臺灣博碩士論文加值系統

陰道鞭毛蟲症是目前世界上最普遍經由性行為感染的疾病。陰道鞭毛蟲基因體全長約170 MB，其含有六萬個基因，是目前已知原蟲當中最多的。由鞭毛體型態轉變成吸附的阿米巴形態對陰道的感染是很重要的步驟。在這個研究中我們利用了比較轉錄體學與蛋白質體學的系統分析方法針對陰道鞭毛蟲滋養體與阿米巴體的分子調控做探討。我們首先建立了陰道鞭毛蟲滋養體的標準二維電泳蛋白圖譜，再利用比較二維電泳結合質譜的方法找出了49個表現量有差異的蛋白。其中在阿米巴體中具有抗氧化壓力活性的蛋白表現量是增加的，而醣類代謝及細胞骨架蛋白表現量則是下降的。轉錄體學則利用大規模表現序列標籤(ESTs)的定序來探討基因表現。由滋養體與阿米巴體的cDNA libraries 分析超過兩萬個ESTs，並依據它們的功能做分類。我們也選取了在阿米巴體時期基因表現量大於5倍(116個基因)及10倍（32個基因）的基因做分析。有趣的是，與壓力有關的基因在阿米巴體時期被大量的表現，表示阿米巴體時期是在壓力的狀態之下。這也暗示這些抗氧化壓力的蛋白在阿米巴體時期扮演重要的角色，且阿米巴體時期對陰道鞭毛蟲來說並不是最佳的生理狀態。這些由轉錄體學及蛋白質體學分析所得到的發現希望可以提供對陰道鞭毛蟲症提供新的觀點並且對此寄生蟲與宿主之間的作用有更基礎的了解。

Trichomoniasis caused by Trichomonas vaginalis is the most common sexual transmitted infection in the world. The 170-MB genome of this protozoan contains 60,000 genes, the largest number of genes ever identified in protozoan. The morphological transformation from a flagellated form to an adherent amoeboid form is crucial to the establishment of infection in vagina. In this study, we established the reference two-dimensional gel electrophoresis (2-DGE) map of the trophozoite stage. The molecular response in the amoeboid and trophozoite stages of T .vaginalis was elucidated by using a systemic comparative transcriptomics and proteomics approach. We identified 49 differentially expressed proteins by 2-DGE and MALDI-TOF MS. Among them, stress proteins with antioxidant properties were up-regulated whereas carbohydrate metabolism and cytoskeletal proteins were down-regulated in the amoeboid form. Transcriptomics analysis at gene expression level was performed by using large scale expressed sequence tags (ESTs) sequencing. More than 20,000 ESTs from trophozoite and amoeboid cDNA libraries were clustered and classified according to their biological functions. We also summarized the genes which expressed 5-fold (116 genes) and 10-fold (32 genes) higher in the amoeboid stage. Interestingly, most of these significantly expressed genes in the amoeboid form are stress-related genes, indicating that the amoeboid stage is under stress. This implied that these antioxidant proteins have important roles in the amoeboid stage and the pathogenic amoeboid stage is not the optimum physiological condition for T. vaginalis. These findings derived from proteome and transcriptome analysis are expected to provide new insights into the pathogenesis of trichomoniasis and will facilitate a more fundamental understanding of the host-parasite interplay.

CONTENTS

Contents---------------------------------------------------------------------------I
致謝--------------------------------------------------------------III
中文摘要-------------------------------------------------------------------------IV
Abstract---------------------------------------------------------------------------V

Chapter 1: Introduction-------------------------------------------------------1
1.1 Trichomonas vaginalis-----------------------------------------------------2
1.2 Life cycle--------------------------------------------------------------------2
1.3 Treatment--------------------------------------------------------------------3
1.4 Cytoadherence--------------------------------------------------------------4
1.5 Transcriptomics-------------------------------------------------------------5
1.6 Proteomics-------------------------------------------------------------------6

Chapter 2 : Materials and Method------------------------------------------8
2.1 T. vaginalis culture conditions---------------------------------------------9
2.2 Protein extraction and 2-DE------------------------------------------------9
2.3 Protein visualization and image analysis---------------------------------10
2.4 In gel digestion and MALDI-TOF-MS analysis------------------------11
2.5 Database Search and Protein Identification------------------------------11
2.6 Complementary DNA construction and EST sequencing-------------12
2.7 Functional annotations and sequence analyses--------------------------13
2.8 Quantitative real-time PCR (qRT-PCR)---------------------------------14

Chapter 3: Results-------------------------------------------------------------15
3.1 A proteome reference map of T.vaginalis-------------------------------16
3.1.1 2-DE protein reference maps and image analysis--------------------16
3.1.2 Protein identification by MALDI-TOF-MS---------------------------16
3.1.3 Discrepancy of theoretical and experimental pI and MW-----------17
3.1.4 Biological functions of the identified proteins------------------------18
3.1.5 Energy production and carbohydrate metabolism--------------------18
3.1.6 Cytoskeletal proteins-----------------------------------------------------20
3.1.7 Defense and stress-related proteins-------------------------------------20
3.1.8 Nucleotide metabolism---------------------------------------------------21
3.1.9 Cysteine proteinases------------------------------------------------------22
3.2 Comparative proteome and transcriptome of trophozoite and amoeboid stages------------------------------------------------------------------22
3.2.1 2-DE Profiling of the amoeboid and trophozoite stages-------------22
3.2.2 Differentially expressed proteins in the amoeboid stage------------24
3.2.3 Functional Classification of the differentially expressed proteins--25
3.2.4 Transcriptional profiling of the amoeboid stage compared to trophozoite------------------------------------------------------------------------26
3.3 Integration of transcriptomic and proteomic data-----------------------27

Chapter 4: Discussion and Conclusion-------------------------------------29

Figures and Tables-------------------------------------------------------------32

References------------------------------------------------------------------------68

Alper, M. M., B. N. Barwin, W. M. McLean, I. J. McGilveray, and S. Sved.1985. Systemic absorption of metronidazole by the vaginal route. Obstet.

Arroyo, R., A. González Robles, A. Martínez Palomo, and J.F. Alderete. 1993. Signaling of Trichomonas vaginalis for ameboid transformation and adhesin synthesis follows cytoadherence. Mol. Microbiol. 7:299.

Arroyo R., Gonzàlez-Robles A., Martìnez-Palomo A., Alderete J.F., Signalling of Trichomonas vaginalis for amoeboid transformation and adhesin synthesis follows cytoadherence, Mol. Microbiol. 7 (1993) 299–309.

Audic, S., and J. M. Claverie. 1997. The significance of digital gene expression profiles. Genome Res. 7:986–995.

Becker K, Gromer S, Schirmer RH, Müller S .2000 Thioredoxin reductase as a pathophysiological factor and drug target. Eur J Biochem 267:6118–6125.

Bonaldo, M. F., G. Lennon, and M. B. Soares. 1996. Normalization and
subtraction: two approaches to facilitate gene discovery. Genome Res.
6:791–806.

Brosson D, Kuhn L, Delbac F, Garin J, P Vivarès C, Texier C. 2006. Proteomic analysis of the eukaryotic parasite Encephalitozoon cuniculi (Microsporidia): a reference map for proteins expressed in late sporogonial stages. Proteomics 6:3625–3635

Caescu CI, Vidal O, Krzewinski F, Artenie V, Bouquelet S .2004. Bifidobacterium longum requires a fructokinase for fructose catabolism. J Bacteriol 186: 6515-6525.

Carlton JM, Hirt RP, Silva JC, Delcher AL, Schatz M, Zhao Q, Wortman JR.2007. Draft genome sequence of the sexually transmitted pathogen Trichomonas vaginalis. Science 315:207-212.

Claverie, J. M. 1999. Computational methods for the identification of differential and coordinated gene expression. Hum. Mol. Genet. 8:1821–1832.

Cotch MF, Pastorek JG 2nd, Nugent RP, Hillier SL, Gibbs RS, Martin DH, Eschenbach DA, Edelman R, Carey JC, Regan JA, Krohn MA, Klebanoff MA, Rao AV, Rhoads GG .1997. Trichomonas vaginalis associated with low birth weights and preterm delivery. Sex Transm Dis 24:353-360.

Crouch ML, Alderete JF. Trichomonas vaginalis interactions with fibronectin and laminin. Microbiology 1999, 145:2835-2843.

Cuervo P, De Jesus JB, Junqueira M, Mendonça-Lima L, González LJ, Betancourt L, Grimaldi G Jr, Domont GB, Fernandes O, Cupolillo E .2007. Proteome analysis of Leishmania (Viannia) braziliensis by two-dimensional gel electrophoresis and mass spectrometry. Mol Biochem Parasitol 154:6–21.

Cunningham, F. E., D. M. Kraus, L. Brubaker, and J. H. Fischer. 1994.
Pharmacokinetics of intravaginal metronidazole gel. J. Clin. Pharmacol.
34:1060–1065

De Jesus JB, Cuervo P, Junqueira M, Britto C, Silva-Filho FC, Soares MJ, Cupolillo E, Fernandes O, Domont GB .2007. A further proteomic study on the effect of iron in the human pathogen Trichomonas vaginalis. Proteomics 12:1961-1972.

Diamond LS, Clark CG, Cunnick CC (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.

Draper D, Donohoe W, Mortimer L, Heine RP. 1998. Cystein proteiases of Trichomonas vaginalis degrade secretary leucocyte protease inhibitor. J Infect Dis 178:815-819

Ewing, B., and P. Green. 2000. Analysis of expressed sequence tags indicates 35,000 human genes. Nat. Genet. 25:232–244.

Fluegge, K., O. Schweier, et al. 2004. Identification and immunoreactivity of proteins released from Streptococcus agalactiae. Eur. J. Clin. Microbiol. Infect. Dis. 23:818-824.

Garcia, A., T.-H. Chang, M. Benchimol, D.J. Klumpp, M.W. Lehker, and J.F. Alderete. 2003. Iron and contact with host cells surface express adhesins of Trichomonas vaginalis. Mol. Microbiol. 47: 1207-1224.

Garcia, A., M. Benchimol, and J.F. Alderete. 2005. Trichomonas vaginalis polyamine metabolism is linked to host cell adherence and cytotoxicity. Infect. Immun. 73: 2602-2610.

Gonzàles-Robles A., Lazaro-Haller A., Espinosa-Castellano M., Anaya-Velazques F., Martìnez-Palomo A., Trichomonas vaginalis: ultrastructural basis of the cytopathic effect, J. Euk. Microbiol. 42 (1995) 641–651.

Gram IT, Macaluso M, Churchill J, Stalsberg H.1992. Trichomonas vaginalis and human papilloma virus infection and the incidence of cervical intraepithelial neoplasia grade III. Cancer Cause Control 3:231-236.

Grodstein F, Goldman MB, Cramer DW .1993. Relation of tubal infertility to a history of sexually transmitted disease. Am J Epidemiol 137:577-584.

Gupta, S., D. Zink, B. Korn, M. Vingron, and S. A. Haas. 2004. Genome
wide identification and classification of alternative splicing based on EST
data. Bioinformatics 20:2579–2585.

Heyworth PG, Gutteridge WE .1984. Pyrimidine metabolism in
Trichomonas vaginalis. FEBS Lett 176:55–60.

Heyworth PG, Gutteridge WE, Ginger CD .1982. Purine metabolism
in Trichomonas vaginalis. FEBS Lett 141:106–110

Hinzmann, and A. Rosenthal. 1999. Exhaustive mining of EST libraries for genes differentially expressed in normal and tumour tissues. Nucleic Acids Res. 27:4251–4260.

Honigberg, B., and V. M. King. 1964. Structure of Trichomonas vaginalis Donne. J. Parasitol. 50:345–364.

Johnson PJ, Lahti CJ, Bradley PJ .1993 Biogenesis of the hydrogenosome
in the anaerobic protist Trichomonas vaginalis. J Parasitol 79:664–670.

Juliano C, Monaco G, Rubino S, Cappuccinelli P .1986. Inhibition of
Trichomonas vaginalis replication by the microtubule stabilizer
taxol. J Protozool 33:255–260

Juliano C, Monaco G, Bandiera P, Tedde G, Cappuccinelli P.1987.
Action of anticytoskeletal compounds on in vitro cytopathic
effect, phagocytosis, and adhesiveness of Trichomonas vaginalis.
Genitourin Med 63:256–263

Krauth-Siegel RL, Coombs GH .1999. Enzymes of parasite thiol
metabolism as drug targets. Parasitol Today 15:404–409

Li, L., B. P. Brunk, J. C. Kissinger, D. Pape, K. Tang, R. H. Cole, J. Martin, T. Wylie, M. Dante, S. J. Fogarty, D. K. Howe, P. Liberator, C. Diaz, J. Anderson, M. White, M. E. Jerome, E. A. Johnson, J. A. Radke, C. J. Stoeckert, Jr., R. H. Waterston, S. W. Clifton, D. S. Roos, and L. D. Sibley.2003. Gene discovery in the apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Res. 13:443–454.

Lindmark, D., and M. Muller. 1973. Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism. J. Biol. Chem. 248:7724–7728.

Lossick, J. G., and H. L. Kent. 1991. Trichomoniasis: trends in diagnosis
and management. Am. J. Obstet. Gynecol. 165:1217–1222.

Miller RL, Miller WH .1986. Purine salvage enzymes in Trichomonas
vaginalis. Adv Exp Med Biol 195:573–575.

Munagala NR, Wang CC .2003. Adenosine is the primary precursor
of all purine nucleotides in Trichomonas vaginalis. Mol Biochem
Parasitol 127:143–149

Nielsen, M., and R. Nielsen. 1975. Electron microscopy of Trichomonas
vaginalis Donne: interaction with vaginal epithelium in human trichomoniasis.Acta Pathol. Microbiol. Scand. Sect. B 83:305–320.

Nielsen, M. 1976. In vitro effect of metronidazole on the ultrastructure of
Trichomonas vaginalis Donne. Acta Pathol. Microbiol. Scand. Sect. B 84: 93–100.

Petrin D, Delgaty K, Bhatt R, Garber G. 1998.Clinical and
microbiological aspects of Trichomonas vaginalis. Clin Microbiol Rev,
11:300-317.

Posthuma CC, Bader R, Engelmann R, Postma PW, Hengstenberg W, Pouwels PH .2002. Expression of the xylulose 5-phosphate phosphoketolase gene, xpka, from Lactobacillus pentotosus MD363 is induced by sugars that are fermented via the phosphoketolase pathway and is repressed by glucose mediated by CcpA and the mannose phosphoenolpyruvate phosphotransferase system. Appl Environ Microbiol 68:831–837.

Schwebke JR, Burgess D. 2004.Trichomoniasis. Clin Microbiol Rev, 17:794-803.

Schena, M., D. Shalon, R. W. Davis, and P. O. Brown. 1995. Quantitative
monitoring of gene expression patterns with a complementary DNA microarray. Science 270:467–470.

Sommer U, Costello CE, Hayes GR, Beach DH, Gilbert RO, Lucas JJ, Singh BN (2005) Identification of Trichomonas vaginalis cysteine proteases that induce apoptosis in human vaginal epithelial cells. J Biol Chem 280:23853–23860.

Wang CC, Cheng HW.1984.Salvage of pyrimidine nucleosides by
Trichomonas vaginalis. Mol Biochem Parasitol 10:171–184.

Wellerson R Jr, Kupferberg AB .1962. On glycolysis in Trichomonas
vaginalis. J Protozool 9:418–424

Wolfsberg, T. G., and D. Landsman. 1997. A comparison of expressed
sequence tags (ESTs) to human genomic sequences. Nucleic Acids Res.
25:1626–1632.

Yoon K, Ryu JS, Min DY .1991. Cytotoxicity of lymphokine activated peritoneal macrophages against Trichomonas vaginalis. Kor J Parasitol 29:381–388.