698-702.
Muller,S., Liebau,E., Walter,R.D., and Krauth-Siegel,R.L. (2003) Thiol-based redox metabolism of protozoan parasites Trends Parasitol. 19: 320-328.
Page-Sharp,M., Behm,C.A., and Smith,G.D. (1996) Tritrichomonas foetus and Trichomonas vaginalis: the pattern of inactivation of hydrogenase activity by oxygen and activities of catalase and ascorbate peroxidase Microbiology. 142: 207-211.
Paget,T.A., Lloyd,D. (1990) Trichomonas vaginalis requires traces of oxygen and high concentrations of carbon dioxide for optimal growth Mol.Biochem.Parasitol. 41: 65-72.
Pereira-Neves,A., Ribeiro,K.C., and Benchimol,M. (2003) Pseudocysts in trichomonads--new insights Protist. 154: 313-329.
Peterson,K.M., Alderete,J.F. (1984) Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors J.Exp.Med. 160: 398-410.
Petrin,D., Delgaty,K., Bhatt,R., and Garber,G. (1998) Clinical and microbiological aspects of Trichomonas vaginalis Clin.Microbiol.Rev. 11: 300-317.
Prickril,B.C., Kurtz,D.M., Jr., LeGall,J., and Voordouw,G. (1991) Cloning and sequencing of the gene for rubrerythrin from Desulfovibrio vulgaris (Hildenborough) Biochemistry. 30: 11118-11123.
Putz,S., Gelius-Dietrich,G., Piotrowski,M., and Henze,K. (2005) Rubrerythrin and peroxiredoxin: two novel putative peroxidases in the hydrogenosomes of the microaerophilic protozoon Trichomonas vaginalis Mol.Biochem.Parasitol. 142: 212-223.
Rendon-Maldonado,J., Espinosa-Cantellano,M., Soler,C., Torres,J.V., and Martinez-Palomo,A. (2003) Trichomonas vaginalis: in vitro attachment and internalization of HIV-1 and HIV-1-infected lymphocytes J.Eukaryot.Microbiol. 50: 43-48.
Solano-Gonzalez,E., Burrola-Barraza,E., Leon-Sicairos,C., Avila-Gonzalez,L., Gutierrez-Escolano,L., Ortega-Lopez,J., and Arroyo,R. (2007) The trichomonad cysteine proteinase TVCP4 transcript contains an iron-responsive element FEBS Lett. 581: 2919-2928.
Sorvillo,F., Kovacs,A., Kerndt,P., Stek,A., Muderspach,L., and Sanchez-Keeland,L. (1998) Risk factors for trichomoniasis among women with human immunodeficiency virus (HIV) infection at a public clinic in Los Angeles County, California: implications for HIV prevention Am.J.Trop.Med.Hyg. 58: 495-500.
Steinbuchel,A., Muller,M. (1986) Anaerobic pyruvate metabolism of Tritrichomonas foetus and Trichomonas vaginalis hydrogenosomes Mol.Biochem.Parasitol. 20: 57-65.
Tsai,C.D., Liu,H.W., and Tai,J.H. (2002) Characterization of an iron-responsive promoter in the protozoan pathogen Trichomonas vaginalis J.Biol.Chem. 277: 5153-5162.
Warton,A., Honigberg,B.M. (1979) Structure of trichomonads as revealed by scanning electron microscopy J.Protozool. 26: 56-62.
Wawer,M.J., Sewankambo,N.K., Serwadda,D., Quinn,T.C., Paxton,L.A., Kiwanuka,N., Wabwire-Mangen,F., Li,C., Lutalo,T., Nalugoda,F., Gaydos,C.A., Moulton,L.H., Meehan,M.O., Ahmed,S., and Gray,R.H. (1999) Control of sexually transmitted diseases for AIDS prevention in Uganda: a randomised community trial. Rakai Project Study Group Lancet. 353: 525-535.
Williams,K., Lowe,P.N., and Leadlay,P.F. (1987) Purification and characterization of pyruvate: ferredoxin oxidoreductase from the anaerobic protozoon Trichomonas vaginalis Biochem.J. 246: 529-536.
World Health Organization. Global Prevalence and Incidence of Selected Curable Sexually Transmitted Diseases: Overview and Estimates. 2001. Ref Type: Report