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(1)台北醫學大學醫學科學研究所醫學檢驗暨生物技術組碩士論文. 陰道鞭毛蟲滋養體及阿米巴體比較蛋白質體學與轉錄體學之研究. Comparative proteomics and transcriptomics of Trichomonas vaginalis trophozoite and amoeboid stages. 指導教授:梁有志博士(Yu-Chih Liang, Ph.D.) 研究生:黃國洋 (Kuo-Yang Huang). 中華民國九十八年七月 July, 2009.

(2) 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 I .

(3) 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. II .

(4) 致謝能順利的完成碩士的學業,首先感謝梁有志老師在我研究所期間給予的各項協助，並且安排各個老師幫我完成口試。另外感謝長庚大學鄧致剛老師給予的協助,碩士的各項實驗皆在長庚大學完成,因此感謝鄧老師給我一個很好的研究環境,在實驗上也幫我解決許多問題，鄧老師給我很大的空間發揮，讓我學習獨立思考及解決問題的能力，這是我覺得在研究所這兩年獲得最多的。這段期間,我學習如何撰寫論文投稿於期刊，最後也順利被接受發表，是一個很好的學習過程及經驗。實驗生活，有時難免枯燥乏味，所以很幸運能進入這個充滿歡樂的實驗室。另外也感謝實驗室夥伴的協助,陪我度過研究所的這兩年生涯。感謝姮臨、維敏、若慈學姊在生活上的照顧；威辰同學在實驗上的幫忙跟討論，讓我學習到很多；儷霖、玉純跟志豪在實驗室的陪伴，讓實驗室的生活更加精采；小白及阿嫚則讓我的生活增加了不少樂趣；還有其他的學弟妹，讓整個實驗室充滿活力，所以能待在這邊完成我的碩士學業，我覺得很幸運。最後感謝我的父母培養我到現在，可以在這個家庭長大，覺得三生有幸；感謝姊姊的照顧及付出，讓我可以專心於學業，沒有後顧之憂。今後我一定會更加努力，在研究的領域中找出屬於自己的一片天。. 黃國洋謹致台北醫學大學醫學科學研究所中華民國九十八年七月. III .

(5) 中文摘要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。. IV .

(6) ABSTRACT 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.. V .

(7) Introduction. 1 .

(8) Chapter 1: INTRODUCTION. 1.. Trichomonas vaginalis. Trichomonas vaginalis, a protozoan parasite, is the causative agent of trichomoniasis, a serious sexually transmitted disease of worldwide distribution. More than 170 million people are infected annually. Women infected during pregnancy are predisposed to premature of the placental membranes, preterm delivery, low-birth-rate infants (Cotch et al. 1997; Minkoff et al. 1984), infertility (Grodstein et al. 1993), and cervical cancer (Gram et al. 1992; Kharsany et al. 1993). Trichomoniasis may also increase the risk of human immunodeficiency virus (HIV) transmission (Draper et al. 1998; Laga et al. 1993). In axenic culture, the shape of the trophozoite is typically pyriform, but amoeboid shapes are evident when attached to vaginal epithelial cells (Arroyo et al. 1993; Heath et al. 1981). In the vagina, T.vaginalis must adhere to host cell to establish and maintain an infection. The T. vaginalis genome is estimated to be 160 Mb and about two-thirds of the genome are repeats and transposable elements (Carlton et al. 2007). A core set of approximately 60,000 protein-coding genes was predicted, almost twice the number of genes identified in human.. 2.. Life cycle. The shape of T. vaginalis in culture is typically pyriform, although 2 .

(9) amoeboid shapes are evident in parasites adhering to vaginal tissues in vivo (Nielsen et al. 1975). T. vaginalis is varies in size, with an average length and width being 10 and 7 mm, respectively. Nondividing organisms have four anterior flagella. One recurrent flagellum and the costa originate in the kinetosomal complex at the anterior of the parasite. Internal organelles include a prominent nucleus and a rigid structure, the axostyle that runs through the cell from the anterior end to the posterior end (Honigberg et al. 1964; Nielsen et al. 1975). Unique energy-producing organelles, the hydrogenosomes (Lindmark et al. 1973), are present as paraxostylar and paracostal chromatic granules by light microscopy and as osmiophilic granules by electron microscopy (Nielsen et al. 1976; Nielsen et al. 1975). The life cycle of T. vaginalis is simple in that the trophozoite is transmitted through coitus and no cyst form is known. The trophozoite divides by binary fission and, in natural infections, gives rise to a population in the lumen and on the mucosal surfaces of the urogenital tracts of humans.. 3.. Treatment. The only curative treatment currently available for T. vaginalis infection in the United States is metronidazole. Usually prescribed as a single or multiple oral doses, metronidazole can also be administered intravenously. Vaginal metronidazole creams and pessaries have also been available but are no longer favored due to their poor rate of cure compared to oral metronidazole (Alper et al. 1985; Cunningham et al. 3 .

(10) 1994). Topical vaginal medications and pessaries can be prescribed for the treatment of T. vaginalis in women. Modern preparations include clotrimazole, povidone-iodine, nonoxynol-9, and arsenical pessaries. These preparations provide local symptom relief, but documentation on their effectiveness as cures has been inconsistent. There are no topical treatments for trichomoniasis in men (Lossick et al. 1991).. 1.4 Cytoadherence. Trichomonas transformation from a free-swimming trophozoite to an adherent amoeba is crucial to parasite establishment in the host vagina and subsequent pathogenesis (Lehker et al. 2000; Schwebke et al. 2004; Crouch et al. 1999; Petrin et al. 1998). Amoebal transformation takes place upon binding to vaginal epithelial cells or to extracellular matrix (ECM) proteins and can be induced in vitro upon binding to ECM components laminin and fibronectin (Crouch et al. 1999). T. vaginalis reacts dramatically to the presence of the target cell. Following cytoadherence the parasite changes its morphology, typically pear-shaped, into amoeboid, and a series of modifications in expression of molecules involved in pathogenesis occur. Amoeboid parasites become tightly adherent, forming numerous cytoplasmic projections interdigitating with the microvilli of the target cell (Arroyo et al. 1993). The morphological transformation leads to formation of areas where parasite and target become tightly associated, and the spaces formed between membranes are isolated from the environment (Gonzàles-Robles et al. 1995). The ability 4 .

(11) to undergo morphological changes is directly related to virulence, since formation of microenvironments is fundamental for pathogenicity: it allows the parasite to modify the local conditions, such as pH and Ca2+ concentration, thus creating the ideal environment for release and action of the pore-forming proteins.. 1.5 Transcriptomics. Living organisms possess notable properties of adaptation to physiological changes or to adverse conditions. Unicellular organisms, in particular, must contend with fluctuations in nutrients, pH, temperature, and external osmolarity, as well as exposure to a range of potentially toxic environmental compounds. The cellular response to these environmental challenges involves drastic changes in gene expression. This reprogramming of gene transcription can be unveiled using high-throughput technologies such as expressed sequence tag (EST) sequencing and DNA microarrays, which provide valuable information about the expression patterns of the cells under determined conditions. Characterization of environmentally triggered gene expression changes provides insights into when and how each gene is expressed and offers a glimpse at the physiological response of the cells to variations in their surroundings. ESTs have historically provided data for gene discovery (Ewing et al. 2000; Schmitt et al. 1999), tissue- or stage-specific gene expression (Audic et al 1997; Bonaldo et al. 1996; Claverie et al. 1999), and alternative splicing (Gupta et al. 2004 , Wolfsberg et al. 1997). In fact, 5 .

(12) EST sequencing is likely to make its greatest impact on understudied genomes where little prior sequence data exist and where full genome sequencing projects may not be undertaken in the near future (Li et al. 2003). DNA microarray technology created in the 1990s, using either whole-genome information or selected ESTs, allowed the simultaneous analysis of thousands of genes, as well as the identification of gene expression patterns related to cellular physiology, revolutionizing gene expression analysis (Schena et al. 1995).. 1.6 Proteomics. Transcriptome level involved mRNA abundance may only represent putative function. On the contrary, investigations at the proteome level can provide a more direct assessment of biological processes by monitoring the expressed proteins performing the regulator, enzymatic, and structural functions. Recently, proteomics is widely accepted to be a key technology in the postgenomic era for investigations of protein synthesis. In addition, proteomic tools are valuable for the research of the basic biology of an organism such as physiology, pathogenesis, potential drug target, and drug resistance mechanisms. Proteomic techniques using two-dimensional electrophoresis (2-DE) combined with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and LC-MS/MS were exploited to elucidate the intracellular protein patterns and their identification of a cell. The proteome of different organisms have been studied extensively, however, 6 .

(13) relatively few works have been reported on the reference proteome of pathogenic protozoan ( Brosson et al. 2006; De Jesus et al. 2007; Cuervo et al. 2007).. 7 .

(14) Materials and Methods. 8 .

(15) Chapter 2: Materials and Methods. 2.1 T. vaginalis culture conditions. T. vaginalis isolate ATCC30236 (JH 31A#4) and isolate TO16 were maintained in YIS medium, pH 5.8, containing 10 % heat-inactivated fetal calf serum at 37oC (Diamond et al. 1995). The number of viable cells was determined based on Trypan blue exclusion hemocytometer counts. Isolate JH 31A#4 grown to a late logarithmic phase with more than 90% viable cells was harvested for the construction of unsynchronized trophozoites EST library (TvE). Isolate TO16 grown in fibronectin coated T-75 flasks for 3 hours at 37°C was harvested for the construction of fibronectin-induced amoeboid form EST library (TvF).. 2.2 Protein extraction and 2-DE. Mid-logarithmic phase parasites cells were harvested by centrifugation at 3,000 rpm for 15 min and washed in normal saline for three times. The cells were disrupted in lysis buffer (8M urea, 4 % CHAPS) containing protease inhibitors (Roche diagnostics). Samples were lysed by ultrasonificaion in an ice bath for eight cycles, each consisting 10 second sonication followed by a 10 second break. The lysates were centrifuged at 13,000 rpm, 4oC for 15 min. The 2D-clean up kit (GE Healthcare) was used to remove impurities from the lysates. Protein concentration was determined using the Bio-Rad Protein Assay 9 .

(16) Kit. Approximately 250 μg of protein was diluted to a final volume of 250 μl in rehydration buffer (8M Urea, 2% CHAPS) containing trace amount of bromophenol blue for first dimension electrophoresis. Samples were applied to 13cm IPG gel strips (GE Healthcare) with a linear separation range of pH 4-7. Rehydration and isoelectric focusing were carried out in an Ettan IPGphor II (GE Healthcare) by using the followings settings: 30 V for 12 h, 50 V for 0.5 h, 100 V for 0.5 h, 250 V for 0.5 h, 500 V for 0.5 h, 1,000 V for 0.5 h, 4,000 V for 0.5 h, and gradient to 8,000 V for 45,000 Vh. The IPG strips were incubated for 15min in equilibration buffer (50 mM Tris-HCl pH 8.8, 6 M urea, 30 % glycerol, 2 % SDS, and a trace of bromophenol blue) containing 1% (w/v) dithiothreitol, followed by 15min in equilibration buffer containing 2.5% (w/v) iodoacetamide. Equilibrated IPG strips were separated across 15% SDS-PAGE gels and sealed with a solution of 0.5% (w/v) agarose containing a trace of bromophenol blue. Gels were run at 35 mA/gel and 4 oC until the tracking dye migrated to the bottom of the gel. Each experiment was performed twice to ensure the accuracy of analyses.. 2.3 Protein visualization and image analysis. The silver-stained 2-DE gels were scanned and analyzed by using the PhoretixTM 2D analysis software. The relative abundance of each protein spot was determined as a percentage of the total spot intensity. Experimental Mr and pI of each spot identified by 2-DE gels were obtained by comparing with a set standard Mr and pI markers. Only those 10 .

(17) spots that changed more than 1.5-fold were selected for MALDI-TOF-MS analysis.. 2.4 In gel digestion and MALDI-TOF-MS analysis. Differentially expressed protein spots were selected and excised from the gels. Then, the gel pieces were transferred to 0.5 ml tube for in gel trypsin digestion. The gel pieces were first destained in a destaining solution which contain 30 mM potassium ferricyanide and 100mM sodium thiosulphate, followed by washing and shrinking steps using 50 mM ammonium bicarbonate and acetonitrile (ACN). The gel pieces were completely dried by vacuum centrifuge and rehydrated in 3-fold volume of trypsin (Promega) solution (20 μg /ml in 25 mM ammonium bicarbonate). To extract peptides, 2 μl of extraction buffer (100% ACN with 1% TFA) was added, and the samples were sonicated for 30 min. Peptides were eluted and then co-crystallized with a saturated solution on an MALDI-TOF sample plate using 0.5 μl of sample and 0.5 μl matrix. The peptides were analyzed by using Ultraflex MALDI-TOF Mass Spectrometer (Bruker Daltonic).. 2.5 Database Search and Protein Identification. We constructed a local MASCOT T. vaginalis protein database based on the genomic sequences provided by The Institute for Genomic 11 .

(18) Research (TIGR), cDNA sequences from the T. vaginalis EST project and protein sequences deposited in National Center for Biotechnology Information (NCBI). The T. vaginalis G3 genome contain more than 60,000 putative open reading frames (ORFs), and the ESTs from different resources contain around 25,000 unigenes. An automated database search was performed by using the Biotool protein analysis software (Bruker Daltonic). The T.vaginalis-specific database was explored by means of MASCOT database search engine. The following search parameters were used: trypsin was used as enzyme, the peptide tolerance window was set to 100 ppm, one missed cleavage was allowed, carbamidomethyl and oxidized methionine were set as fixed and variable modification, respectively.. 2.6 Complementary DNA construction and EST sequencing. We constructed two cDNA libraries from mRNA isolated under following conditions: (1) Unsynchronized trophozoites (TvE) in nutrient-rich medium. T. vaginalis isolate ATCC30236 (JH 31A#4) was maintained in YIS medium, pH 5.8, containing 10 % heat-inactivated fetal calf serum at 37oC. (2) Fibronectin-induced amoeboid form (TvF). T. vaginalis isolate TO16 was grown in fibronectin coated T-75 flasks for 3 hours at 37°C.Total RNA was extracted from harvested cells using a commercial kit (Pharmacia) and contaminating genomic DNA was digested with DNase I. PolyA+ RNA was isolated using the PolyA+ tract mRNA isolation kit (Promega). Complementary DNA primed with 12 .

(19) oligo-dT was synthesized using a ZAP-cDNA synthesis kit and directionally cloned into the EcoRI and XhoI sites of Uni-ZAP XR (Stratagene). Single and well-separated plaques were cored out from agar plates and transferred into 96-well microtiter plates containing SM buffer. The phage stocks were used as templates for cDNA insert amplification with the T3 and T7 primers (1 nM for each primer). Amplified products were separated in 1.5% agarose gels and clones that yielded single PCR-amplified bands were collected for sequencing. Single-pass sequencing from the 5’-end of the cDNA insert was carried out with T3 primer by using the ABI PRISM BigDye Terminator Cycle Sequencing Kit (Applied Biosystems). The sequencing products were resolved and analyzed either on an ABI PRISM 377 (Applied Biosystems) or a MEGABACE DNA Sequencer (GE). The nucleotide sequences obtained were processed with the Phred/Phrap/Consed package.. 2.7 Functional annotations and sequence analyses. Assembled sequence contigs were compared with BLAST tools to putative open-reading-frames provided by the Institute of Genomic Research, as well as NCBI’s non-redundant (nr) nucleotide (E-value 10-15) and protein database, Swiss-Prot (E-value 10-10). Interpro version version 2005 and Gene Ontology version 27.00 were used to relate genes in different functional categories. The contigs with identification against the databases with identities greater than 60% of the contig length were assigned to annotated and assigned in KEGG pathways. Orthrologous 13 .

(20) genes in other sequenced protozoan genomes, including Plasmodium falciparum, Trypanosoma brucei, Tryapnisima cruzi , Leishmania Mexicana , Entamoeba histolytica and Giardia lamblia , were also identified by BLASTP. All BLAST results with significant hits were examined manually to assure identities and exclude errors.. 2.8 Quantitative real-time PCR (qRT-PCR). Reverse transcription was carried out in a reaction mixture containing total RNA, 50 nM RT primer, 0.25 mM dNTPs, 0.75 U/µl ThermoScriptTM III reverse transcriptase, 0.2 U/µl RNase out, and 0.05 M DTT (ThermoScript. TM. III RT-PCR System, Invitrogen). The RT reaction. mixture was incubated at 25oC 5 min, 50oC for 30 min and then stopped by 70 oC for 15min. RT-PCR was performed using a Ampliqon III RealQ-PCR master mix kit on an MX3000 System (Stratagene). The 20 µl PCR mixture contain1µg reverse transcription product, master mix, 0.5 µM real-time forward and reverse primers. The reactions were incubated in a 96-well plate at 95oC for 10 min, followed by 40 cycles of 95 oC for 30 sec, 50 oC for 1 min, and 72 oC for 30 sec. 60S rRNA was used as an internal control for normalization in all experimental groups.. 14 .

(21) Results. 15 .

(22) Chapter 3: Results. 3.1 A proteome reference map of T.vaginalis 3.1.1 2-DE protein reference maps and image analysis. To obtain a global view of the protein expression profile of T.vaginalis trophozoite, soluble proteins isolated from log phase culture was separated by 2DGE using wide range IPG strip of pH 3-10 (Fig. 1). We observed that majority of the expressed proteins are acidic as predicted by bioinformatics approach. In order to increase the resolution of acidic proteins, IPG strip of pH range 4-7 was used for all subsequent studies. Multiple reproducible 2-DE gels separated at pH 4-7 were conducted. A representative gel was showed as the 2-DE reference map of T.vaginalis in Fig. 3. Approximately 500 protein spots were detected in this silver-stained reference map. The majority of proteins spots detected have molecular weights between 11 KDa and 72 KDa. In addition, we also separated the basic proteins of T. vaginalis in the range of pH 6-11 (Fig. 2) and about 30 protein spots were detected. The presence of abundant acidic proteins in the T. vaginalis proteome may reflect an adaptation to the acidic microenvironment of vagina.. 3.1.2 Protein identification by MALDI-TOF-MS. To obtain the peptide mass fingerprinting (PMF) of the proteins on silver-stained 2-DE gels, 329 and 24 protein spots were excised from the 16 .

(23) pH 4-7 (Fig. 3) and pH6-11 (Fig. 2) 2-DE reference gels, respectively. In-gel trypsin digested peptides were submitted to MALDI-TOF PMF analyses. Of the 353 protein spots processed, 247 protein spots were successfully identified, representing 164 unique proteins. The accession numbers, protein scores, theoretical and experimental pI and molecular weights, and sequence coverage of all identified proteins in the T.vaginalis proteome were provided as Table 1 and Table 2. The most abundant proteins identified in our proteome study are glycolytic proteins, cytoskeleton proteins, and Hsp70. This protein profile is similar to the protein profile of T. vaginalis grown in iron rich and iron depleted medium (De Jesus et al. 2007).. 3.1.3 Discrepancy of theoretical and experimental pI and MW. The MW and pI values of the identified proteins on 2-DE gels were estimated by the Phoretix™ 2D analysis software and then compared with their theoretical MW and pI values obtained by MASCOT search results. The experimental MW and pI matched well with those obtained from the theoretical predictions, although some discrepancies were detected. This can be explained by differential posttranslational processing and modification such as processing of signal sequence, acetylation, and phosphorylation. In some cases, the same protein identified on the 2-DE gels was present in different positions, which generally can be divided into two different classes. Some proteins exhibited different MW and pI. Other spots appeared at a similar MW but 17 .

(24) with different pI, such as cytosolic heat shock protein 70 (spots 219, 220, 221), actin (spots232, 233, 234), and phosphoenolpyruvate carboxykinase (spots 260, 261, 262, 264) which may indicate different degree of posttranslational modifications.. 3.1.4 Biological functions of the identified proteins. Putative functional annotations for the identified proteins were classified into the following categories: energy production and carbohydrate metabolism, cytoskeleton, stress-related responses, nucleotide metabolism, and other identified proteins (Fig. 4). The metabolism-related proteins, especially energy and carbohydrate metabolism proteins composed a high percentage of identified proteins in the T.vaginals proteome. Similar results have been reported in T. vaginalis grown at iron-rich medium (De Jesus et al. 2007) and other microorganisms (Caescu et al. 2004; Posthuma et al. 2002). Obviously, energy and carbohydrate proteins were necessary for sustaining life. Not surprisingly, metabolic enzymes were also the most frequently identified ESTs in the transcriptomes (http://TvXpress.cgu.edu.tw). Moreover, we also identified a number of proteins involved in the pathogenesis and antioxidant system, such as cysteine proteinase (spot 198) and methionine gamma-lyase.(spot 297).. 3.1.5 Energy production and carbohydrate metabolism. 18 .

(25) T. vaginalis has features that are common to anaerobic organisms in terms of its carbohydrate and energy metabolism. Energy metabolism of T. vaginalis relies on fermentative carbohydrate catabolism located in cytosolic and hydrogenosomal compartments. Glycolytic enzymes are found in the cytosol but those involved in pyruvate oxidation are localized in hydrogenosome (Johnson et al.1993). Several glycolytic enzymes have been described in the previous biochemical studies (Wellerson and Kupferberg 1962). In the present study, eight proteins (fructose-1,6-bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoenolpyruvate carboxykinase, lactate dehydrogenase, malic enzyme) were assigned to the carbohydrate metabolism category which accounting for 24.7% of total proteins identified. Glycolytic enzymes represented the most abundant category in the T. vaginalis proteome. Within this class, fructose-1, 6-bisphosphate aldolase is the highest expressed protein in the T. vaginalis 2-DE reference map. In addition to glycolysis, T. vaginalis also uses a variety of amino acids as energy resources. Under normal culture conditions, T. vaginalis consumes large amount of arginine and small amounts of methionine for energy production (Yoon et al. 1991). We identified several proteins which may participate in amino acid metabolism. For example, carbamate kinase (spot 135) involved in the arginine dihydrolase pathway, adenosylhomocysteinase (spot 248) and methionine gamma-lyase (spot 297), which participate in methionine metabolism.. 19 .

(26) 3.1.6 Cytoskeletal proteins. Many biological processes, such as cell motility and morphological transformation, require remodeling of cytoskeleton in response to intracellular and extracellular signals. The ability to undergo morphological changes is directly related to virulence and pathogenesis in T. vaginalis. Anticytoskeletal drugs are able to interfere with the cytopathic effect of amoeboid form on target cells (Juliano et al. 1986; Juliano et al. 1987). Around 9.72% of the T. vaginalis proteome was composed of cytoskeletal proteins. Actin, beta tubulin, cofilin/tropomysin-type actin binding protein, actinin, and EF hand family were among the identified proteins involved in cell structure in our study. The identification of these cytoskeleton proteins provided a venue for future studies on the role of cytoskeletal proteins on the pathogenicity of this parasite.. 3.1.7 Defense and stress-related proteins. T. vaginalis is an aerotolerant anaerobic protozoan. During tissue invasion, the trophozoites are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS) (Coombs et al. 2004; Sen et al. 2007). Toxic molecules such as superoxide (O2. -) and hydrogen peroxide (H2O2) can alter membrane properties, disrupt membrane-bound proteins and biological macromolecules, leading to cell death (Sen et al. 2007). Therefore T. vaginalis has to maintain its redox homeostasis for 20 .

(27) survival. Several defense proteins which play important roles in cell survival during oxidative stress such as [Fe]-dependent superoxide dismutase (SOD) (spot 348), thioredoxin peroxidase (spots 181, 199, 347), thioredoxin reducase (TrxR) (spot 155), and methionine-γ-lyase (spot 297) were identified in our experiment. In the absence of glutathione and catalase, TrxR is a strong antioxidant which protects T. vaginalis from oxidative attack by the activated host phagocyte and monocyte during tissue adherence and invasion (Coombs et al. 2004; Krauth-Siegel et al. 1999; Becker et al. 2000). These defense proteins can be used as targets for drug design for treatment of trichomonasis.. 3.1.8 Nucleotide metabolism. T.vaginalis is incapable of de novo synthesis of purine and and pyrimidine nucleotides and depends solely on the salvage of preformed nucleosides (Heyworth et al.1984; Wang et al. 1984). The purine salvage system is highly simplified and depends entirely on the functions of purine nucleoside phosphorylase (PNP) and a nucleoside kinase. We identified several PNPs (spots 171, 212, 213) and cytidine deaminase (spot 10). T. vaginalis PNP catalysed conversion of adenine and guanine to their corresponding nucleosides (Miller et al. 1986; Heyworth et al. 1982). It has been shown that an effective inhibition of PNPs activity is anticipated to deplete the nucleotides from T.vaginalis (Munagalaet et al.2002).. 21 .

(28) 3.1.9 Cysteine proteinases. Cysteine proteinases (CPs) (spot 198) are thought to play significant roles in the pathogenesis of trichomoniasis and they seem to be essential for efficient adhesion molecules-mediated adhesion of T.vaginalis to target cells. It has been studied that a secreted cysteine proteinase fraction from T.vaginalis may induce apoptosis in human vaginal epithelial cells (Sommer et al. 2005). Additional studies have reported that CPs secreted by T.vaginalis degrade IgG, IgM, and IgA, which allows the parasite to survive the antibody response.. 3.2 Comparative proteome and transcriptome of trophozoite and amoeboid stages. The aim of this study is to identify possible molecular factors involved in the pathogenesis of T .vaginalis. We performed a systemic analysis of molecular responses at gene and protein levels using parallel transcriptomics and proteomics-based approaches. We use comparative proteomic approaches to identify differentially expressed proteins in the flagellated and amoeboid form. Our study provided insight into molecular components particularly expressed in the adherent stage of T. vaginalis and will help to elucidate the roles of identified proteins and genes, hopefully leading to uncovering the pathogenesis of trichomoniasis.. 3.2.1 2-DE Profiling of the amoeboid and trophozoite stages 22 .

(29) Although many studies have been conducted to elucidate the interaction between T .vaginalis and host cell, the mechanisms of pathogenesis are still unclear. Proteomics analysis platform have been applied to study the proteome of many organisms, but relatively few proteomic analysis of pathogenic protozoan have been reported. To better understand the pathogenesis of trichomoniasis, we conducted a comparative proteomic analysis between trophozoite and amoeboid stage. In the previous study, we established the proteome reference maps of T.vaginalis and a total of 247 spots representing 164 non-redundant proteins.To investigate the differentially expressed proteins in the pathogenesis-related amoeboid form and trophozoite, comparative proteomic analysis from these different stages was performed using 13 cm IPG strip and 15% homogeneous SDS PAGE. The representative 2-DE gels stained with silver nitrate are shown in Figure 3. To access changes in proteins profiles in the amoeboid and trophozoite stages, normalized spot volume was calculated using PhoretixTM 2D analysis software. A total of 63 protein spots were selected for MALDI-TOF MS analysis. As shown in Figure 5, we identified 49 differentially expressed proteins in the trophozoite and amoeboid stage. Among them, 34 spots were down-regulated in the amoeboid form and 15 spots were up-regulated in the amoeboid stage. The accession numbers, protein scores, theoretical and experimental pI and molecular weights, and sequence coverage of all identified proteins were provided (Table 3; Table 4). 23 .

(30) 3.2.2 Differentially expressed proteins in the amoeboid stage. In the previous section, we indicated that carbohydrate metabolism represented the most abundant category in the T. vaginals reference proteome. In addition, approximately 10% of T. vaginals reference proteome was composed of cytoskeletal proteins. These proteins involved in the energy production and cell motility are continuously expressed in the trophozoite under normal condition. However, among the 34 down-regulated protein spots in the amoeboid stage, 14 and 10 spots were classified into glycolysis and cytoskeletal proteins (Figure 8). Cysteine proteinases (CP) are the major proteolytic enzymes expressed by T.vaginalis and some of them are known to be involved in cytotoxicity , hemolysis , immune response evasion , and cytoadherence . Therefore, CPs seems to be an essential factor for efficient adhesion molecules-mediated adhesion of T.vaginalis to target cells and thought to play significant roles in the pathogens is of trichomoniasis. In the present study, we observed that CP was of greater abundance in the amoeboid stage compared to trophozoite in our proteomic study. (Figure 9). It has been reported that CP profiles of T.vaginalis isolates exhibited high- and low- virulence phenotypes and differences in CP expression indicated that papain-like CPs are the main factors in the cellular damage. During tissue invasion, T.vaginalis are exposed to oxidative stress and need to deal with highly toxic reactive oxygen species (ROS) (Coombs et al. 2004; Sen et al. 2007).The detoxification of ROS is 24 .

(31) mediated by cellular antioxidant systems and many proteins with known antioxidant functions has been identified in T.vaginalis. In the present study, 7 stress-related proteins were up-regulated in the amoeboid stage. For example, a protein identified as tryparedoxin peroxidase (TXNPx) (spot 57 ; Fig 9) increased ~5.5 f old in the amoeboid stage compared to trophozoite. In Trypanosoma cruzi, there is increasing evidence that antioxidant enzymes play a significant immune evasion roles by protecting the parasite against macrophage-derived ROS. In Leishmania dovani, TXNPx is not only crucial for survival during oxidative stress, but also enhances the infective abilities. In addition, thioredoxin peroxidase (spot 33) was observed to increase ~ 1.9 fold in the amoeboid stage. Thioredoxin reductase (spot 59), which functions together with thioredoxin and thioredoxin peroxidase to detoxify potentially damaging oxidants, was only expressed in the amoeboid stage. TrxR is a strong antioxidant which protects T. vaginalis from oxidative attack by the activated host phagocyte and monocyte during tissue adherence and invasion (Coombs et al. 2004; Krauth-Siegel et al. 1999; Becker et al. 2000). Other proteins with antioxidant functions showing increased expression in the amoeboid stage included thiol peroxidase (spots 29, 30, 60) and flavodoxin family protein (spot 61). Our proteome results suggest a global increase in antioxidant enzyme in the amoeboid stage. This may implied that pathogenic stage is not the optimum physiological condition for T. vaginalis.. 3.2.3 Functional Classification of the differentially expressed proteins 25 .

(32) Putative functional annotations for the 49 identified proteins were classified into the following categories: glycolysis, protein binding, proteolysis, stress proteins, amino acid biosynthesis, tricarboxylic acid cycle, hypothetical proteins. Of the 34 proteins with lower abundance in the amoeboid form, glycolysis (41%) represented the most abundant category, followed by protein binding (28 %) and hypothetical proteins (9%) ,and last proteolysis (6%) ,amino acid biosynthesis (3%) ,stress proteins (3%), tricarboxylic acid cycle (3%) and others(6%) (Figure 8).On the contrary, of the 15 proteins with higher abundance in the amoeboid form, stress proteins (53 %) represents the major class of protein.. 3.2.4 Transcriptional profiling of the amoeboid stage compared to trophozoite. A total of 20,704 ESTs were obtained from the unsynchronized trophozoites (TvE) and the fibronectin-induced amoeboid form (TvF) cDNA libraries (Table 5). Assembly of the ESTs generated 4365 and 1854 unigenes from TvE and TvF, respectively. In addition, data from TvF showed lower EST % in singletons (10.79 %) compared to TvE (28.78 %), indicating less gene expression diversity in the ameoboid form .Table 6 showed the 100 most highly expressed ESTs from the TvF cDNA library. A high percentage of these genes encoding proteins related to antioxidant properties, protein biosynthesis and carbohydrate 26 .

(33) metabolism , such as TXNPx, SOD, TRXR, and ribosomal proteins. Based on the TvE and TvF EST datasets, we summarized the genes which expressed more than 5-fold (116 genes) and 10-fold (32 genes) in the amoeboid stage over the trophozoite as control and these genes were further categorized according to their biological function (Figure 11).The results showed that the most significantly changed genes in the amoeboid form were stress proteins (42 %).. 3.3 Integration of transcriptomic and proteomic data. We also investigated whether these differentially expressed proteins are regulated at the mRNA expression level. The transcrips selected for measurement by qRT-PCR were G3PD, TXNPx, thioredoxin reductase, thiol peroxidase, flavodoxin family protein, PDase: cathepsin L-like cysteine peptidase, SOD, and lactate dehydrogenase. The primers pairs used for qRT-PCR are shown in Table 7. For most of the differentially expressed proteins, the alteration of mRNA expression level was in parallel with the protein expression level. With respect to enzymes with antioxidant activities, our qRT-RCR results (Figure 12) correlate well with our proteome-level observations. It is clear that these antioxidant enzymes are up-regulated in the gene expression level and protein expression level in the amoeboid stage as supported by our 2-DE, ESTs and qRT-PCR results. However, there have been reports of non-correlation between the transcriptome and proteome data. For example, the abundance of the transcript of lactate dehydrogenase was 27 .

(34) observed to increase significantly in the amoeboid stage but the protein expression level was observed to decrease, suggesting the occurrence of post-translational modification.. 28 .

(35) Discussion and Conclusion. 29 .

(36) CHAPTER 4: Discussion and Conclusion Although proteomics analysis platform have been applied to study the proteome of many organisms, relatively few reference proteome of pathogenic protozoan have been reported. The reference proteome of Leishmania (Viannia) braziliensis contain 101 proteins representing 75 protein entries (Cuervo et al. 2007). In Encephalitozoon cuniculi, a set of 177 unique proteins was identified (Brosson et al. 2006). In T. vaginalis, 116 spots in the pH range 4–7 representing 67 unique proteins were identified in cells grown at different conditions (De Jesus et al.2007). In the present study, we established the proteome reference maps of T. vaginalis with different pH ranges (pH 3-10, pH 4-7, pH 6-11). A total of 247 spots representing 164 different proteins were identified. Approximately 70% of the proteins reported in the previous T. vaginalis proteomic study (De Jesus et al. 2007) were also identified in our proteome database (S1, S2). This work presented the largest proteome dataset of T. vaginalis to date. Information generated from proteome analysis not only provided a new insight on the biochemistry and physiology of T.vaginalis, but also served as a basis for comparative proteomic analysis on the pathogenesis, virulence factors, and drug interaction of this parasite. T .vaginalis transformation from a free-swimming flagellated form to an adherent amoeboid form is crucial to parasite establishment in the host vagina and subsequent pathogenesis. Thus, to understand systematical responses in the amoeboid form of T.vaginalis, we combined the results from transcriptomics and proteomics approaches. Comparative proteomics analysis from amoeboid form and trophozoites demonstrated quantitative and qualitative differences in the protein profile of 30 .

(37) T.vaginalis. Global gene expression analysis using EST sequencing provided valuable information about the expression patterns in the pathogenesis adherent amoeboid stage. Both our proteome- and transcriptome-level results suggest that the amoeboid stage of T.vaginalis significantly expressed proteins with antioxidant activities. Stress responses in the amoeboid stage suggest that (i) such antioxidant proteins have important roles in the amoeboid stage and (ii) that the amoeboid stage is not the optimum physiological condition for T. vaginalis. However, it is necessary to investigate the detailed information about the differential expression changes obtained from our proteomics and transcriptomics results. Moreover, we observed that there were some discrepancies between mRNA level and protein level. Taken together, our work provided insight into molecular components particularly expressed in the amoeboid stage of T. vaginalis and elucidated the roles of identified proteins and genes, hopefully leading to uncovering the pathogenesis of trichomoniasis.. 31 .

(38) Figures and Tables. 32 .

(39) FIG GURES. Fig 1 Tw wo-dimensioonal gel elecctrophoresiss (2-DE) maap of T. vaginaliss soluble prooteins separrated at pH range r 3–10.. 33 .

(40) Fig 2 Two-dimensionnal gel electrrophoresis (2-DE) ( mapp of T. vaginalis soluble s proteins separatted at pH raange 6-11.. 34 .

(41) Figg. 3 The refference two--dimensionaal gel electrrophoresis ((2-DE) map p of T. vaginaliss soluble prroteins separrated at pH 4-7. The prroteins weere separatedd in the firsst dimension n in the pH range 4-7 aand in the seccond dimennsion on a 155% polyacrrylamide gell. Silver-staained spots ideentified by MALDI M -TO OF PMF aree numberedd and detailss of their ideentification are given inn S1.. 35 .

(42) Fig. 4 Fuunctional claassification of T. vaginnalis proteom me. T. vaginaliss proteins iddentified by MALDI -T TOF PMF w were classifiedd according to the Genee Ontology index (http://ww ww.geneonntology.org/)).. 36 .

(43) Figu ure 5. Two-dimensional electrophhoresis maps of proteinns from (A) trophozoitees and (B) fibroneectin-induceed amoeboidd form of T.vaginalis. T T first dim The mension waas perfformed withh 250μg off proteins ussing 13 cm pH 4-7 stripps, followedd by 15% polyyacrylamidee gel for thee second dim mension. Prroteins weree visualized by silver stainning. Differrentially exppressed prottein spots in n the amoebboid form coompared to tropphozoite aree marked byy arrows andd numbers, Red arrowss show spotss of greater abunndance andd blue arrow ws show spots of lower abundance in the amoeeboid stage... 37 .

(44) Figure 6. Magnified regions of the 2-DE maps show the differentially expressed protein spots in the trophozoite and amoeboid form of T.vaginalis. Panel (A) show the reference markers in these two stages. Panel (B) and (C) show the protein spots which were down-regulated and up-regulated in the amoeboid stage compared to trophozoite.. 38 .

(45) Figure 7. Results of TXNPx as the representative of protein identification using MALDI-TOF-MS/MS.A,a, Enlarged 2-DE images of TXNPx which was upregulated in the amoeboid stage of T.vaginalis. b, Expression patterns of TXNPx in 2-DE. (B) Annotated mass spectrum for TXNPx. (C) output of the data by the MASCOT database search engine for the identification of TXNPx. The matched peptides were shown in bold red.. 39 .

(46) Figu ure 8. (A) Graphical G prresentation of the down n-regulatedd proteins inn the amoebo oid stagge of T.vaginnalis. Proteeins differenntially expreessed are nuumbered andd each bar reprresents the relative r abuundance exppressed as th he normalized spot voluume calculaated T TM usinng Phoretix 2D analyysis softwarre. Details of o their idenntification w were shown in the Table 1.(B)) Functionall categorizaation of the down-regul d lated proteinns, identifieed by MALDI-TO M OF PMF were classifiedd according g to the Genee Ontology index (httpp://www.geeneontologyy.org/).. 40 .

(47) Figu ure 9. Grapphical presenntation of thhe up-regulated proteinns in the am moeboid stag ge of T.vaaginalis. Prooteins differrentially expressed are numbered and each baar representts the relative abuundance exppressed as thhe normalizzed spot vollume calculaated using PhooretixTM 2D analysis sooftware. Dettails of theirr identificatiion were shhown in the Tabble 1.. 41 .

(48) C. Figure 10. Transcriptional profiling of the amoeboid stage compared to trophozoite.. 42 .

(49) A. B. Figure 11. Functional categorization of the up-regulated genes in the amoeboid stage compared to trophozoite of T.vaginalis. (A) Differentially expressed genes which expressed more than 5-fold (A) and 10-fold (B) in the amoeboid stage over the trophozoite.. 43 .

(50) Figure 12.RT-PCR analysis of representative antioxidant enzymes in the amoeboid stage and trophozoite of T.vaginalis. Changes in the relative abundance of transcripts of (A) TXNPx (B) SOD (C) Thioredoxin reductase.. 44 .

(51) TABLES Table 1 Identification of T.vaginalis proteins in the range of pH 4-7 by MALDI-TOF peptide mass figerprinting Spot Protein Name. Accession No.. Volume. Score Coverage. ％. MW / pI. Exp MW / pI. 2. Hypothetical protein. 86412.t00188. 0.85%. 39. 32%. 12.7 / 4.63. 13.0 / 4.28. 3. Hypothetical protein. 86943.t00003. 0.82%. 58. 43%. 11.5 / 5.10. 13.1 / 4.41. 4. Hypothetical protein. 86431.t00136. 0.08%. 100. 68%. 17.7 / 4.65. 16.8 / 4.62. 5. Hypothetical protein. 132230.t00005. 0.24%. 34. 26%. 13.1 / 5.24. 15.8 / 4.69. 7. CNPV220 N1R/p28-like protein-related (KilA, N-terminal) 123415.t00005. 0.09%. 47. 59%. 13.8 / 8.76. 14.9 / 4.80. 8. Hypothetical protein. 90655.t00137. 0.91%. 39. 51%. 15.1 / 5.06. 14.6 / 4.86. 9. Hypothetical protein. 92204.t00048. 1.28%. 41. 56%. 15.4 / 4.97. 11.8 / 4.64. 10. cytidine deaminase. 84774.t00085. 0.76%. 56. 62%. 15.6 / 5.48. 12.8 / 4.78. 11. Hypothetical protein. 86239.t00005. 0.60%. 42. 43%. 11.8 / 7.03. 12.3 / 4.89. 12. Cofilin/tropomyosin-type actin-binding protein. 86673.t00308. 0.36%. 81. 55%. 16.1 / 5.17. 15.3 / 4.98. 13. endonuclease-related. 139654.t00006. 0.50%. 32. 22%. 15.9 / 5.02. 16.0 / 5.20. 15. Hypothetical protein. 82952.t00118. 0.13%. 28. 28%. 15.2 / 6.11. 16.3 / 5.58. 17. Cofilin, Actin depolymerizing factor (Tv_ADF2). 94534.t00169. 0.30%. 86. 60%. 16.2 / 5.54. 13.1 / 5.51. (His-Me finger endonucleases). 45 . ％. Theore.

(52) 18. Hypothetical protein. 88712.t00357. 0.48%. 23. 36%. 14.9 / 5.46. 12.6 / 5.56. 20. Cofilin/tropomyosin-type actin-binding protein. 86673.t00308. 0.22%. 72. 48%. 16.1 / 5.17. 14.0 / 5.61. 22. Hypothetical protein. 97196.t00092. 0.35%. 55. 24%. 20.6 / 5.42. 14.8 / 5.78. 24. Hypothetical protein. 97007.t00158. 0.27%. 63. 52%. 15.3 / 5.93. 11.7 / 5.75. 95218.t00073. 0.40%. 48. 24%. 37.3 / 6.51. 14.2 / 5.93. 25. Guanine nucleotide-binding protein beta subunit-like protein.-related. 26. QXW lectin repeat family protein (Ricin B). 89002.t00198. 1.34%. 122. 81%. 15.0 / 5.95. 13.3 / 5.88. 28. Hypothetical protein. 84693.t00004. 0.34%. 42. 46%. 12.7 / 5.06. 12.7 / 5.92. 29. thiol peroxidase. 85876.t00193. 0.08%. 74. 46%. 18.1 / 6.05. 15.6 / 6.20. 30. thiol peroxidase. 85876.t00193. 0.81%. 195. 83%. 18.1 / 6.05. 16.7 / 6.38. 31. Nucleoside diphosphate kinase family protein. 92069.t00285. 0.43%. 52. 43%. 15.3 / 6.09. 12.8 / 6.27. 33. Hypothetical protein. 96252.t00273. 0.61%. 37. 62%. 12.3 / 5.17. 12.6 / 6.62. 35. Hypothetical protein. 101133.t00002. 0.53%. 44. 44%. 10.6 / 6.27. 11.5 / 6.49. 35. glyceraldehyde 3-phosphate dehydrogenase. 86677.t00069. 0.53%. 57. 52%. 18.4 / 6.41. 11.5 / 6.49. 36. Hypothetical protein. 123489.t00004. 0.16%. 22. 33%. 16.6 / 4.71. 14.8 / 4.63. 43. Hypothetical protein. 96252.t00273. 0.45%. 42. 48%. 12.3 / 5.17. 14.0 / 5.13. 44. Hypothetical protein. 96252.t00273. 0.23%. 46. 68%. 12.3 / 5.17. 13.6 / 5.09. 45. Hypothetical protein. 88712.t00357. 0.12%. 44. 53%. 14.9 / 5.46. 14.4 / 5.18. 46. Hypothetical protein. 91588.t00004. 0.32%. 35. 47%. 13.4 / 5.19. 13.8 / 5.22. 47. Hypothetical protein. 96252.t00273. 0.23%. 46. 49%. 12.3 / 5.17. 13.2 / 5.28. 49. Hypothetical protein. 88885.t00009. 0.32%. 30. 36%. 11.8 / 5.84. 11.5 / 5.83. 50. endoribonuclease L-PSP. 81529.t00530. 0.37%. 63. 70%. 13.8 / 6.07. 11.9 / 5.96. 46 .

(53) 52 54. Profilin Heterogeneous nuclear ribonucleoprotein D-like (hnRPD-like protein) (RNA recognition motif, RNP-1. 85736.t00011. 0.79%. 44. 69%. 13.4 / 7.66. 10.4 / 6.00. 85728.t00153. 0.53%. 109. 69%. 21.2 / 10.30. 20.5 / 4.01. 58. EF hand family protein, Centrin/Caltractin. 81272.t00019. 0.13%. 30. 29%. 18.4 / 4.69. 20.0 / 4.55. 59. translation initiation factor eIF-5A family protein. 89692.t00480. 0.46%. 49. 25%. 18.6 / 5.01. 18.2 / 4.49. 60. Hypothetical protein. 82471.t00131. 0.27%. 32. 22%. 22.0 / 4.73. 18.7 / 4.59. 61. Hypothetical protein. 96405.t00007. 0.17%. 39. 27%. 27.4 / 5.01. 31.0 / 4.86. 62. actin. 102170.t00002. 0.15%. 97. 38%. 33.3 / 5.17. 31.3 / 5.14. 63. beta-tubulin. 85300.t00020. 0.25%. 85. 41%. 28.5 / 5.21. 25.1 / 4.72. 64. Hypothetical protein. 140110.t00006. 0.27%. 41. 30%. 26.7 / 5.00. 26.1 / 4.78. 65. Hypothetical protein. 122281.t00003. 0.48%. 37. 36%. 27.4 / 5.24. 26.3 / 5.20. 66. Peptidase C1A, cysteine peptidase. 97121.t00200. 0.25%. 39. 20%. 29.7 / 5.57. 27.4 / 5.38. 68. Ras GTPase superfamily, RAC. 86673.t00331. 0.08%. 38. 25%. 24.0 / 4.99. 23.5 / 4.90. 69. Hypothetical protein. 84139.t00016. 0.52%. 64. 34%. 23.9 / 9.24. 23.0 / 4.79. 70. Ras GTPase superfamily, RAC. 86673.t00331. 0.19%. 30. 19%. 24.0 / 4.99. 21.6 / 4.75. 71. Hypothetical protein. 84005.t00003. 0.24%. 55. 39%. 21.1 / 9.25. 21.2 / 4.74. 73. actin. 111150.t00005. 0.14%. 101. 53%. 26.7 / 5.02. 18.0 / 4.70. 75. actin. 111150.t00005. 0.27%. 101. 43%. 26.7 / 5.02. 25.2 / 6.11. 76. actin. 111150.t00005. 0.25%. 106. 52%. 26.7 / 5.02. 20.7 / 4.84. 78. actin. 111150.t00005. 0.31%. 60. 25%. 26.7 / 5.02. 19.5 / 4.84. 79. Kv channel-interacting protein 4 (kchip4.2). 95912.t00282. 0.49%. 62. 52%. 21.2 / 4.97. 19.0 / 4.79. 80. Hypothetical protein. 123800.t00002. 0.14%. 42. 33%. 24.5 / 5.33. 23.7 / 5.02. 47 .

(54) 81. actin. 111150.t00005. 0.12%. 150. 55%. 26.7 / 5.02. 23.5 / 5.03. 82. Hypothetical protein. 102158.t00002. 0.14%. 53. 48%. 24.2 / 9.26. 22.9 / 5.01. 83. proteasome alpha subunit. 90073.t00060. 0.23%. 105. 60%. 27.6 / 5.26. 22.9 / 5.07. 84. actin. 111150.t00005. 0.46%. 71. 40%. 26.7 / 5.02. 21.8 / 5.00. 85. actin. 111150.t00005. 0.28%. 76. 41%. 26.7 / 5.02. 21.2 / 4.98. 86. Hypothetical protei. 98212.t00003. 0.16%. 60. 31%. 24.0 / 9.26. 20.5 / 4.98. 87. Hypothetical protein. 97196.t00092. 0.17%. 63. 26%. 20.6 / 5.42. 19.7 / 4.96. 89. actin. 83372.t00049. 0.68%. 66. 32%. 42.1 / 5.05. 18.5 / 4.94. 90. actin. 111150.t00005. 0.18%. 56. 26%. 26.7 / 5.02. 23.7 / 5.17. 92. Hypothetical protein. 104979.t00002. 0.16%. 40. 35%. 24.2 / 9.26. 22.9 / 5.23. 94. Ras GTPase superfamily, Rab5b. 82578.t00573. 0.12%. 31. 21%. 21.5 / 5.10. 20.7 / 5.20. 95. Hypothetical protein. 94952.t00002. 0.16%. 52. 35%. 21.4 / 7.57. 20.3 / 5.17. 96. Hypothetical protein. 82759.t00076. 0.14%. 46. 32%. 23.7 / 9.05. 20.0 / 5.17. 98. actin. 82114.t00023. 0.88%. 62. 31%. 42.1 / 5.12. 18.5 / 5.15. 99. Hypothetical protein. 80594.t00008. 0.14%. 40. 33%. 18.3 / 7.63. 17.6 / 5.06. 101. Triosephosphate isomerase (TIM). 83581.t00073. 0.37%. 39. 20%. 27.7 / 5.76. 22.3 / 5.50. 103. Hypothetical protein. 84943.t00148. 0.20%. 42. 31%. 22.8 / 7.79. 21.5 / 5.44. 104. Hypothetical protein. 88267.t00002. 0.52%. 43. 41%. 24.1 / 4.86. 20.8 / 5.43. 105. Ankyrin-repeat protein MM0045.-related. 97480.t00027. 0.32%. 34. 30%. 20.6 / 5.32. 18.9 / 5.41. 106. Hypothetical protein. 82490.t00289. 0.40%. 52. 41%. 21.7 / 8.65. 17.5 / 5.38. 108. skeletrophin. 80466.t00081. 0.18%. 32. 29%. 18.3 / 5.17. 17.7 / 5.49. 111. Hypothetical protein. 84212.t00005. 0.14%. 42. 38%. 19.7 / 9.08. 17.6 / 6.21. 48 .

(55) 112. Hypothetical protein. 108454.t00003. 0.12%. 45. 36%. 29.6 / 8.98. 24.9 / 5.33. 113. heat shock protein 70 (cytoplasmic). 94003.t00003. 0.11%. 103. 46%. 33.0 / 5.82. 24.7 / 5.66. 116. Hypothetical protein. 87388.t00151. 0.12%. 53. 28%. 21.5 / 6.49. 21.0 / 5.06. 117. Hypothetical protein. 91562.t00095. 0.15%. 38. 34%. 18.3 / 6.46. 16.7 / 5.19. 118. Hypothetical protein. 81907.t00051. 0.10%. 42. 28%. 30.6 / 8.96. 28.4 / 4.04. 119. Tvp14, Zinc finger, CCCH-type. 88613.t00095. 0.22%. 56. 48%. 15.1 / 6.20. 13.9 / 6.41. 120. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.07%. 53. 28%. 36.4 / 5.79. 21.5 / 5.55. 121. Hypothetical protein. 85163.t00033. 0.10%. 48. 31%. 24.0 / 5.06. 20.8 / 5.55. 87130.t00079. 0.27%. 29. 23%. 25.0 / 5.46. 22.7 / 5.65. 122. Lipid-binding START (STeroidogenic Acute Regulatory related lipid Transfer). 123. Hypothetical protein. 102041.t00002. 0.31%. 48. 24%. 24.9 / 5.24. 21.7 / 5.61. 124. Ankyrin-repeat protein MM0045.-related. 97480.t00027. 0.25%. 44. 30%. 20.6 / 5.32. 21.7 / 5.64. 125. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.18%. 66. 32%. 36.4 / 5.79. 21.0 / 5.61. 126. Ankyrin-repeat protein MM0045.-related. 97480.t00027. 0.26%. 41. 30%. 20.6 / 5.32. 21.1 / 5.63. 127. Hypothetical protein. 89741.t00033. 0.15%. 68. 39%. 23.7 / 9.26. 19.8 / 5.62. 128. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.71%. 80. 35%. 36.4 / 5.79. 19.5 / 5.65. 129. Hypothetical protein. 97196.t00092. 0.55%. 41. 22%. 20.6 / 5.42. 20.0 / 5.68. 130. L-lactate/malate dehydrogenase. 86045.t00059. 0.36%. 81. 26%. 37.3 / 5.77. 20.7 / 5.70. 131. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.18%. 64. 23%. 36.4 / 5.79. 27.9 / 5.62. 132. fructose-1,6-bisphosphate aldolase. 92775.t00058. 0.19%. 67. 28%. 36.4 / 5.88. 28.3 / 5.66. 82578.t00479. 0.42%. 88. 31%. 33.6 / 6.03. 26.6 / 5.65. 133. C2 calcium-dependent membrane targeting/calcium-dependent phospholipid binding. 49 .

(56) protein 135. Carbamate kinase. 89002.t00281. 0.35%. 64. 38%. 40.5 / 5.48. 28.2 / 5.74. 136. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.59%. 189. 50%. 36.4 / 5.79. 28.6 / 5.79. 137. fructose-1,6-bisphosphate aldolase. 92775.t00058. 0.61%. 148. 44%. 36.4 / 5.88. 28.7 / 5.83. 138. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.75%. 155. 47%. 36.4 / 5.79. 28.7 / 5.90. 139. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.51%. 85. 39%. 36.5 / 6.02. 27.4 / 5.84. 142. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.23%. 68. 33%. 36.4 / 5.79. 21.9 / 5.74. 81845.t00167. 0.33%. 31. 24%. 21.8 / 5.38. 21.2 / 5.74. 143. PHD/FYVE/RING-type Zinc-finger family protein with cysteine peptidase active site. 144. Triosephosphate isomerase. 83581.t00073. 0.73%. 54. 19%. 27.7 / 5.76. 23.7 / 5.82. 145. Triosephosphate isomerase. 83581.t00073. 0.63%. 124. 40%. 27.7 / 5.76. 23.2 / 5.82. 146. Triosephosphate isomerase. 83581.t00073. 0.39%. 140. 50%. 27.7 / 5.76. 22.8 / 5.80. 147. Ras GTPase superfamily, Rab12. 96555.t00137. 0.16%. 35. 28%. 21.0 / 5.43. 22.8 / 5.86. 148. Hypothetical protein. 92404.t00006. 0.13%. 45. 32%. 23.4 / 9.05. 22.8 / 5.88. 149. Ankyrin-repeat protein MM0045.-related. 97480.t00027. 0.14%. 43. 30%. 20.6 / 5.32. 21.8 / 5.85. 150. Hypothetical protein. 93762.t00006. 0.15%. 40. 29%. 21.0 / 9.55. 20.6 / 5.83. 151. Hypothetical protein. 82759.t00076. 0.18%. 46. 36%. 23.7 / 9.05. 20.1 / 5.82. 153. fructose-1,6-bisphosphate aldolase. 92775.t00058. 0.27%. 143. 41%. 36.4 / 5.88. 30.9 / 6.01. 154. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.78%. 90. 35%. 36.5 / 6.02. 28.0 / 6.02. 155. thioredoxin reductase. 96723.t00098. 0.54%. 143. 62%. 32.8 / 6.02. 28.1 / 6.06. 156. Myosin II heavy chain, non muscle.-related. 85686.t00138. 0.10%. 51. 23%. 30.5 / 5.90. 30.8 / 6.14. 157. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.36%. 71. 36%. 36.5 / 6.02. 30.0 / 6.18. 50 .

(57) 158. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.16%. 70. 38%. 36.4 / 5.79. 28.7 / 6.15. 159. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.26%. 79. 33%. 36.4 / 5.79. 29.2 / 6.30. 160. Hypothetical protein. 125956.t00003. 0.19%. 34. 23%. 27.2 / 7.08. 27.9 / 6.61. 161. Hypothetical protein. 93835.t00218. 0.06%. 37. 30%. 27.3 / 6.25. 29.4 / 6.65. 163. heat shock protein 70 (cytoplasmic). 94003.t00003. 0.28%. 135. 54%. 33.0 / 5.82. 25.2 / 5.94. 165. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.18%. 104. 44%. 36.4 / 5.79. 25.1 / 6.01. 166. Hypothetical protei. 130472.t00002. 0.32%. 42. 35%. 24.2 / 5.60. 25.1 / 6.04. 167. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.22%. 80. 40%. 36.4 / 5.79. 24.3 / 6.02. 168. Hypothetical protein. 86319.t00280. 0.17%. 64. 28%. 34.5 / 5.25. 23.4 / 6.04. 169. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.10%. 48. 26%. 36.4 / 5.79. 22.1 / 6.02. 170. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.34%. 68. 29%. 36.4 / 5.79. 21.3 / 5.92. 171. Purine nucleoside phosphorylase. 95946.t00093. 0.13%. 65. 33%. 25.9 / 5.97. 22.5 / 5.96. 172. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.55%. 57. 40%. 36.4 / 5.79. 22.2 / 5.99. 173. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.33%. 78. 41%. 36.4 / 5.79. 21.5 / 5.99. 174. Ras GTPase superfamily, Rab17. 93905.t00131. 0.46%. 45. 35%. 22.1 / 5.04. 22.2 / 6.04. 175. Hypothetical protein. 113682.t00002. 0.27%. 67. 44%. 21.0 / 9.48. 25.2 / 6.11. 176. fructose-1,6-bisphosphate aldolase. 92210.t00096. 0.40%. 52. 26%. 36.5 / 6.06. 24.7 / 6.11. 177. Triosephosphate isomerase (TIM). 83581.t00073. 0.93%. 69. 41%. 27.7 / 5.76. 23.3 / 6.11. 178. Hypothetical protein. 81238.t00191. 0.24%. 56. 46%. 27.2 / 6.47. 22.3 / 6.09. 179. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.48%. 42. 36%. 36.4 / 5.79. 21.3 / 6.10. 180. Hypothetical protein. 137411.t00003. 0.22%. 47. 37%. 19.3 / 6.47. 18.6 / 6.15. 181. thioredoxin peroxidase. 84105.t00110. 0.07%. 43. 37%. 22.2 / 6.31. 19.3 / 6.20. 51 .

(58) 182. tryparedoxin peroxidase. 95981.t00016. 0.34%. 87. 59%. 22.1 / 6.06. 19.9 / 6.23. 183. Hypothetical protein. 83996.t00294. 0.13%. 90. 47%. 28.3 / 6.62. 27.3 / 6.83. 184. Hypothetical protein. 83996.t00294. 0.06%. 49. 30%. 28.3 / 6.62. 26.8 / 6.80. 185. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.46%. 104. 43%. 36.5 / 6.02. 25.6 / 6.66. 190. histone-lysine N-methyltransferase, bat/ehmt. 93865.t00184. 0.34%. 53. 51%. 21.1 / 6.44. 21.8 / 6.75. 191. Hypothetical protein. 80644.t00035. 0.08%. 42. 31%. 30.4 / 6.27. 26.6 / 6.41. 192. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.15%. 68. 37%. 36.4 / 5.79. 25.5 / 6.35. 195. Cell cycle control protein cwf16.-related. 82178.t00142. 0.42%. 46. 30%. 28.7 / 6.56. 23.5 / 6.46. 196. Hypothetical protein. 93129.t00083. 0.17%. 33. 21%. 23.8 / 6.82. 22.5 / 6.45. 197. Hypothetical protein. 88645.t00174. 0.08%. 36. 27%. 22.0 / 9.31. 22.0 / 6.41. 198. Peptidase C1A, cysteine peptidase CP2. 82181.t00175. 0.59%. 70. 28%. 35.2 / 8.40. 21.5 / 6.53. 199. thioredoxin peroxidase. 97029.t00065. 0.79%. 104. 54%. 22.1 / 6.31. 19.8 / 6.52. 92069.t00224. 0.27%. 54. 33%. 26.5 / 6.31. 22.3 / 6.67. 200. proteasome alpha subunit, threonine endopeptidase, alpha type 7. 201. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.29%. 86. 35%. 36.5 / 6.02. 25.1 / 6.24. 202. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.49%. 72. 37%. 36.5 / 6.02. 24.4 / 6.24. 203. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.21%. 69. 26%. 36.5 / 6.02. 23.8 / 6.24. 205. Ankyrin-repeat protein MM0045.-related. 97480.t00027. 0.39%. 52. 36%. 20.6 / 5.32. 22.7 / 6.37. 207. Hypothetical protein. 115828.t00003. 0.24%. 48. 36%. 24.9 / 7.81. 21.6 / 6.23. 210. Hypothetical protein. 81295.t00073. 0.11%. 46. 40%. 21.8 / 5.92. 23.4 / 6.18. 211. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.10%. 77. 28%. 36.5 / 6.02. 22.7 / 6.14. 212. Purine nucleoside phosphorylase. 84433.t00164. 0.19%. 53. 22%. 26.2 / 5.89. 21.9 / 6.17. 52 .

(59) 213. Purine nucleoside phosphorylase. 95946.t00093. 0.63%. 133. 63%. 25.9 / 5.97. 22.5 / 6.20. 215. heat shock protein 70 (endoplasmic reticulum ). 83450.t00067. 0.12%. 100. 22%. 71.6 / 5.06. 45.5 / 4.63. 216. heat shock protein 70 (endoplasmic reticulum ). 83450.t00067. 0.11%. 89. 19%. 71.6 / 5.06. 45.7 / 4.67. 217. heat shock protein 70 (endoplasmic reticulum ). 83450.t00067. 0.07%. 123. 29%. 71.6 / 5.06. 64.6 / 4.96. 218. heat shock protein 70 (endoplasmic reticulum ). 83450.t00067. 0.30%. 234. 40%. 71.6 / 5.06. 74.4 / 5.07. 219. heat shock protein 70 (cytoplasmic). 85859.t00323. 0.59%. 182. 36%. 71.4 / 5.29. 67.0 / 5.13. 220. heat shock protein 70 (cytoplasmic). 85859.t00323. 0.37%. 198. 39%. 71.4 / 5.29. 66.9 / 5.19. 221. heat shock protein 70 (cytoplasmic). 86492.t00134. 0.15%. 40. 13%. 71.4 / 5.29. 67.1 / 5.25. 225. Vacuolar ATP synthase subunit A. 94782.t00175. 0.18%. 76. 21%. 68.3 / 5.14. 48.2 / 4.88. 228. actinin. 88273.t00344. 0.10%. 96. 15%. 128.5 / 4.90. 43.9 / 4.84. 230. heat shock protein 70 (cytoplasmic). 139762.t00002. 0.09%. 75. 20%. 46.5 / 5.25. 39.3 / 4.82. 232. actin. 83372.t00049. 0.28%. 94. 32%. 42.1 / 5.05. 40.6 / 4.90. 233. actin. 85746.t00231. 0.99%. 153. 47%. 42.2 / 5.05. 40.4 / 4.97. 234. actin. 83372.t00049. 0.78%. 105. 30%. 42.1 / 5.05. 40.6 / 5.05. 235. actin. 85746.t00231. 0.60%. 197. 67%. 42.2 / 5.05. 37.8 / 4.89. 238. actin. 102170.t00002. 0.58%. 59. 27%. 33.3 / 5.17. 35.6 / 5.07. 239. actin. 102170.t00002. 0.14%. 30. 21%. 33.3 / 5.17. 33.4 / 4.98. 240. Hypothetical protein. 93001.t00003. 0.16%. 38. 20%. 35.2 / 6.28. 33.7 / 4.99. 241. actin. 102170.t00002. 0.27%. 44. 30%. 33.3 / 5.17. 33.7 / 5.10. 242. actin. 102170.t00002. 0.69%. 106. 55%. 33.3 / 5.17. 35.9 / 5.18. 243. Hypothetical protein. 80596.t00227. 0.28%. 52. 30%. 31.6 / 4.80. 33.8 / 5.23. 244. actin. 91931.t00037. 0.22%. 80. 33%. 42.2 / 5.12. 35.8 / 5.30. 53 .

(60) 247. Hypothetical protein. 83289.t00032. 0.18%. 36. 16%. 38.0 / 5.77. 33.8 / 5.49. 248. adenosylhomocysteinase. 94232.t00223. 0.20%. 71. 25%. 53.9 / 5.53. 54.4 / 5.56. 249. heat shock protein 70 (cytoplasmic). 100090.t00002. 0.25%. 133. 46%. 41.4 / 5.40. 44.3 / 5.65. 251. Hypothetical protein. 102064.t00002. 0.55%. 41. 28%. 37.2 / 5.47. 36.6 / 5.61. 252. Ketopantoate reductase PanE/ApbA family protein. 112876.t00003. 0.54%. 81. 38%. 37.8 / 5.69. 36.7 / 5.80. 253. Aldose 1-epimerase family protein. 84306.t00061. 0.26%. 72. 20%. 39.7 / 5.64. 40.0 / 5.90. 256. Hypothetical protein. 91798.t00143. 0.44%. 43. 20%. 37.1 / 6.07. 36.7 / 6.03. 258. adenosinetriphosphatase (ATPase, V1 complex, subunit B ) 91362.t00072. 0.15%. 102. 32%. 55.8 / 5.77. 57.4 / 6.00. 260. Phosphoenol pyruvate carboxykinase [GTP]. 87399.t00118. 0.44%. 118. 30%. 67.9 / 5.98. 66.7 / 6.28. 261. Phosphoenol pyruvate carboxykinase [GTP]. 87399.t00118. 0.44%. 99. 27%. 67.9 / 5.98. 66.0 / 6.40. 262. Phosphoenol pyruvate carboxykinase [GTP]. 87399.t00118. 0.40%. 88. 25%. 67.9 / 5.98. 65.4 / 6.53. 263. heat shock protein 70 (mitochondrial). 92066.t00128. 0.51%. 78. 29%. 68.9 / 6.76. 65.5 / 6.70. 264. Phosphoenol pyruvate carboxykinase [GTP]. 96829.t00054. 0.43%. 65. 25%. 67.9 / 6.55. 66.1 / 6.85. 271. Hypothetical protein. 96252.t00244. 0.08%. 50. 21%. 62.0 / 6.58. 54.0 / 6.22. 272. myosin heavy chain. 83337.t00009. 0.09%. 35. 16%. 48.3 / 5.90. 50.6 / 6.20. 274. severin kinase. 80378.t00240. 0.08%. 44. 20%. 43.1 / 4.87. 52.8 / 6.42. 97193.t00084. 0.53%. 47. 23%. 46.8 / 5.96. 42.3 / 6.38. 276. Clan MH, family M18, aspartyl aminopeptidase-like metallopeptidase. 277. Lysophosphatidic acid phosphatase type 6 precursor. 87617.t00023. 0.27%. 41. 22%. 45.7 / 6.33. 40.8 / 6.42. 278. coiled-coil domain-containing protein, putative. 80671.t00422. 0.26%. 52. 33%. 45.6 / 5.82. 42.0 / 6.50. 279. Hypothetical protein. 80671.t00422. 0.41%. 47. 25%. 45.6 / 5.82. 42.7 / 6.52. 283. malic enzyme. 97241.t00125. 0.26%. 119. 32%. 42.8 / 6.39. 40.7 / 6.60. 54 .

(61) 288 290. Hypothetical protein Succinyl-CoA ligase beta-chain (AP51), hydrogenosomal precursor. 82174.t00112. 0.13%. 46. 22%. 40.4 / 8.65. 39.4 / 6.17. 88864.t00118. 0.23%. 58. 16%. 44.1 / 6.53. 36.3 / 6.13. 291. Phospholipid/glycerol acyltransferase. 86001.t00241. 0.15%. 56. 15%. 37.6 / 8.74. 36.6 / 6.19. 292. Phospholipid/glycerol acyltransferase. 86001.t00241. 0.18%. 40. 12%. 37.6 / 8.74. 36.1 / 6.22. 293. glyceraldehyde 3-phosphate dehydrogenase. 85241.t00213. 0.50%. 55. 18%. 39.7 / 7.98. 36.6 / 6.24. 294. Casein kinase 1, alpha 1. 83007.t00211. 0.14%. 42. 20%. 34.1 / 6.05. 36.9 / 6.27. 295. 60S ribosomal protein L4/L1 family protein. 84488.t00127. 0.20%. 43. 17%. 40.6 / 9.99. 37.9 / 6.26. 296. Alcohol dehydrogenase (Adh), zinc-containing. 87955.t00248. 0.59%. 62. 21%. 39.5 / 6.14. 37.7 / 6.32. 297. methionine/cysteine gamma-lyase. 85284.t00031. 0.60%. 98. 23%. 43.5 / 6.04. 37.8 / 6.36. 299. Phosphoglycerate kinase (Tv_PGK1). 89154.t00220. 0.24%. 56. 16%. 45.5 / 6.58. 35.4 / 6.35. 300. hyaluronan mediated motility receptor. 85884.t00047. 0.19%. 57. 22%. 41.2 / 5.81. 34.5 / 6.16. 301. fructose-1,6-bisphosphate aldolase. 81812.t00080. 1.03%. 179. 47%. 36.4 / 5.79. 32.5 / 6.13. 302. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.16%. 47. 14%. 36.4 / 5.79. 24.4 / 6.24. 303. L-lactate/malate dehydrogenase. 86045.t00046. 0.14%. 55. 19%. 37.3 / 5.96. 34.4 / 6.25. 306. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.15%. 153. 40%. 36.4 / 5.79. 31.9 / 6.20. 307. fructose-1,6-bisphosphate aldolase. 92775.t00058. 0.23%. 182. 45%. 36.4 / 5.88. 32.2 / 6.32. 309. fructose-1,6-bisphosphate aldolase. 90332.t00087. 0.57%. 150. 37%. 36.5 / 6.02. 31.6 / 6.52. 310. fructose-1,6-bisphosphate aldolase. 92210.t00096. 0.11%. 104. 26%. 36.5 / 6.06. 31.1 / 6.51. 311. fructose-1,6-bisphosphate aldolase. 81812.t00080. 0.31%. 89. 21%. 36.4 / 5.79. 32.9 / 6.63. 314. Hypothetical protein_81528.m00048. 81528.t00048. 0.12%. 44. 18%. 39.3 / 6.38. 36.7 / 6.84. 318. glyceraldehyde 3-phosphate dehydrogenase. 85241.t00213. 0.22%. 171. 47%. 39.7 / 7.98. 36.6 / 5.61. 55 .

(62) 321. Phosphoglycerate kinase (Tv_PGK2). 93588.t00089. 0.21%. 71. 21%. 45.5 / 6.82. 35.4 / 6.56. 322. Alcohol dehydrogenase (Adh), zinc-containing. 87955.t00248. 0.90%. 148. 60%. 39.5 / 6.14. 37.8 / 6.50. 323. Alcohol dehydrogenase (Adh), zinc-containing. 87955.t00248. 0.64%. 119. 41%. 39.5 / 6.14. 36.9 / 6.46. 324. glyceraldehyde 3-phosphate dehydrogenase. 85241.t00213. 0.65%. 89. 38%. 39.7 / 7.98. 36.2 / 6.47. 325. glyceraldehyde 3-phosphate dehydrogenase. 85241.t00213. 0.49%. 131. 45%. 39.7 / 7.98. 36.2 / 6.44. 56 .

(63) Table 2 Identification of T.vaginalis proteins in the range of pH 6-11 by MALDI-TOF peptide mass figerprinting Spot Protein Name. Accession No.. Volume Score ％. ％. Theore MW / pI. Exp MW / pI. 331 phosphoenol pyruvate carboxykinase. 90655.m00168. 1.38%. 147. 35%. 67.8 / 6.71. 67.2 / 6.57. 332 phosphoenol pyruvate carboxykinase. 90655.m00168. 1.92%. 98. 35%. 67.8 / 6.71. 67.1 / 6.70. 333 phosphoenol pyruvate carboxykinase. 96829.m00054. 1.83%. 94. 28%. 67.9 / 6.55. 67.3 / 6.85. 335 glyceraldehyde 3-phosphate dehydrogenase. 92321.m00066. 4.52%. 63. 28%. 39.7 / 7.03. 40.3 / 6.67. 336 glyceraldehyde 3-phosphate dehydrogenase. 92321.m00066. 6.78%. 131. 55%. 39.7 / 7.03. 40.4 / 6.91. 338 glyceraldehyde 3-phosphate dehydrogenase. 85241.m00213. 9.77%. 152. 60%. 39.7 / 7.98. 39.9 / 7.45. 339 glyceraldehyde 3-phosphate dehydrogenase. 85241.m00213. 7.30%. 93. 40%. 39.7 / 7.98. 39.8 / 7.69. 340 branched chain amino acid transferase, putative. 84943.m00201. 1.24%. 130. 51%. 37.8 / 6.71. 36.4 / 6.77. 341 Hypothetical ANK-repeat protein MM0045.-related. 86088.m00409. 1.34%. 41. 36%. 28.2 / 6.38. 28.7 / 6.32. 342 hypothetical protein. 83996.m00294. 1.90%. 143. 54%. 28.3 / 6.62. 28.3 / 6.50. 346 hypothetical protein. 112044.m00002. 1.24%. 38. 50%. 24.8 / 6.75. 23.8 / 7.34. 347 thioredoxin peroxidase. 84105.m00110. 1.75%. 117. 58%. 22.2 / 6.31. 20.6 / 6.23. 348 iron superoxide dismutase-. 91931.m00031. 4.53%. 95. 52%. 21.6 / 6.55. 20.0 / 6.56. 349 chloroplastic iron superoxide dismutase. 81958.m00091. 1.73%. 73. 43%. 22.4 / 6.79. 20.6 / 6.77. 350 Probable thiol peroxidase. 85876.m00193. 2.20%. 40. 23%. 18.1 / 6.05. 17.4 / 6.11. 352 Profilin 1.-related. 85736.m00011. 29.96%. 66. 79%. 13.4 / 7.66. 11.1 / 7.11. 353 profilin A-related. 96092.m00156. 4.63%. 53. 64%. 13.4 / 8.44. 10.9 / 7.98. 0.53%. 80. 29%. 46.9 / 7.96. 44.6 / 7.64. 354 Hydrogenosomal oxygen reductase. 81529.m00518. 57 . Coverage.

(64) Table 3 List of down-regulated proteins identified by MALDI-TOF MS in the amoeboid stage compared to trophozoite. Spot no. Protein Name. Accession No.. Score. Coverage. Theore MW/ pI. ％ 5.0. hypothetical protein. 94782.m00175. 78. 17%. 68.3 / 5.14. 7.0. Peptidase family M20/M25/M40 containing protein. 87848.m00053. 87. 29%. 51.9 / 5.03. 11.0. Ubiquitin family protein. 46. 14%. 43.0 / 5.15. 12.0. hypothetical protein. 94782.m00175. 109. 29%. 68.3 / 5.14. 17.1. actin. 85746.m00231. 108. 38%. 42.2 / 5.05. 18.0. actin. 83372.m00049. 86. 34%. 42.1 / 5.05. 19.0. actin. 85746.m00231. 155. 56%. 42.2 / 5.05. 20.0. actin. 82114.m00023. 90. 17%. 42.1 / 5.12. 21.0. Peptidase family M20/M25/M40 containing protein. 87848.m00053. 96. 37%. 51.9 / 5.03. 22.0. alpha-tubulin 1. 86772.m00061. 120. 32%. 50.7 / 4.9. 23.0. alpha-tubulin 1. 86772.m00062. 92. 33%. 50.7 / 4.9. 26.0. actinin. 85661.m00070. 87. 25%. 69.6 / 4.89. 35.0. Hsp90 protein. 83450.m00067. 76. 20%. 71.6 / 5.06. 40.0. fructose-1,6-bisphosphate aldolase. 92775.m00058. 66. 34%. 36.4 / 5.79. 41.0. fructose-1,6-bisphosphate aldolase. 81812.m00080. 87. 41%. 36.4 / 5.79. 42.0. fructose-1,6-bisphosphate aldolase. 81812.m00080. 50. 29%. 36.4 / 5.79. 43.0. fructose-1,6-bisphosphate aldolase. 90332.m00087. 88. 48%. 36.5 / 6.02. 44.0. fructose-1,6-bisphosphate aldolase. 81812.m00097. 54. 38%. 36.5 / 5.84. 83629.m00178. 58 .

(65) 46.0. fructose-1,6-bisphosphate aldolase. 81812.m00097. 124. 49%. 36.5 / 5.84. 47.0. L-latate dehydrogenase. 86045.m00059. 111. 45%. 37.3 / 5.77. L-latate dehydrogenase. 86045.m00046. 111. 45%. 37.3 / 5.96. Phosphoglycerate kinase. 89154.m00220. 77. 25%. 45.5 / 6.58. Phosphoglycerate kinase. 93588.m00089. 77. 25%. 45.5 / 6.82. 49.0. fructose-1,6-bisphosphate aldolase. 90332.t00087. 110. 48%. 36.5 / 6.02. 50.0. fructose-1,6-bisphosphate aldolase. 81812.m00097. 78. 47%. 36.5 / 5.84. 63.0. Beta-tubulin. 85300.t00020. 85. 41%. 28.5 /. 73.0. Actin. 85746.m00231. 189. 53%. 42.2 / 5.05. 74.0. Actin. 85746.m00231. 274. 67%. 42.2 / 5.05. 75.0. Actin. 82114.m00023. 149. 53%. 42.1 / 5.12. 129.0. Hypothetical protein. 97196.m00092. 41. 22%. 20.6 /. 5.42. 131.0. Fructose-1,6-bisphosphate aldolase. 81812.m00080. 64. 23%. 36.4 /. 5.79. 132.0. Fructose-1,6-bisphosphate aldolase. 92775.m00058. 67. 28%. 36.4 /. 5.88. 135.0. Carbamate kinase. 89002.m00281. 64. 38%. 40.5 /. 5.48. 136.0. Fructose-1,6-bisphosphate aldolase. 81812.m00080. 189. 50%. 36.4 /. 5.79. 249.0. Heat shock protein 70. 100090.m00002. 133. 46%. 41.4 /. 5.40. 252.0. Ketopantoate reductase. 112876.m00003. 81. 38%. 37.8 /. 5.69. 48.0. 59 . 5.21.