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Biochemical Characterization of Echinococcus multilocularis Antigen B3 Reveals Insight into Adaptation and Maintenance of Parasitic Homeostasis at the Host-parasite Interface Chun-Seob Ahn, Jeong-Geun Kim, Xiumin Han, YoungAn Bae, Woo-Jae Park, Insug Kang, Hu Wang, and Yoon Kong J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.6b00799 • Publication Date (Web): 13 Dec 2016 Downloaded from http://pubs.acs.org on December 14, 2016

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Journal of Proteome Research

Biochemical Characterization of Echinococcus multilocularis Antigen B3 Reveals Insight into Adaptation and Maintenance of Parasitic Homeostasis at the Host-parasite Interface

Chun-Seob Ahn†,*, Jeong-Geun Kim†,*, Xiumin Han‡, Young-An Bae||, Woo-Jae Park§, Insug Kang⊥, Hu Wang||, Yoon Kong†,**

†

Department of Molecular Parasitology, Sungkyunkwan University School of Medicine,

Suwon 16419, Korea, ‡Qinghai Province Institute for Endemic Diseases Prevention and Control, Xining 811602, China, ||Department of Microbiology, Gachon University Graduate School of Medicine, Incheon 21936, Korea, §Department of Biochemistry, Gachon University Graduate School of Medicine, Incheon 21936, Korea, ⊥Department of Molecular Biology and Biochemistry, School of Medicine, Kyung Hee University, Seoul 02447, Korea

*These authors contributed equally to the work.

**Correspondence: Dr. Yoon Kong, Department of Molecular Parasitology, Sungkyunkwan University School of Medicine, Suwon 16419, Korea. E-mail: [email protected]; Fax: +82-31-299-6269

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ABSTRACT: Alveolar echinococcosis (AE) caused by Echinococcus multilocularis metacestode is frequently associated with deleterious zoonotic helminthiasis. The growth patterns and morphological features of AE, such as invasion of the liver parenchyme and multiplication into multivesiculated masses, are similar to those of malignant tumors. AE has been increasingly detected in several regions of Europe, North America, Central Asia, and northwestern China. An isoform of E. multilocularis antigen B3 (EmAgB3) showed a specific immunoreactivity against patient sera of active-stage AE, suggesting that EmAgB3 might play important roles during adaptation of the parasite to hosts. However, expression patterns and biochemical properties of EmAgB3 remained elusive. Protein profile and the nature of component proteins of E. multilocularis hydatid fluid (EmHF) have never been addressed. In this study, we conducted proteome analysis of EmHF of AE cysts harvested from immunocompetent mice. We observed the molecular and biochemical properties of EmAgB3, including differential transcription patterns of paralogous genes, macromolecular protein status by self-assembly, distinct oligomeric states according to individual anatomical compartment of the worm, and hydrophobic-ligand-binding protein activity. We also demonstrated tissue expression patterns of EmAgB3 transcript and protein. EmAgB3 might participate in immune response and recruitment of essential host lipids at the host-parasite interface. Our results might contribute to an in-depth understanding of the biophysical and biological features of EmAgB3, thus providing insights on the design of novel targets to control AE.

KEYWORDS: Echinococcus multilocularis, alveolar echinococcosis, hydatid fluid, proteome, antigen B3, expressional regulation, multimeric states, hydrophobic-ligandbinding protein

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INTRODUCTION Alveolar echinococcosis (AE) is a tissue invasive larval cestodiasis caused by Echinococcus multilocularis metacestode. AE is a relatively rare disease compared to E. granulosus cystic echinococcosis (CE).1 However, AE has been increasingly detected in several enclaves of Europe, North America, Central Asia, and northwestern China.2-5 In some areas of the Qinghai-Tibetan plateaus in China, the prevalence of AE has been reported to be ranging from 3.4% to 6.8%.6 AE invokes chronic and fatal zoonotic helminthiasis and is recognized as one of major neglected tropical diseases by the World Health Organization (WHO) due to its significant impacts on public health (http://www.who.int/neglected_diseases/diseases/en/). Humans are the intermediate hosts of E. multilocularis and are infected by incidental contract with parasite’s eggs that pass out with canine stool. Once ingested, activated oncospheres penetrate the intestinal wall and are liberated into the circulation. They mostly egress in the liver through portal circulation, invade the liver parenchyme, and asexually multiply to form multivesiculated cystic masses filled with fluid (hydatid fluid; HF) and protoscoleces.7 HF consists mainly of diverse proteinaceous and non-proteinaceous components secreted by the parasite. HF also contains some host-derived resources absorbed during the metabolic processes.8 The growth patterns and morphological features of AE are similar to those of malignant tumors since the masses typically show combined lesions of central necrosis and peripheral proliferation with indistinct irregular margins.9,10 Moreover, metastatic lesions in nearby/distant tissues and organs are frequently observed.9,11 Human AE is characterized by a long asymptomatic period. If an appropriate diagnosis is not made and treatment is not initiated before the lesions overtly grow or before distant metastatic lesions are found, the prognosis is grave.9,11 Early diagnosis and proper management may significantly reduce disability-adjusted life years associated with AE. The practical diagnosis of AE relies largely on concurrent interpretation of imaging findings of 3



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ultrasonography (US), computed tomography (CT), and/or magnetic resonance imaging (MRI), together with data provided by serological tests. Serological tests are popular in AE endemic areas due to their ease of use and relatively high reproducibility.12 Antigen B proteins (EgAgB1-5) were initially described from E. granulosus hydatid fluid (EgHF).13,14 The proteins have been extensively studied owing to their reliable diagnostic performance. Later studies have demonstrated that these proteins are multifunctional lipoproteins. Biological functions of EgAgB proteins include modulating Th2 biased host immune responses, and impeding dendritic cell maturation and neutrophil migration.13-16 EgAgBs have hydrophobic-ligand-binding protein (HLBP) activity. They participate in the uptake and sequestration of hydrophobic substances.17,18 EgAgBs are synthesized from a multigene family with marked genetic versatility.19-21 Eight genes and five isoforms have been detected in the draft genome (http://www.genedb.org/homepage) and proteome database (http://www.ncbi.nlm.nih.gov/protein) of E. granulosus with term of Echinococcus granulosus (txid6210),22-24 suggesting that the expression profiles of multiple EgAgB isoforms are highly complicated along with transcriptional activities of their paralogous genes. In addition, EgAgBs have particular structural conformations. EgAgB1-5 exist mostly as monomer (8 kDa), dimer (16 kDa), and trimer (24 kDa) in EgHF under reducing conditions.13,25,26 However, those proteins have large molecular masses (between 140 and 550 kDa) as a result of homomultimer and/or heteromultimer formation when analyzed by molecular sieve chromatography or by dynamic light scattering studies.13,25 Multimerization seems not to be associated with cysteine-mediated disulfide bond since these multimers are highly resistant to denaturation and/or reducing conditions.25 Enzymes involved in fatty acid anabolism have not been detected in the transcriptome or genome of E. granulosus, E. multilocularis or Taenia solium.22-24 Taeniid parasites should be equipped with a variety of lipid uptake/transport systems. HLBPs, such as 4


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EgAgBs, and 7 and 10 kDa of T. solium metacestode (TsM), which are subunits of the 150 kDa protein complex, are uniquely expressed in cestode parasites.17,18,27 Those HLBPs are small α-helix rich lipid binding proteins (7-10 kDa) and mediate various cellular events, including detoxification for cellular protection, and transport of phospho-/glycolipids, fatty acids, and other hydrophobic molecules. The excretory-secretory TsM HLBP can cargo lipid molecules from the host and deliver these molecules into the parasite across syncytial membrane.27 Taeniidae HLBPs might play pertinent roles during the acquisition of fatty acids from the environments. They might be intimately involved in long-term survival of parasites in the hosts.27 High sequence identity shared between EgAgBs and EmAgBs22 strongly suggests that EmAgB proteins might constitute HLBP operating in E. multilocularis metacestode, but their genuine HLBP activity has not been investigated. We have previously demonstrated that an EmAgB3 isoform has a specific immunoreactivity against patient sera of active stage AE with negligible cross-reactions with sera from other parasitic infections and other causes of hepatic space-occupying lesions, including primary hepatocellular carcinoma.28 A recent transcriptome analysis has shown that distinct isoforms of EmAgB3 are differentially activated during oncosphere and early developing metacestode stages.29 These results collectively suggest that EmAgB3 might be intimately involved in adaptation of the parasite to the hosts and inducing immunobiological alterations. However, expression patterns and biochemical properties of EmAgB3 remained elusive. More importantly, protein profile and the nature of component proteins of E. multilocularis HF (EmHF) have never been addressed. Since AE mass(es) survive and invade human tissues for considerable periods of time, the parasite will have to continuously synthesize bioactive molecules to adapt to the cytopathic host environments and modulate host immune defenses. EmHF might contain diverse secretory proteins, in addition to EmAgB molecules. 5



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In the present study, we undertook proteome analysis of EmHF of AE cysts harvested from experimentally infected mice. We observed molecular and biophysical/biochemical properties of EmAgB3 which included differential transcription patterns of paralogous EmAgB3s, multimeric macromolecular formation, distinct oligomeric states according to individual anatomical compartments of the worm, and differential HLBP activity compared to its E. granulosus ortholog (EgAgB3). We also demonstrated tissue expression patterns of EmAgB3 transcript and protein. Our results suggested strongly that EmAgB3 might participate in recruitment of essential host lipids, thus contributing to the adaptation and maintenance of parasitic homeostasis at the host-parasite interface. The multifunctional EmAgB3 might constitute novel targets for intervention strategies against life-threatening AE.

MATERIALS AND METHODS Parasite and Sample Preparation Fifty Kunming mice (6-week-old females; Lanzhou Institute of Biological Products, Lanzhou, China) were inoculated intraperitoneally with 1000 viable AE protoscoleces collected from naturally infected voles (Microtus fuscus) in an AE endemic area (Dari County, Qinghai Province, China). Mice were killed at 9-month postinfection. Approximately 50 intact AE masses clustered with various sized metacestode vesicles (approximately 0.3-5 cm in diameter) were harvested from the liver surface and peritoneal cavity. The metacestode vesicles were carefully decorticated without host tissue contaminants and washed with physiological saline for more than 10 times. EmHF was aseptically drawn from individual cysts using 26 gauge needles. The fluid was pooled in the presence of protease inhibitor cocktail (25 ml per 1 tablet; Roche, Mannheim, Germany). Protoscoleces were removed by centrifugation at 500 g for 2 min. We collected 6


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approximately 50 ml of EmHF from 36 mice. Crude EmHF was centrifuged at 20000 g for 1 h to remove insoluble particles. Supernatant was dialyzed against phosphate buffered saline (PBS; 100 mM, pH 7.4) at 4°C for 4 h, concentrated by lyophilization, and stored at -80°C. Empty germinal layers were soaked and washed with physiological saline to separate membrane bound protoscoleces. Germinal layer fraction was kept separately. Protoscoleces were collected by chopping the whole AE masses followed by filtering through 300 µm poresized mesh and washing more than 10 times with physiological saline. The protoscoleces were individually harvested under a dissecting microscope. The protoscolex and germinal layer were separately homogenized with a Teflon-pestle homogenizer in PBS supplemented with protease inhibitor cocktail. The respective homogenates were centrifuged at 20000 g for 1 h and supernatants were used as respective extracts. A fertile single ovine CE2 cyst (approximately 15 cm in diameter) collected from an abattoir in Xining (Qinghai Province, China) was also used.26 EgHF and each extract (germinal layer and protoscolex) were separately prepared. All procedures for sample preparation were done at 4°C unless otherwise specified. Fresh intact AE cysts were put into RNAlater solution (Thermo Fisher Scientific, Waltham, MA, USA). The protoscolex and germinal layer were separately harvested under a dissecting microscope. Total RNA was extracted from whole AE, protoscolex, and germinal layer using RNA extraction kit (iNtRON, Seongnam, Korea) according to the manufacturer’s instructions. All study protocols were approved by the Institutional Review Committee of Qinghai Province Institute for Endemic Diseases Prevention and Control (protocol 2013-722).

Electrophoreses Protein samples mixed with native-PAGE sample buffer (62.5 mM Tris-glycine, 25% 7



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glycerol, and 0.01% bromophenol blue; pH 6.8) were separated by 4-20% Mini-PROTEAN TGX gels (Bio-Rad, Hercules, CA, USA) at 20 mA constant current. Samples were subjected to 10% Tricine SDS-PAGE or 15% SDS-PAGE under reducing conditions. EmHF (200 µg) was electrofocused on immobilized pH gradient strips (IPG, pH 4-7 and pH 6-11; GE Healthcare, Little Chalfont, UK) for 25 kVh and further separated by 10% Tricine SDSPAGE (2-dimensional gel electrophoresis; 2-DE). For blue native-PAGE, proteins were separated by 4-20% native-PAGE in the first dimension. The gel was cut into strips, incubated with 1% SDS for 15 min, and washed with deionized distilled water. The strip was run on 10% Tricine SDS-PAGE in the second dimension. Gels were either stained with Coomassie brilliant blue G-250 (CBB) or processed for immunoblotting.

Protein Identification by Mass Spectrometry and Bioinformatics EmHF separated by 2-DE was subjected to mass spectrometry (MALDI-TOF MS; AB SCIEX TOF/TOF 4800 PLUS) for protein identification. Internal standards were tryptic autodigestion peaks (m/z = 842.5099 and 2211.1046). Monoisotopic peptide masses were selected between 600 and 3500 Da, and the proteins were identified by peptide mass fingerprinting (PMF) with search program Mascot (http://www.matrixscience.com). Mass tolerance was ± 50 ppm. One missed cleavage site was allowed. Identification was accepted when PMF revealed at least two identified peptides (> 99% probability). For LC-MS/MS, proteins were diluted in denaturing buffer. Disulfide bonds were reduced with dithiothreitol (DTT) and carbamidomethylated with iodoacetamide. Tryptic digested peptides were desalted and analyzed using an on-line 1200 nano-flow system (Agilent Technologies, Santa Clara, CA, USA) connected to a LTQ linear ion trap mass spectrometer (Thermo Fisher Scientific). Peptides were eluted in a linear gradient of acetonitrile (10-40%) over 65 min. MS survey was conducted at 300-2000 m/z with three 8


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data-dependent MS/MS scans isolation width (1.5 m/z), normalized collision energy (25%), and dynamic exclusion duration (180 sec). MS data were generated in RAW file format (Thermo Fisher Scientific) employing Xcalibur1.4 with Tune1.0. Peptide peaks were introduced into MS/MS ions search using the Mascot program mentioned above. Mass values were selected with monoisotopic masses. Peptide and MS/MS tolerances were ± 1.2 and ± 0.6 Da, respectively. Protein identifications of individual ions with scores exceeding 43 were considered as significant homology (P < 0.05). Cysteine carbamidomethylation and methionine oxidation were considered during the analyses. Duplicated biological samples were independently analyzed. Signal peptide and non-classical secretions were predicted with PSORT (http://psort.hgc.jp/) and SignalP (http://www.cbs.dtu.dk/services/SignalP/). MS database search was carried out for merged files of non-redundant and expressed sequence tags (EST) against NCBI database (http://www.ncbi.nlm.nih.gov), and gene/protein database of E. granulosus and E. multilocularis (http://www.genedb.org/homepage). The proteins identified were functionally categorized using Blast2GO ver3.2 (http://www.blast2go.com) and BLASTp with NCBInr database and Blast Expectation Value (E-Value: 1.0E-3). Based on BLASTp results, InterProScan, mapping, annotation, and graphical analysis were sequentially processed. Histograms of biological process, molecular function, and cellular component were generated using the second-level of GO hierarchy.

N-terminal Amino Acid Sequencing EmHF was separated by 2-DE, transferred to a polyvinylidene difluoride membrane (PVDF; Santa Cruz), and stained with CBB. Spots of 6 and 8 kDa EmAgB3 were cut and applied to an ABI model 477A protein sequencer and an ABI model 120A PTH analyzer (Perkin Elmer, Foster City, CA, USA). N-terminal amino acid sequence was analyzed by Edman 9



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degradation.

Cloning and Expression of Recombinant EmAgB Isoforms and EgAgB3 The mature domains of EmAgB1-5 and EgAgB3 genes were amplified with gene-specific primers tagged with BamHI (forward) and XhoI (reverse) (Supplementary Table S1), and total RNA (200 ng) extracted from the AE mass or a single fertile ovine CE2 cyst. Thermal cycling conditions were: 45°C for 30 min, 95°C for 5 min, 35 cycles of 95°C for 1 min, 60°C for 45 sec, 72°C for 1 min, followed by final extension at 72°C for 5 min. Amplicons were ligated with pGEX-6P-1 expression vector (Novagen, Cambridge, MA, USA) and transformed into Escherichia coli BL21 (DE3). Bacterial cells were cultured in Luria-Bertani medium supplemented with ampicillin (50 µg/ml). Recombinant proteins were induced with 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) for 4 h at 37°C. Cells were harvested, sonicated, and recombinant proteins were purified using a glutathione (GST)-sepharose-4B affinity column (GE Healthcare). The GST-tag was removed using PreScission protease (GE Healthcare) and dialyzed against PBS at 4°C for 4 h. Recombinant proteins concentrated by lyophilization were reconstituted with PBS. To observe multimeric state, native protein eluted from immunoprecipitation and GST-removed delipidated recombinant proteins were incubated at 25°C for 2 h.

Generation of Mono-specific Antibodies Polyclonal mono-specific antibodies against respective EmAgB isoforms were generated by immunizing BALB/c mice with rEmAgB1-5 (each 30 µg) mixed with Freund’s complete adjuvant (Sigma-Aldrich, St. Louis, MO, USA). Subsequent immunization was done using Freund incomplete adjuvant twice at 2-week interval. Final booster was an intravenous injection of 10 µg protein (protocol no. 14-12). Blood was collected by heart puncture and 10


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centrifuged at 3000 g for 10 min. IgG fractions were purified using Protein G affinity column (Peptron, Seoul, Korea) and stored at -80°C.

Immunoprecipitation EmHF (50 µg) was incubated with anti-rEmAgB3 mono-specific antibody (5 µl) overnight at 4°C and further incubated with 30 µl of Protein G-coupled agarose 4B (Peptron) for 6 h at 4°C. The immune complex was washed with PBS and recovered by centrifugation at 20000 g for 5 min. Bound proteins were eluted with glycine buffer (50 mM, pH 3.0). The pH of the eluent was immediately adjusted to 7.2 by adding 1 M Tris. In each experiment, freshly prepared eluents were used.

Immunoblotting Proteins separated by electrophoreses were transferred to nitrocellulose membranes (Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1-6 h at 4°C. Membranes were incubated with Tris-buffered saline (100 mM, pH 8.0) containing 0.05% Tween 20 and 3% skim milk for 1 h followed by incubation with 1:2000 diluted respective antibodies at 4°C overnight. Membranes were further incubated with 1:4000 diluted horseradish peroxidase (HRP)conjugated anti-mouse IgG (Cappel, West Chester, PA, USA) at room temperature for 2 h. All signals were detected by West-Q Pico enhanced chemiluminescence (ECL; GenDEPOT, Dallas, TX, USA) after 2 min exposure for quantitative analysis. Images were digitalized with a Perfection V700 Photo Scanner (Epson America, San Jose, CA, USA).

Reverse Transcription (RT)-PCR and Quantitative Real-time PCR (qPCR) Total RNA was extracted from respective AE samples as described above. Gene-specific primers for each EmAgB and actin (internal control; EmuJ_000407400) were constructed 11



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(Supplementary Table S1). RT-PCR was done using RT-PCR PreMix kit (iNtRON). Thermal cycler profiles included 45°C for 30 min, 95°C for 5 min, 30 cycles for 95°C for 45 sec, 60°C for 45 sec, 72°C for 1 min, and a final extension at 72°C for 2 min. PCR products separated by 1.5% agarose gel electrophoresis were stained with ethidium bromide. For qPCR, total RNA (200 ng) was reverse transcribed to cDNA using a first-strand cDNA synthesis system (Thermo Fisher Scientific). Gene-specific primers were optimized by qPCR with Rotor-Gene Q using Rotor-Gene SYBR Green PCR kit (Qiagen, Valencia, CA, USA). qPCR cycling parameters were 95°C (10 min) followed by 45 cycles of 95°C (15 sec), 60°C (30 sec), and 72°C (30 sec) with melting curve analysis. E. multilocularis actin gene was used for normalization. The relative expression was calculated by differences in cycle threshold (Ct) between control and EmAgB genes using Rotor-Gene Screen Clust HRM Software (Qiagen).

Analysis of Genomic Loci of EmAgB3 Genes Partial EmW_scaffold_07 sequences of E. multilocularis draft genome (nucleotide positions between 610940 and 688045; LN902847.1) containing EmAgB1-5 genes were retrieved from NCBI database (http://www.ncbi.nlm.nih.gov/nuccore/961439103). A schematic diagram including exon-intron architectures and relative direction of transcription was created according to the genetic information obtained. Homologous repeat regions were scanned by self-to-self comparison of the sequence using Align Sequences Nucleotide BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Nucleotide sequences encompassing two identical EmAgB3 genes were amplified from E. multilocularis genomic DNA by PCR employing gene-specific primers (5′-GGTGGAAGCTACTGA-3′ and 5′-GGTGCTCTTTGCAGT-5′ for EmuJ_000381600; 5′-AAGCTCTCAGTGCCA-3′ and 5′-ATAGGGGTTAGAGAG-3′ for EmuJ_000381700) designed from up- and down-stream regions of each gene. PCR 12


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conditions included denaturation of template genomic DNA at 95°C for 10 min, 35 cycles of 95°C for 1 min, 52°C for 1 min, 72°C for 3 min, followed by a final extension at 72°C for 10 min. PCR products were subjected to paired-end sequencing using ABI BigDye Terminator ver3.1.

Fluorescence In Situ Hybridization (FISH) and Immunohistochemical Staining Worm sections (4-µm in thickness) were hybridized with fluorescent Cy5-labeled probes (antisense, 5'-GGGTGACTTCATCATCATCAGCACGAGCAACG-3'; sense, 5'CGTTGCTCGTGCTGATGATGATGAAGTCACCC-3') at 55°C for 16 h. Section slides were washed with 2× SSC containing 20% formamide at 30°C for 30 min. The slides were counterstained with 4',6-diamidino-2-phenolindole (DAPI, Thermo Fisher Scientific) at 4°C for 5 min in a dark chamber and mounted on an oxygen depleted medium. Staining intensities were observed with a LSM710 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). AE sections (4-µm thick) were treated with 3% hydrogen peroxide for 5 min and incubated with PBS supplemented with 3% bovine serum albumin and 0.05% Tween 20 for 1 h at room temperature. The sections were further incubated with 1:400 diluted specific antibody at 4°C overnight and subsequently incubated with 1:1000 diluted fluorescein isothiocyante (FITC)-conjugated goat anti-mouse IgG (Abcam, San Francisco, CA, USA). The slides were counterstained with DAPI. Preimmune mouse serum diluted at the same ratio was used as a control. Images were photographed with an Axioplot light/fluorescent microscope (Carl Zeiss).

Delipidation of Bacterial Lipids Bacterial lipid fractions were delipidated using an Octyl-Sepharose 4 Fast flow reverse-phase 13



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hydrophobic interaction chromatography according to the manufacturer’s instructions (GE Healthcare). GST-tag removed recombinant proteins were resuspended in 50 mM phosphate buffer (PB, pH 7.0) and mixed with ammonium sulfate up to 1.7 M. Proteins were bound to Octyl-Sepharose beads by flow-through into the column. Delipidated proteins were eluted with a linear gradient (from 0 to 100%) of PB. Protein delipidation was also done by incubating proteins with Sephadex-LH beads (Sigma-Aldrich) at room temperature for 2 h. Delipidated proteins were obtained by centrifugation at 12000 g for 1 min and dialyzed against PBS (100 mM, pH 7.4) at 4°C for 4 h. The lipid remnants in delipidated proteins were determined using free fatty acid (fFA) quantification colorimetric assay kit (BioVision, Milpitas, CA, USA). Proteins (each 2 µg/µl) were sequentially allocated (0-10 µl) into wells of 96-well flat-bottom microtiter plate (Thermo Fisher Scientific) in a total volume of 50 µl and incubated at 37°C for 30 min. Assay mixture (50 µl) was then added to each well and incubated at 37°C in darkness for additional 30 min. Free FAs were spectrometrically quantitated at 570 nm. Concentration of fFAs was calculated following the manufacturer’s instructions using the following equation: fFA = Fa/Sv (nM), where Fa was the FA amount (nM) in the well obtained from standard curve and Sv was the sample volume (µl) added to the sample well. Palmitic acid (PA; 1 nM/ µl) (Sigma-Aldrich) was used as a control to establish standard curve. All assays were independently done in triplicate.

Assay of Hydrophobic-ligand-binding Activity The fluorescent FA analogs/naturally fluorescent ligands used included 11-([5dimethylaminonaphthalene-1-sulfonyl]amino) undecannoic acid (DAUDA), dansyl-DL-αaminocaprylic acid (DACA), 1-anilinonapthalene 8-sulfonic acid (1,8-ANS), 16-(9anthroyloxy) palmitic acid (16-AP), and cis-parinaric acid (cPnA) (Molecular Probes, Eugene, 14


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OR, USA). Saturated and unsaturated FAs, such as palmitic acid (PA), myristic acid (MA), oleic acid (OA), linoleic acid (LA), and arachidonic acid (AA; Sigma-Aldrich) were also used. All stock chemicals (10 mM) diluted in ethanol were stored at -20°C in darkness and freshly diluted to 0.1 mM just prior to use. Differing concentrations of delipidated recombinant proteins (2-16 µg) were added to ligand solutions (each 10 µM). TsM 150 kDa HLBP complex was purified by monoclonal antibody-based immunoaffinity chromatography, delipidated, and used as a positive control.27 Fluorescence was detected after incubation at 25°C for 5 min. Fluorescence emission spectra were acquired using black 96-well Microfluor 1 plates (Thermo Electron) with an Infinite M-200 automated multi-detector (Tecan GmbH, Grödig, Austria). The excitation (Exmax)/emission (Emmax) wavelengths for respective ligands were as follows: DAUDA (345/519 nm), DACA (350/519 nm), 1,8-ANS (370/460 nm), cPnA (315/420 nm), and 16-AP (360/460 nm). Raman scattering by solvent was corrected using appropriate blank solution. The equilibrium dissociation constants (Kd) of proteins bound to 1,8-ANS, cPnA, and 16-AP were determined by adding increasing concentrations (0-80 µM) of each ligand to a reaction mixture containing the recombinant proteins (2 µg/200 µl, equivalent to 4 µM). Spectrometric data were normalized to the peak intensity and corrected for background fluorescence of the ligand at each concentration. Data were assayed with the one-site saturation model and best fit algorithm contained in SigmaPlot10.0.1 software (Systat, San Diego, CA, USA) using the following equation: y = VmaxX/Km+X, where y was relative fluorescence and X was the concentration of lipid ligand. Vmax could be substituted as Fmax (maximum fluorescence). Scatchard-plot was analyzed using the same data of saturation experiments. All assays were independently done for 3-5 times.

RESULTS 15



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Proteome Analysis of E. multilocularis Hydatid Fluid (EmHF) and Functional Annotation of Gene Ontology Terms Global expression patterns of EmHF proteins of AE cysts grown for 9-month in immunologically competent mice was determined. More than 90 protein spots were observed on 2-DE gels through pH 4-7 and 6-11 (Figure 1A and B). We were able to identify 60 proteins by MALDI-TOF MS, including 40 parasite- and 20 host-derived proteins. Among parasite proteins, two major groups were evident: i) proteins with high molecular weights (Mr) ranging from 70-130 kDa between isoelectric points (pI) of 5.5 and 8, and ii) those with low Mr of 6-15 kDa between pI 7 and 9.8. The first group (high Mr acidic/neutral proteins) was largely segregated into nine N-acetylated α-linked acidic dipeptidase 2 (NAALAD2; spots 15-23) and two protease inhibitor I25 (cystatin; spots 8 and 9). The second group (low Mr proteins with neutral/alkaline pHs) mostly represented diverse EmAgB isoforms; EmAgB1 (3 molecules; spots 41, 44, and 47), EmAgB2 (4 species; spots 34, 40, 45, and 46), EmAgB3 (7 entries; spots 33, 42, 43, 48, 49, 59, and 60), and EmAgB4 (5 proteins; spots 32 and 36-39) (Figure 1C). We also identified two species of glycoprotein antigen 5 (spots 29 and 30) orthologous to E. granulosus 38 kDa antigen 5.30 One antioxidant glutathione transferase (spot 10) and a mitochondrial thioredoxin (spot 31) were also recognized. Other proteins included carbohydrate metabolizing fructose 1,6-bisphosphate aldolase (spot 57), vesicular amine transporter (spot 2) mediating the transport of acethylcholine and biogenic amines, touch receptor-related mechanosensory protein 2 (spot 28), and hypothetical proteins (3 molecules: spots 11, 12, and 14). The 20 host-derived proteins were clustered mainly into serum components, such as albumin (9 species), serotransferrin precursor (3 molecules), αfetoprotein (3 proteins), and β-globin (1 molecule). In addition, oxytocin-neurophysin, γaminobutyric acid receptor-A (GABA-A), proteasome activator complex subunit 2 (isoform 2), and unnamed protein were annotated (Table 1 and Supplementary Table S2). 16


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Blast2GO systematic analysis was performed using gene ontology terms assigned to the identified proteins based on similarity pattern.31 The majority of the ontologies of parasite proteins were allocated to molecular function with binding and catalytic activity (17 entries), suggesting that dynamic molecular biochemical and cellular events are actively occurring within EmHF to maintain cellular homeostasis. In biological processes (28 entries), similar proportions of host (15 entries) and parasite proteins (13 entries) were distributed. Cellular components (16 entries) largely constituted host extracellular/secreted and luminal proteins (Figure 2), which might have absorbed during metabolism or leaked into EmHF during sample preparation.

Genome-wide Survey and Expression Analysis of EmAgB3 Isoforms We retrieved EmAgB genes by mining the E. multilocularis draft genome (http://www.genedb.org/Homepage/Emultilocularis). EmAgB1 (EmuJ_000381200), EmAgB2 (EmuJ_000381100), EmAgB4 (EmuJ_000381400), and EmAgB5 (EmuJ_000381800) existed as a single copy. EmAgB3 was multiplied into three copies (EmuJ_000381500, EmuJ_000381600, and EmuJ_000381700). The coding DNA sequences (CDS) of these EmAgBs were intervened by a single intron and displayed 53.9-100% and 38.1-100% sequence identities to one another at mRNA and protein levels, respectively. Most strikingly, the nucleotide sequences of EmuJ_000381600 and EmuJ_000381700, both of which coded for EmAgB3,22 appeared to be identical to each other including those in the intervening intron (Supplementary Table S3). The individual identities of EmuJ_000381600 and EmuJ_000381700 were further analyzed at chromosome level. The genes were mapped to a 77-kb region of a genomic scaffold (EmW_scaffold_07, LN902847.1) flanked by prohormone 4 and metastasis associated protein 1 genes (Figure 3). EmAgB3 paralogs were tandemly arrayed between 17



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EmAgB4 and EmAgB5 with an identical direction of transcription. BL2Seq analysis (E-value = 0.0) of the nucleotide sequence in positions between 654901 and 665173 detected highly homologous regions that separately corresponded to each of the EmAgB3 gene (inset, Figure 3). Adding to CDS and intron, the 217- and 434-bp sequences in the direct upstream and downstream sequences of EmuJ_000381600 and EmuJ_000381700 were also identical to each other. Homologous segments amplified from E. multilocularis genomic DNA by PCR with primer pairs specific to EmuJ_000381600- and EmuJ_000381700-occupying loci showed a similarity pattern identical to that of EmW_scaffold_07 sequence (Figure 3 and Supplementary Figure S1). The expression levels of diverse EmAgB proteins in EmHF collected from 9-monthold AE cysts were determined. EmAgB3 (7 species) synthesized from EmuJ_000381500 (4 proteins) and EmuJ_000381600 and/or EmuJ_000381700 (3 molecules) revealed the highest levels, followed by EmAgB4 (5 proteins), EmAgB2 (4 species), and EmAgB1 (3 molecules) (Table 1 and Figure 4A). EmAgB5 protein was not detected. Since we were unable to differentiate EmuJ_000381600 from EmuJ_000381700 due to their identical sequences, we hereafter designated EmAgB3 proteins synthesized from EmuJ_000381600 and EmuJ_000381700 as EmAgB3b and EmAgB3c, respectively. We named EmAgB3 protein from EmuJ_000381500 as EmAgB3a. The total transcription levels of EmAgB3 isoforms (those of EmAgB3a and EmAgB3b/c) were the highest. EmAgB2 and EmAgB4 were also abundantly transcribed, but EmAgB5 transcripts were weakly detected. Transcription levels of EmAgB1 were the lowest among EmAgB isoforms (Figure 4B and C).

EmAgB3b/c Shows Differential Oligomeric Assembly Patterns in Distinct Histological Compartments of E. multilocularis Metacestode High transcription levels of EmAgB3a during the early stages of AE development from 18


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oncosphere to 5-month-old have been reported.29 In this study, abundant transcripts and proteins of paralogous EmAgB3s in 9-month-old AE cysts were also detected (Table 1 and Figure 4A), which suggested that EmAgB3s might play crucial roles during adaptation and subsequent survival of AE in the host environments. Furthermore, EmAgB3b/c has a specific antibody reactivity against patients’ sera from active-stage AE.28 Those observations prompted us to characterize the biological and biochemical properties of EmAgB3b/c. We examined whether respective antibodies generated against each recombinant protein had cross reactivity to other EmAgB isoforms. Each antibody specifically reacted to the corresponding rEmAgB protein, but not with other isoforms (Supplementary Figure S2). Employing this mono-specific antibody reactive to rEmAgB3b/c, we comparatively delineated expression patterns of EmAgB3b/c in individual histological compartments of E. multilocularis and E. granulosus metacestodes. The GST-removed recombinant protein detected at 8 kDa accorded well with its theoretical molecular mass and demonstrated that the immune signal observed at 8 kDa represented a genuine monomeric EmAgB3b/c protein (Figure 5A). In the protoscolex, EmAgB3b/c appeared as a 64 kDa oligomeric form (possibly octamer). The germinal layer, which contained membrane-bound protoscoleces to some extents, exhibited a strong reactivity at 24 kDa protein and a weak reactivity at 64 kDa. A high immunoreactive signal at 24 kDa compared to that of protoscolex suggests strongly that large proportions of EmAgB3b/c might exist as a trimer form in the germinal layer. EmAgB3b/c predominantly existed as 6 and 8 kDa proteins in the EmHF. Faintly smeared bands of 16, 64, and ≈ 140 kDa, which might represent different oligomeric states, were also recognized. Conversely, proteins extracted from individual compartments of E. granulosus metacestode did not exhibit any positive reaction against anti-rEmAgB3b/c antibody. These collective results indicated that EmAgB3b/c is immunologically distinctive from E. granulosus ortholog (EgAgB3) and has distinct oligomeric structure depending on 19



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anatomical compartments of E. multilocularis metacestode under reducing conditions. We detected 6 kDa EmAgB3 through proteome analysis of EmHF (spot 48, Figure 1C). The molecule had a concurrent response to anti-rEmAgB3b/c antibody (Figure 5A and B). However, the gene(s) possibly encoding 6 kDa protein could not be retrieved from the transcriptome or the genome of E. multilocularis.22,24 We thought that the 6 kDa protein might be a degradation product or a partial proteolytic fragment of the 8 kDa molecule. We separated these two proteins by 2-DE and analyzed internal amino acid sequences. Nterminal amino acid sequences of the 6 and 8 kDa proteins were determined to be ISEIKHFF and DDEVTQT, respectively, which corresponded to amino acid positions of 36-43 and 2228 of the EmAgB3b/c polypeptide. Their Mr and pI values matched well with those predicted by in silico analysis (5581 Da with pI 9.46 and 7525 Da with pI 9.34, respectively; Figure 5C).

EmAgB3b/c Constitutes a Macromolecular Protein by Self-assembly We determined subunit compositions and oligomeric states of native EmAgB3b/c. Immunologically purified protein was confirmed to contain only EmAgB3b/c by LC-MS/MS. Four EmAgB3b/c specific peptides were identified, but none of which could potentially originated from other EmAgB isoforms including EmAgB3a (Table 2 and Supplementary Figure S3A-D). The protein disclosed two bands at 6 and 8 kDa under reducing conditions. A faintly stained 16 kDa band was additionally observed (EmAgB3b/c-ip, Figure 6A), which was in agreement with Western blot analysis (Figure 5A). Interestingly, only native EmAgB3b/c produced the 6 kDa fragment. When multimerized rEmAgB3b/c protein was separated under the same conditions, a single 8 kDa protein was observed (rEmAgB3b/c-m, Figure 6A). Immunoprecipitation analysis of multimerized rEmAgB3b/c with antirEmAgB3b/c antibody also revealed the 8 kDa protein, but not the 6 kDa species 20


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(rEmAgB3b/c, Figure 6A). We subsequently analyzed multimeric architectural structure of both native and recombinant EmAgB3b/c. EmHF separated by native-PAGE revealed two major bands at ≈ 75 kDa and ≈ 360 kDa, together with broad bands from ≈ 360 to > 520 kDa. Major fractions of the native EmAgB3b/c migrated to ≈ 360 kDa with smearing bands between ≈ 360 and 520 kDa (left panel, Figure 6B). Multimerized rEmAgB3b/c also showed migration pattern similar to that of the native protein. When we probed the same blot with anti-rEmAgB3b/c antibody, both proteins demonstrated strong immunoreactive signals at ≈ 360 kDa (right panel, Figure 6B). The multimerized rEmAgB3b/c also showed a single peak at ≈ 380 kDa by molecular sieve chromatography (data not shown). These results demonstrated that EmAgB3 might be able to form a multimer by self-aggregation. We assessed whether native macromolecular EmAgB3b/c could concurrently self-dissociate into the 6 and 8 kDa species by blue native-PAGE. The 6 and 8 kDa proteins dissociated from the 360 kDa macromolecule were evident (Figure 6C). When multimerized rEmAgB3b/c was separated by blue native-PAGE, however, a single 8 kDa species dissociated from the 360 kDa was detected (Supplementary Figure S4).

Tissue Distribution of EmAgB3b/c mRNA Transcript and Protein The tissue distribution pattern of EmAgB3b/c transcript was analyzed using Cy5-labeled specific sense and antisense primers (Figure 7). Strong positive signals were detected at the parenchymal regions of the protoscolex. Weak positive signals were also observed at the germinal layer with a punctuate fashion. When mRNA transcription levels of EmAgB3b/c in each compartment were detected, the transcripts were expressed mainly in the protoscolex. The germinal layer exhibited significantly lowered levels (P < 0.001) (Figure 7B and C). We next examined histological localization of EmAgB3b/c protein. AE sections 21



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treated with anti-rEmAgB3b/c antibody created a well-demarcated strong immune signal at tegumental areas of the protoscolex (e-h, Figure 8A. See also highlight view of g and h, Figure 8B). Germinal layer demonstrated a weak positive reaction (h, Figure 8B). In contrast, E. granulosus tissue sections incubated with anti-rEmAgB3b/c antibody did not exhibit any detectable positive reaction (i-l, Figure 8A and l, Figure 8B). Preimmune mouse serum (negative control) also did not show any positive signal (a-d, Figure 8A and d, Figure 8B).

EmAgB3b/c has Differential Hydrophobic-ligand-binding Specificity Compared to E. granulosus Ortholog Bacterial lipid contents were removed through Octyl-Sepharose 4 (C8)-coupled or LH-beads (C3) reverse-phase hydrophobic interaction column. When lipid concentrations of mock E. coli vector and rEmAgB3b/c induced E. coli extracts were assayed, considerable amounts of free FA were detected (11.1 ± 0.73 and 9.7 ± 0.58 pM/µg protein). Those of GSH-affinity purified rEmAgB3b/c were 2.8 ± 0.28 pM/µg protein. Delipidation using C8 hydrophobic interaction completely abolished bacterial free FA contents. Significant amount of lipid remnants were also depleted by LH-beads (0.3 ± 0.04 pM/µg protein) (Figure 9). The precleared rEmAgB3b/c showed somewhat lowered binding activity toward 16-AP, which estimated to be approximately 80-85% of delipidated rEmAgB3b/c through either LH-beads or Octyl-Sepharose 4 column (Supplementary Figure S5A-D). Those delipidated proteins were used for HLBP activity assay employing a panel of FA analogues. The C8-column delipidated rEgAgB3 was used as a control for comparison. We also included TsM 150 kDa protein complex with strong HLBP activity against several molecular ligands as a positive control.27 Our results indicate that rEmAgB3b/c bound to 1,8ANS, cPnA, and 16-AP. Such binding significantly increased the fluorescent emission spectra in a dose-dependent manner (Figure 10A-C). However, rEmAgB3b/c did not bind to 22


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DAUDA or DACA (Supplementary Figure S5E and F). rEgAgB3 only displayed binding activity toward 16-AP (Figure 10D) while TsM 150 kDa HLBP (2 µg) had strong binding activities toward all substrates examined (Figure 10A-D and Supplementary Figure S5). The FA binding specificity of rEmAgB3b/c was further analyzed by evaluating the displacement effect of non-fluorescent saturated and unsaturated FAs. When rEmAgB3b/c bound to 1,8-ANS or 16-AP was subjected to competitive displacement, protein-substrate binary complex was displaced by oleic acid in a dose-dependent fashion. rEgAgB3:16-AP showed similar displacement activity by oleic acid (Figure 10E-G). Linoleic acid also demonstrated competitive binding activity similar to oleic acid, but its effect was lower than that of oleic acid (Supplementary Figure S6A-C). Conversely, saturated FAs (myristic acid and palmitic acid) did not induce competitive displacement (Supplementary Figure S6D-G). The Kd value of 1,8-ANS, cPnA, and 16-Ap bound to rEmAgB3b/c was calculated as 8.4 ± 0.24, 10.8 ± 0.68, and 0.34 ± 0.01 µM, respectively. That of rEgAgB3 against 16-AP was 0.36 ± 0.01 µM. The number of binding sites (n) per monomeric rEmAgB3b/c against 1,8ANS, cPnA, and 16-AP was estimated to be 3.20 ± 0.24 (r = 0.9917), 4.42 ± 0.32 (r = 0.9826), and 0.36 ± 0.03 (r = 0.9923), respectively (Figure 11A-D). Scatchard-plot derived from titration experiments exhibited a straight line, depicting a single binding site mode. Kinetic parameters for the binding reaction of rEmAgB3b/c and rEgAgB3 are summarized in Table 3.

DISCUSSION Several EgAgB proteins have been extensively investigated to understand their highly complicated gene expression patterns and multifaceted biochemical and biological properties.15-21,26,32 However, those of EmAgBs have limitedly been addressed, which might be attributable to extreme difficulty in collecting sufficient amount of relevant biological 23



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samples. Whether EmAgBs are present in EmHF obtained in vitro or in vivo remains contentious issue. EmHF derived from in vivo or in vitro cultures in the presence of or absence of feeder cells/fetal calf serum exhibited different protein expression patterns, as well as different immune recognition profile against infection sera.33,34 EmHF cultured in vivo or in vitro under the influence of host factors exhibited antibody responses against lowmolecular weight proteins, although presence of EmAgB isoforms was not determined. EmHF obtained from in vitro culture without host factors did not show antibody responses against low-molecular weight proteins. In vitro cultured E. multilocularis metacestodes seemed not secret EmAgB isoforms,8 while EmHF collected from in vivo cultured AE cysts harbor and secret significant amounts of diverse EmAgBs.28 We conducted proteome analysis of the EmHF employing fully mature AE cysts grown for 9-month in immunocompetent mice for the first time. We identified 40 parasite proteins, in which EmAgB isoforms constituted the most abundant population (19/40 spots, 47.5%). Presence of diverse antigen B isoforms was similarly detected what observed in the EgHF proteome.24,26,35,36 However, the expression patterns of EmAgB isoforms differed from those of EgAgBs. In this study, EmAgB3 constituted the major portion, while EmAgB1 composed a relatively minor fraction, which was completely reversed for EgAgBs.26,36 EmAgB5 protein was hardly observed along with its low transcription levels. Both EgAgB5 and EmAgB5 were found to be abundantly expressed in the adult, but not in the metacestode, stage.35,37,38 Other proteins identified included NAAALAD2, protease inhibitor, glycoprotein antigen 5, carbohydrate metabolizing enzymes, and antioxidant proteins. A recent transcriptome analysis of activated oncosphere, and 1- and 5-month-old metacestode vesicles demonstrated that different EmAgB isoforms were differentially regulated during those periods.29 The order of their expression levels from high to low was: EmAgB3a>EmAgB1>EmAgB4>EmAgB3b/c>EmAgB2>EmAgB5. Among EmAgB3 paralogs, 24


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expression levels of EmAgB3a was 20-40 times higher than those of EmAgB3b and EmAgB3c.29 These transcription patterns appeared not to match well with the present results of EmAgBs. For example, as is shown in Figure 4B, higher expression of EmAgB3b/c than EmAgB3a was evident by qPCR and RT-PCR analyses. The transcription level of EmAgB1 was minimal, which was reportedly to be abundant during the early developmental stages.29 These apparent discrepancies might be attributable to AE samples used. We utilized fully mature 9-month-old metacestode vesicles; at that stage, transcriptional patterns were rearranged with a high level relation of EmAgB2>EmAgB4>EmAgBb/c>EmAgB3a>EmAgB5>EmAgB1. Relative abundance of respective EmAgB isoforms in EmHF also correlated well with this transcription level order, except for EmAgB3a. However, despite lowered transcription of EmAgB3a, AE metacestode vesicles might possess considerable amounts of EmAgB3a protein by continuous accumulation of previously synthesized protein within EmHF (Figure 4A). The continuously abundant expression of EmAgB3 through switching of its closelyrelated paralogous genes might be associated with important biological roles, including induction of specific antibody responses28 and recruitment of essential lipids at the hostparasite interface (Figure 10). EgAgB isoforms, except for EgAgB3, are synthesized mainly in the germinal layer. EgAgB3 is produced in the protoscolex,39 but minimal amounts are secreted into EgHF, resulting in negligible antibody responses against sera of CE patients.26 In contrast to EgAgB3, the expression of EmAgB3a may commence from an early stage of metacestode development while the expression of EmAgB3b and/or EmAgB3c may start following maturation of the metacestode vesicle (i.e. after formation of brood capsule).28,29 Indeed, AE metacestode vesicles grown for 3-month produced developing immature and mature protoscoleces within brood capsules.28,40 Antibody responses culminated to EmAgB3b/c have been shown to begin after 6-month postinfection in murine AE system.28 25



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A total of 19 EmAgB isoforms detected in EmHF are originated from four homologous EmAgB1-EmAgB4 genes (Table 1 and Figure 3). Together with EmAgB5, those EmAgB homologs are tandemly arrayed in narrow chromosomal segments. Analysis of the genomic sequences showed that EmAgB3s were multiplied into three copies in a relatively recent evolutionary time. Interestingly, two of them (EmAgB3b and c) had identical sequences in their intervening intron and short 5′- and 3′-flanking regions including untranslated regions and CDS (Supplementary Figures S1 and S2). They could not be differentiated from each other at transcription level. Gene duplication is a genetic/genomic event widely observed in diverse eukaryotic genomes. However, genetic redundancy resultant from gene duplication is harmful to cells/organisms. The event may undergo rapid deletion of a single copy or short-period neutral evolution to increase sequence divergence.41,42 Relative expression levels should be intensely different between duplicated genes. Co-expression of duplicated genes has occasionally been observed if the duplication event has occurred during the most recent evolutionary time.43 Further analysis to define the start and end points of the first and second exons of paralogous EmAgB3b/c, which might include some diverged nucleobases is needed to characterize the expression levels of duplicated genes with identical sequences in CDS. Revelation of molecular mechanism underlying differential transcription regulation of those closely-related paralogous genes should form the basis for future studies. It was noteworthy that we detected abundant numbers of NAALAD2 (9 proteins) in the EmHF proteome. NAALAD2 is involved in exacerbation of cancer microenvironments and metastasis of lesions.44 One of the typical features of AE is its capacity of invading adjacent tissue and distant metastasis. The relatively large amounts of NAALAD2 expression might be associated with clinical characteristics of AE which resembles primary hepatocellular carcinoma. 26


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The macromolecular EmAgB3b/c protein consisted mainly of ≈ 360 kDa populations. The protein demonstrated dissimilar oligomeric states according to distinct anatomical compartments of E. multilocularis metacestode. Those oligomeric forms were tightly linked and could not be readily dissociated into monomer form and suggested strongly that EmAgB3b/c might have different stability or resistance to reducing or denaturing conditions within individual compartments (Figure 5A). However, EmAgB3b/c is reportedly expressed as an 8 kDa subunit under reducing conditions, irrespective of its different cellular distribution in AE metacestode.37 We surmised that such an unexpected observation might have originated from incomplete reduction of protein samples during preparation. We examined the effects of various reducing agents with different reducing conditions. Protein profiles of respective compartments using soluble proteins prepared with final concentrations of 100 mM DTT, 10% 2-mercaptoethanol, 4% CHAPS, 4 M urea, or 10 mM reduced glutathione failed to reveal 6 or 8 kDa protein in the protoscolex or germinal layer fraction (Supplementary Figure S7). Those two 6 and 8 kDa proteins were only observable in EmHF. This seeming inconsistency is difficult to explain properly. However, one of the major differences between the two studies is the use of differently prepared parasite materials. The previous study used protoscolex and AE cysts obtained from severely combined immunodeficient (NOD/Shi-scid) mice,45 in which host-derived active immune molecules that affect host-parasite specific interactions could be deficient. Under such circumstances, expression and behavior patterns of bioactive genes/proteins might be altered because functional differentiation and development of metacestode vesicles would be substantially different in response to host stimuli.28,33,46 The parasite cannot survive quite a long period, but maintained for only a few weeks in the absence of host factors.47To clearly elucidate this ambiguous issue, more comprehensive studies are warranted using AE materials derived from diverse culture conditions. 27



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We currently do not know whether EmAgB3b/c exists as an independent monomeric form in physiological in vivo conditions. When considering anaerobic reducing states of the host environment featuring, it might be conceivable. The native EmAgB3b/c protein was readily dissociated into the 6 and 8 kDa monomers in EmHF under reducing conditions. However, when we separated self-multimerized rEmAgB3b/c under the same conditions, the 6 kDa protein was not detected. Only the 8 kDa species was evident. This result suggested that cleavage of 8 kDa into 6 kDa fragment might not be a simple abiotic degradation process, but rather might be mediated by specific cleavage machinery. We could not identify proteolytic enzymes during EmHF proteome analysis, but several species of cathepsins (B, D, and L) are present in the transcriptome of E. multilocularis metacestode (http://www.genedb.org/Query/quickSearch?pseudogenes=true&product=true&allNames=tru e&searchText=cathepsin&taxons=Emultilocularis). Those cysteine/aspartic proteases might play roles in post-translational modification of EmAgB3b/c. To assess the biochemical properties of lipid-binding proteins of helminth parasites, such as HLBPs,17,18,27 fatty-acid-binding proteins (FABPs),48 and polyprotein allergen,49 proper delipidation is a prerequisite. If lipids contaminated the proteins, binding capacity should be decreased by competitive displacement of ligand binding sites by preoccupied lipids. We used two independent methods for delipidation of rEmAgB3b/c, rEgAgB3, and TsM HLBP. rEmAgB3b/c delipidated by either C8- or C3-based hydrophobic interaction showed exactly the same Kd values and number of binding sites per monomer (Table 3 and Figure 11). These results demonstrated that, although C8 chromatography might have better capacity in eliminating lipids than C3 beads,49 C3 hydrophobic interaction is also suitable for delipidation of bacterial lipid contents. Lipids are internalized by transporter proteins, such as HLBPs and FABPs. The recruited lipidic molecules play important roles in parasitic pathobiology.27,48,50 HLBPs of 28


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cestode parasites have some interesting features. Firstly, they typically display overlapping and/or non-overlapping properties toward different types of ligands. EgAgB1-3 bind to 16AP.17,18 A 7 kDa subunit protein of the TsM HLBP (TsMRS1) binds to DAUDA and DACA, but not to 16-AP. Conversely, 10 kDa subunits of the TsM HLBP (CyDA, b1, and m13h) interact only with 16-AP.50 An excretory 10 kDa HLBP of T. solium adult (TsHLBP1) binds to 1,8-ANS, cPnA, and 16-AP, but not to DAUDA or DACA.51 A 8 kDa HLBP of Moniezia expansa has shown binding affinity to DAUDA, DACA, 1,8-ANS, and 16-AP.52 rEmAgB3b/c had differential ligand binding activity; i.e. rEmAgB3b/c bound to 1,8-ANS, cPnA, and 16-AP, but not DAUDA or DACA (Figure 10 and Supplementary Figure S5). rEmAgB3b/c also demonstrated differential displacement effects against FA analogs. Secondly, those proteins have high affinity to long-chain unsaturated FAs, except for arachidonic acid (20:4) which had negligible affinity to saturated FAs. Arachidonic acid did not bind to cestode HLBPs. The parasites might not take up arachidonic acid as they are able to convert linoleic acid to arachidonic acid.53 Alternatively, these parasites might utilize another transporter system, such as FABPs.48,54 The differential binding affinity might be related to the characteristic feature of the FA composition of their host organisms because the parasites take up considerable amounts of lipid molecules from their hosts.27,55 The diverse primary structures of those orthologous/homologous proteins might also have influenced their specialized functions during the evolution of the proteins.48,50 The unique features of lipid moiety of several cestode HLBPs deserve more attention to properly evaluate their biochemical and biological impacts. These HLBPs may be useful as target for developing novel chemotherapeutics or vaccines by impeding worm’s lipid metabolism, which are pertinent to maintain parasitic homeostasis within the hosts.

29



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Conclusions AE represents one of the most frequently chronic and fatal enzootic helminthiasis. This study determined the EmHF proteome and analyzed expression patterns and biochemical properties of EmAgB3b/c, which appeared to be a specific biomarker for active stage AE.28 Among 60 proteins identified, 40 proteins were found to be derived from the parasite, in which EmAgBs (EmAgB1-4) constituted most abundant 19 isoforms. Expression profiles of EmAgB isoforms were quite different from those of EgAgBs. EmAgB3s comprised seven isoforms (four EmAgB3a and three EmAgB3b/c), whose expression levels were differentially regulated during the development of AE cysts. EmAgB3 isoforms were profoundly expressed by switching closely-related, but distinct paralogous genes. EmAgB3b/c was synthesized mainly in the parenchyme of the protoscolex and subsequently migrated to the tegumental surface followed by secretion into the EmHF. The protein is expressed partly in the germinal layer. EmAgB3b/c could be self-polymerized into a high-order macromolecule (≈ 360 kDa). In distinct compartments of the metacestode vesicle, EmAgB3b/c might undergo different conformational changes and shows dissimilar oligomeric states, which was highly resistant to reducing or denaturing conditions. EmAgB3b/c bound to 1,8-ANS, cPnA, and 16-AP, but not DAUDA or DACA, and demonstrated differential displacement against FA analogs. EmAgB3b/c preferred long-chain unsaturated fatty acids. EmAgB3b/c might be importantly involved in the recruiting and sequestration of hydrophobic substances, as well as in inducing specific antibody responses, thereby contributing to molecular and cellular functions at the host-parasite interface. Delineating the biochemical features and action mechanisms of bioactive molecule(s) involved in parasitic homeostasis offers insight into exploring novel intervention strategies against life-threatening AE.

ASSOCIATED CONTENT 30


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Supporting Information Table S-1: Oligonucleotide primers used in this study. Table S-2: Identification of proteins from hydatid fluid of E. multilocularis metacestode by MALDI-TOF MS. Table S-3: Identity values (%) among 8 kDa EmAgB isoforms (RNA/protein). Figure S-1: Comparison of nucleotide sequences of EmuJ_000381600 (EmAgB3b) and EmuJ_000381700 (EmAgB3c) retrieved from the draft genome database of Echinococcus multilocularis (ori) and empirically determined from the genomic DNA (seq). Figure S-2: The mono-specific antibody reactivity of polyclonal antibodies generated against rEmAgB1-5. Figure S-3: Identification of EmAgB3b/c specific peptide fragments of the native EmAgB3b/c purified by immunoprecipitation with LC-ESI-MS/MS. Figure S-4: Subunit composition of the multimerized rEmAgB3b/c determined by blue native-PAGE. Figure S-5: Changes of hydrophobic-ligand-binding activity of rEmAgB3b/c following delipidation process. Figure S-6: Displacement of 1,8-ANS and 16-AP from rEmAgB3b/c:fatty acid binary complexes. Figure S-7: Subunit compositions of EmAgB3b/c in distinct anatomical compartments of E. multilocularis metacestode under different reducing conditions.

AUTHOR INFORMATION **email address: [email protected], Tel: +82-31-299-6251, Fax: +82-31-299-6269 Author Contributions YK & CSA – conceived of the study, designed experiments, and prepared the manuscript, 31



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which was approved by all authors. CSA, JGK & WJP – performed molecular biological and biochemical experiments. YAB & IK – conducted genomic analysis. XH & HW – experimentally infected AE protoscolex into mice. CSA, JGK, YAB, IK & YK – analyzed data. Funding Sources This work was supported by NRF-2013R1A1A2059462. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENT The authors are sincerely grateful to Prof. Y. Nawa for his suggestions and critical review of the manuscript.

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39



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Table 1. Identification of parasite proteins in hydatid fluid of E. multilocularis metacestode by MALDI-TOF MS (EmAgB isoforms are bolded). Spot

MSa

SCb

MPc

Access. no.d Protein name

Mr/pI

no.

SPe

(EmuJ_)

2

75

34

10/51

49531/5.04

000317300

VAT

-

8

73

17

26/95

31345/5.72

000849600

I25

+

9

70

65

13/106

31345/5.72

000849600

I25

+

10

39

32

6/78

21359/5.18

000538000

GST

-

11

70

56

7/70

23961/5.36

000904500

Conserved protein

-

12

129

65

14/93

22758/5.00

000315600

Expressed protein

+

14

35

44

4/95

8644/5.60

000121100

Hypothetical protein

-

15

266

53

35/87

85250/6.61

000908900

NAALAD2

+

16

309

56

37/80

85250/6.61

000908900

NAALAD2

+

17

346

54

41/80

85250/6.61

000908900

NAALAD2

+

18

188

45

26/80

85250/6.61

000908900

NAALAD2

+

19

169

37

25/80

85250/6.61

000908900

NAALAD2

+

20

189

37

27/80

85250/6.61

000908900

NAALAD2

+

21

126

32

21/80

85250/6.61

000908900

NAALAD2

+

22

204

45

28/80

85250/6.61

000908900

NAALAD2

+

23

156

35

24/80

85250/6.61

000908900

NAALAD2

+

28

48

23

11/83

53827/8.51

000523300

MP2

-

29

112

41

17/80

55614/6.21

000184900

GPAg5

+

30

67

38

14/126

55614/6.21

000184900

GPAg5

+

31

39

40

6/81

15871/8.29

000360300

Trx

-

32

100

76

11/80

10460/8.39

000381400

Antigen B4

+

40


Page 41 of 62

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

42

63

6/69

10137/8.91

000381600

Antigen B3

+

34

45

64

8/87

10330/9.48

000381100

Antigen B2

+

36

63

70

6/74

10460/8.39

000381400

Antigen B4

+

37

68

64

8/65

10460/8.39

000381400

Antigen B4

+

38

104

96

9/75

10460/8.39

000381400

Antigen B4

+

39

51

61

7/80

10460/8.39

000381400

Antigen B4

+

40

57

60

7/78

10330/9.48

000381100

Antigen B2

+

41

42

69

5/62

9844/9.25

000381200

Antigen B1

+

42

52

52

9/79

10137/8.91

000381600

Antigen B3

+

43

47

78

7/84

10066/9.36

000381500

Antigen B3

+

44

80

65

12/75

9844/9.25

000381200

Antigen B1

+

45

40

55

6/93

10330/9.48

000381100

Antigen B2

+

46

42

69

4/52

10330/9.48

000381100

Antigen B2

+

47

46

75

6/78

9844/9.25

000381200

Antigen B1

+

48

68

71

10/57

9920/9.36

000381600

Antigen B3

+

49

59

72

9/75

10066/9.36

000381500

Antigen B3

+

57

58

39

9/75

40099/8.03

000905600

FBA

-

59

27

39

4/47

10066/9.36

000381500

Antigen B3

+

60

45

64

8/87

10066/9.36

000381500

Antigen B3

+

Mascot score, protein scores greater than 43 are significant (P < 0.05).

b c

33

Sequence coverage (%)

Matched peptides/Total peptides

d

Accession numbers were identified from NCBI DB (http://www.ncbi.nlm.nih.gov/) and

Sanger DB (http://www.genedb.org/Homepage/Emultilocularis). e

Signal peptide 41



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Abbreviations used: VAT, vesicular amine transporter; I25, proteinase inhibitor I25; NAALAD2, N-acetylated alpha-linked acidic dipeptidase 2; GST, glutathione S-transferase, MP2, mechanosensory protein 2; GPAg5, glycoprotein Antigen 5; Trx, mitochondrial thioredoxin; FBA, fructose 1,6 bisphosphate aldolase.

42


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Table 2. Identification of native EmAgB3b/c by LC-ESI-MS/MSa. Category

Description

Mascot score

107

Sequence coverage (%)

50

No. of matched peptide

4

Theoretical Mw/pI

9920/9.36

Protein ID

Tapeworm specific antigen B

Accession no.

EmuJ_000381600 (CDS36679)/EmuJ_000381700 (CDS36680)

Matched sequence

52LVEVMKEVGSVCQMVR67, 21DDDEVTQTK29, 72MALKEYVR79, 41HFFQSDPLGK50

a

Individual ion scores > 43 indicate significant or extensive homology (P < 0.05).

43



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Table 3. Kinetic parameters for the binding reaction of rEmAgB3b/c and rEgAgB3 toward 1anilinonapthalene 8-sulfonic acid (1,8-ANS), 16-(9-anthroyloxy) palmitic acid (16-AP), and cis-parinaric acid (cPnA). Ligand

Parameters

rEmAgB3b/c

rEgAgB3

1,8-ANS

Vmaxa

2329.2 ± 24.6

NDb

Kd (µM)

8.4 ± 0.24

ND

Vmax/Kd

276.8 ± 7.3

ND

Vmax

272.2 ± 1.64

289.2 ± 0.4

Kd (µM)

0.34 ± 0.01

0.36 ± 0.01

Vmax/Kd

801.1 ± 20.8

803.7 ± 19.4

Vmax

2779.4 ± 12.2

ND

Kd (µM)

10.8 ± 6.8

ND

Vmax/Kd

259.0 ± 14.2

ND

16-AP

cPnA

a

The rate was calculated by measuring relative fluorescence of each reaction.

b

Not-detected.

44


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Legend for Figures

Figure 1. Proteome analysis of E. multilocularis hydatid fluid (EmHF). (A and B) EmHF (200 µg) was electrofocused on IPG strips (pH 4-7 and pH 6-11; each 13 cm-long) and further separated by 10% Tricine SDS-PAGE. Gels were stained with Coomassie brilliant blue G-250 (CBB). The spots were subjected to in-gel trypsin digestion followed by MALDITOF MS analysis. Proteins identified are marked by Arabic numbers (1-60) and are summarized in Table 1 and Supplementary Table S1. (C) Diverse isoforms of E. multilocularis antigen B (EmAgB1-4) (marked by dotted box in B) are shown in highlight view. Mr, molecular weight in kDa; pI, isoelectric points.

Figure 2. Analysis of gene ontology of E. multilocularis hydatid fluid (EmHF) proteins by functional categories. The percentage of identified proteins in each functional group is shown in histogram. The numerals within bars indicate the number of each protein. Terms associated with cellular components, molecular functions, and biological processes were tailored from Blast2GO on the basis of similarity pattern employing the second-level of GO hierarchy.31

Figure 3. Chromosomal map of E. multilocularis antigen B (EmAgB) genes. The nucleotide sequence between 654901 and 665173 (dotted-box) encompassing three EmAgB3 paralogs were used for self-to-self comparison with BL2Seq program. Graphical result is shown in inset. Dotted-arrows indicate transcriptional direction. Red arrows marked with PCR (1) and (2) indicate regions amplified by PCR to empirically determine their nucleotide sequences (See also Supplementary Figure S2). We could not differentially identify EmuJ_000381600 and EmuJ_000381700 due to their identical sequences. We designated EmuJ_000381600 as EmAgB3b (marked by 600) and EmuJ_000381700 as EmAgB3c (denoted as 700). 45



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

EmuJ_000381500 was named EmAgB3a (marked as 500). MTA1, metastasis associated protein 1.

Figure 4. Expression profile of E. multilocularis antigen B (EmAgB) isoforms at 9-month postinfection. (A) Comparison of relative spot intensity of diverse EmAgB isoforms. Density ratio of image spots was quantified with ImageJ software (http://imagej.nih.gov/ij/). Relative spot density of sum of paralogous EmAgB3 was regarded as 100%. mRNA expression levels of individual EmAgBs were amplified by qPCR (B) and RT-PCR (C) employing genespecific primers. The relative expression levels of EmAgB genes compared to control (actin) were calculated based on differences in threshold cycles (Ct) by qPCR. E. multilocularis actin gene (EmuJ_000407400) was used as an internal control for the normalization of qPCR. Error bars represent standard deviations of three independent experiments. *P < 0.05. Gene numbers used: EmAgB1, EmuJ_000381200; EmAgB2, EmuJ_000381100; EmAgB3a, EmuJ_000381500; EmAgB3b/c, EmuJ_000381600/EmuJ_000381700; EmAgB4, EmuJ_000381400; EmAgB5, EmuJ_000381800; Actin, EmuJ_000407400.

Figure 5. Differential expression patterns of E. multilocularis antigen B3b/c (EmAgB3b/c) in distinct anatomical compartments of the metacestodes. (A) Expression patterns of EmAgB3b/c in individual compartments of E. multilocularis (Em) and E. granulosus (Eg) metacestodes. Proteins (10 µg each) were separated by 10% Tricine SDS-PAGE under reducing conditions, electroblotted to nitrocellulose membrane, probed with antirEmAgB3b/c antibody (1:2000 dilution), and subsequently incubated with HRP-conjugated anti-mouse IgG antibody (1:4000 dilution). Signals were detected by ECL. EmHF, Em hydatid fluid; PSC, protoscolex; GL, germinal layer; EgHF, Eg hydatid fluid; rEmAgB3b/c, GST-removed delipidated rEmAgB3b/c. Mr, relative molecular mass in kDa. (B) Two46


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DE/immunoblotting of EmAgB3b/c. EmHF (200 µg) was electrofocused on IPG strip (pH 611, 13 cm-long), separated by 10% Tricine-SDS-PAGE, and transferred to nitrocellulose membrane. The blot was probed with anti-rEmAgB3b/c antibody, after which immune signal was detected with ECL. Spot numbers are the same as described in Figure 1. (C) Protein spots of 6 and 8 kDa separated by 2-DE were subjected to N-terminal amino acid sequencing. ISEIKHFF from the 6 kDa and DDEVTQT from the 8 kDa (gray italicized letters) were obtained by Edman degradation. Signal peptide is underlined. Molecular weights and isoelectric point of each fragment were theoretically deduced with ExPASy program (http://web.expasy.org/compute_pi/).

Figure 6. Subunits and multimeric states of native and recombinant E. multilocularis antigen B3b/c (EmAgB3b/c). (A) SDS-PAGE of native and recombinant EmAgB3b/c under reducing conditions. EmHF (10 µg), native EmAgB3b/c eluted by immunoprecipitation (EmAgB3b/cip; 3 µg), GST-removed, delipidated rEmAgB3b/c multimerized by incubation at 25°C for 2 h (rEmAgB3b/c-m; 3 µg), and rEmAgB3b/c (3 µg) immunologically precipitated with antirEmAgB3b/c antibody were separated by 10% Tricine SDS-PAGE and stained with CBB. Closed and open circles, IgG heavy- and light-chains, respectively. Mr, molecular weight in kDa. (B) Macromolecular status of native and recombinant EmAgB3b/c by native-PAGE. EmHF (10 µg), native EmAgB3b/c purified by immunoprecipitation (EmAgB3b/c-ip; 3 µg), and multimerized rEmAgB3b/c (rEmAgB3b/c-m; 3 µg) were electrophoresed on 4-20% native-PAGE and visualized by CBB staining (left panel). The proteins were transblotted to nitrocellulose membrane and probed with anti-rEmAgB3b/c antibody (right panel). (C) Subunit compositions of the native EmAgB3b/c eluted through immunoprecipitation (3 µg) were determined by blue native-PAGE followed by immunoblotting. First dimension electrophoresis was done on 4-20% native PAGE and the gel strip was further separated by 47



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

10% Tricine SDS-PAGE (second dimensional electrophoresis). Proteins were transferred to nitrocellulose membrane, probed with anti-rEmAgB3b/c antibody (1:2000 dilution), and further incubated with 1:4000 diluted HRP-conjugated anti-mouse IgG antibody. The signals were detected by ECL. Mr, relative molecular mass in kDa.

Figure 7. In situ hybridization and transcriptional profiles of E. multilocularis antigen B3b/c (EmAgB3b/c) in different tissue compartments. (A) Tissue sections of E. multilocularis metacestode (4-µm thick) were hybridized with Cy5-labeled EmAgB3b/c-specific sense and antisense probes. The mRNA transcripts of EmAgB3b/c were detected by antisense probe (red). Counterstaining was done using 4',6-diamidino-2-phenolindole (DAPI; blue). The signals were observed by confocal laser scanning microscopy. Bar denotes 50 µm. (B) Expression levels of EmAgB3b/c in different histological compartments of AE metacestode at 9-month postinfection. Total RNAs (200 ng) extracted separately from whole worm (WW), protoscolex (PSC), and germinal layer (GL) were analyzed by RT-PCR. (C) Comparison of expression levels of EmAgB3s in different compartments of AE cysts grown for 9-month was quantitated by RT-PCR. Graphs show mean ± S.D. of three independent experiments. ***, P < 0.001.

Figure 8. Immunohistochemical localization of E. multilocularis antigen B3b/c (EmAgB3b/c). (A) Tissue sections (4-µm thick) of the E. multilocularis (e-h) and E. granulosus metacestodes (i-l) were incubated with anti-rEmAgB3b/c antibody (1:400 dilution) followed by incubation with FITC-conjugated anti-mouse IgG (1:1000 dilution). Slides were counterstained with 4',6-diamidino-2-phenolindole (DAPI). Preimmune mouse serum was used for control at the same dilution ratio (a-d). GL, germinal layer; PSC, protoscolex. Bar denotes 100 µm. (B) Each region marked by white-dotted boxes in A is 48


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shown in magnified view. PSC, protoscolex; GL, germinal layer; SU, sucker; TG, tegument; H, hooklet.

Figure 9. Colorimetric assay of bacterial lipid remnants of delipidated recombinant E. multilocularis antigen B3b/c (rEmAgB3b/c). Delipidation of bacterial fatty acids was done through Octyl-Sepharose 4 reverse-phase hydrophobic column (C8) or Sephadex-LH beads (C3). Each extract (2 µg/µl protein) was serially incubated in 96-well microtiter plate (0-10 µl) as described in the Materials and Methods (n = 3, mean ± S.D.). Closed circle, mock E. coli extract; open circle, rEmAgB3b/c induced E. coli extract; open triangle, GST-tag cleaved rEmAgB3b/c (precleared rEmAgB3b/c); closed rectangle, rEmAgB3b/c treated with Sephadex-LH beads; open rectangle, rEmAgB3b/c delipidated with Octyl-Sepharose 4 column. *P < 0.05; **P < 0.01. (Inset) Palmitic acid (1 nM/µl) control was used to establish standard curve.

Figure 10. In vitro hydrophobic-ligand-binding activity and displacement of protein-ligand binary complex with oleic acid (OA). (A-D) Increasing doses of rEmAgB3b/c or rEgAgB3 were incubated each with 10 µM 1-anilinonapthalene 8-sulfonic acid (1,8-ANS), cisparinaric acid (cPnA), or 16-(9-anthroyloxy) palmitic acid (16-AP) for 2 min. Fluorescence emission spectra were recorded at 25°C using black 96-well plates (Exmax/Emmax = 370/460 nm for 1,8-ANS, Exmax/Emmax = 315/420 nm for cPnA, and Exmax/Emmax = 360/460 nm for 16-AP). TsM 150 kDa HLBP was used as a positive control. (E-G) Displacement of 1,8-ANS and 16-AP bound to rEmAgB3b/c or rEgAgB3 by competitive binding of OA (2-8 µM). The displacement by OA is shown in histogram (n = 3-5, mean ± S.D.).

49



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Figure 11. Steady-state kinetics of 1,8-ANS (A), cPnA (B), and 16-AP (C) bound to rEmAgB3b/c and 16-AP bound to rEgAgB3 (D). Changes in fluorescence intensity of reaction mixtures containing rEmAgB3b/c (2 µg) and increasing doses of 1anilinonapthalene 8-sulfonic acid (1,8-ANS; 0.1-80 µM), cis-parinaric acid (cPnA; 0.05-20 µM), or 16-(9-anthroyloxy) palmitic acid (16-AP; 0.1-80 µM) were monitored. The curve was used to derive the equilibrium dissociation constant (Kd) for 1,8-ANS:rEmAgB3b/c, cPnA:rEmAgB3b/c, 16-AP:rEmAgB3b/c, and 16-AP:rEgAgB3 interactions. The curved lines represent theoretical binding with Kd by fitting experimental curves to a single-site binding model. The number of binding sites (n) per monomer is also shown.

50


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for TOC only

51



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 1. Proteome analysis of E. multilocularis hydatid fluid (EmHF). (A and B) EmHF (200 µg) was electrofocused on IPG strips (pH 4-7 and pH 6-11; each 13 cm-long) and further separated by 10% Tricine SDS-PAGE. Gels were stained with Coomassie brilliant blue G-250 (CBB). The spots were subjected to in-gel trypsin digestion followed by MALDI-TOF MS analysis. Proteins identified are marked by Arabic numbers (160) and are summarized in Table 1 and Supplementary Table S1. (C) Diverse isoforms of E. multilocularis antigen B (EmAgB1-4) (marked by dotted box in B) are shown in highlight view. Mr, molecular weight in kDa; pI, isoelectric points. 176x167mm (300 x 300 DPI)


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Figure 2. Analysis of gene ontology of E. multilocularis hydatid fluid (EmHF) proteins by functional categories. The percentage of identified proteins in each functional group is shown in histogram. The numerals within bars indicate the number of each protein. Terms associated with cellular components, molecular functions, and biological processes were tailored from Blast2GO on the basis of similarity pattern employing the second-level of GO hierarchy.31 184x151mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 3. Chromosomal map of E. multilocularis antigen B (EmAgB) genes. The nucleotide sequence between 654901 and 665173 (dotted-box) encompassing three EmAgB3 paralogs were used for self-to-self comparison with BL2Seq program. Graphical result is shown in inset. Dotted-arrows indicate transcriptional direction. Red arrows marked with PCR (1) and (2) indicate regions amplified by PCR to empirically determine their nucleotide sequences (See also Supplementary Figure S2). We could not differentially identify EmuJ_000381600 and EmuJ_000381700 due to their identical sequences. We designated EmuJ_000381600 as EmAgB3b (marked by 600) and EmuJ_000381700 as EmAgB3c (denoted as 700). EmuJ_000381500 was named EmAgB3a (marked as 500). MTA1, metastasis associated protein 1. 263x100mm (300 x 300 DPI)


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Figure 4. Expression profile of E. multilocularis antigen B (EmAgB) isoforms at 9-month postinfection. (A) Comparison of relative spot intensity of diverse EmAgB isoforms. Density ratio of image spots was quantified with ImageJ software (http://imagej.nih.gov/ij/). Relative spot density of sum of paralogous EmAgB3 was regarded as 100%. mRNA expression levels of individual EmAgBs were amplified by qPCR (B) and RT-PCR (C) employing gene-specific primers. The relative expression levels of EmAgB genes compared to control (actin) were calculated based on differences in threshold cycles (Ct) by qPCR. E. multilocularis actin gene (EmuJ_000407400) was used as an internal control for the normalization of qPCR. Error bars represent standard deviations of three independent experiments. *P < 0.05. Gene numbers used: EmAgB1, EmuJ_000381200; EmAgB2, EmuJ_000381100; EmAgB3a, EmuJ_000381500; EmAgB3b/c, EmuJ_000381600/EmuJ_000381700; EmAgB4, EmuJ_000381400; EmAgB5, EmuJ_000381800; Actin, EmuJ_000407400. http://imagej.nih.gov/ij/ 185x120mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 5. Differential expression patterns of E. multilocularis antigen B3b/c (EmAgB3b/c) in distinct anatomical compartments of the metacestodes. (A) Expression patterns of EmAgB3b/c in individual compartments of E. multilocularis (Em) and E. granulosus (Eg) metacestodes. Proteins (10 µg each) were separated by 10% Tricine SDS-PAGE under reducing conditions, electroblotted to nitrocellulose membrane, probed with anti-rEmAgB3b/c antibody (1:2000 dilution), and subsequently incubated with HRP-conjugated anti-mouse IgG antibody (1:4000 dilution). Signals were detected by ECL. EmHF, Em hydatid fluid; PSC, protoscolex; GL, germinal layer; EgHF, Eg hydatid fluid; rEmAgB3b/c, GST-removed delipidated rEmAgB3b/c. Mr, relative molecular mass in kDa. (B) Two-DE/immunoblotting of EmAgB3b/c. EmHF (200 µg) was electrofocused on IPG strip (pH 6-11, 13 cm-long), separated by 10% Tricine-SDS-PAGE, and transferred to nitrocellulose membrane. The blot was probed with anti-rEmAgB3b/c antibody, after which immune signal was detected with ECL. Spot numbers are the same as described in Figure 1. (C) Protein spots of 6 and 8 kDa separated by 2-DE were subjected to N-terminal amino acid sequencing. ISEIKHFF from the 6 kDa and DDEVTQT from the 8 kDa (gray italicized letters) were obtained by Edman degradation. Signal peptide is underlined. Molecular weights and isoelectric point of each fragment were theoretically deduced with ExPASy program (http://web.expasy.org/compute_pi/). http://web.expasy.org/compute_ 168x125mm (300 x 300 DPI)


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Figure 6. Subunits and multimeric states of native and recombinant E. multilocularis antigen B3b/c (EmAgB3b/c). (A) SDS-PAGE of native and recombinant EmAgB3b/c under reducing conditions. EmHF (10 µg), native EmAgB3b/c eluted by immunoprecipitation (EmAgB3b/c-ip; 3 µg), GST-removed, delipidated rEmAgB3b/c multimerized by incubation at 25°C for 2 h (rEmAgB3b/c-m; 3 µg), and rEmAgB3b/c (3 µg) immunologically precipitated with anti-rEmAgB3b/c antibody were separated by 10% Tricine SDS-PAGE and stained with CBB. Closed and open circles, IgG heavy- and light-chains, respectively. Mr, molecular weight in kDa. (B) Macromolecular status of native and recombinant EmAgB3b/c by native-PAGE. EmHF (10 µg), native EmAgB3b/c purified by immunoprecipitation (EmAgB3b/c-ip; 3 µg), and multimerized rEmAgB3b/c (rEmAgB3b/c-m; 3 µg) were electrophoresed on 4-20% native-PAGE and visualized by CBB staining (left panel). The proteins were transblotted to nitrocellulose membrane and probed with anti-rEmAgB3b/c antibody (right panel). (C) Subunit compositions of the native EmAgB3b/c eluted through immunoprecipitation (3 µg) were determined by blue native-PAGE followed by immunoblotting. First dimension electrophoresis was done on 4-20% native PAGE and the gel strip was further separated by 10% Tricine SDS-PAGE (second dimensional electrophoresis). Proteins were transferred to nitrocellulose membrane, probed with anti-rEmAgB3b/c antibody (1:2000 dilution), and further incubated with 1:4000 diluted HRP-conjugated anti-mouse IgG antibody. The signals were detected by ECL. Mr, relative molecular mass in kDa. 261x125mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 7. In situ hybridization and transcriptional profiles of E. multilocularis antigen B3b/c (EmAgB3b/c) in different tissue compartments. (A) Tissue sections of E. multilocularis metacestode (4-µm thick) were hybridized with Cy5-labeled EmAgB3b/c-specific sense and antisense probes. The mRNA transcripts of EmAgB3b/c were detected by antisense probe (red). Counterstaining was done using 4',6-diamidino-2phenolindole (DAPI; blue). The signals were observed by confocal laser scanning microscopy. Bar denotes 50 µm. (B) Expression levels of EmAgB3b/c in different histological compartments of AE metacestode at 9month postinfection. Total RNAs (200 ng) extracted separately from whole worm (WW), protoscolex (PSC), and germinal layer (GL) were analyzed by RT-PCR. (C) Comparison of expression levels of EmAgB3s in different compartments of AE cysts grown for 9-month was quantitated by RT-PCR. Graphs show mean ± S.D. of three independent experiments. ***, P < 0.001. 202x141mm (300 x 300 DPI)


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Figure 8. Immunohistochemical localization of E. multilocularis antigen B3b/c (EmAgB3b/c). (A) Tissue sections (4-µm thick) of the E. multilocularis (e-h) and E. granulosus metacestodes (i-l) were incubated with anti-rEmAgB3b/c antibody (1:400 dilution) followed by incubation with FITC-conjugated anti-mouse IgG (1:1000 dilution). Slides were counterstained with 4',6-diamidino-2-phenolindole (DAPI). Preimmune mouse serum was used for control at the same dilution ratio (a-d). GL, germinal layer; PSC, protoscolex. Bar denotes 100 µm. (B) Each region marked by white-dotted boxes in A is shown in magnified view. PSC, protoscolex; GL, germinal layer; SU, sucker; TG, tegument; H, hooklet. 341x182mm (300 x 300 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 9. Colorimetric assay of bacterial lipid remnants of delipidated recombinant E. multilocularis antigen B3b/c (rEmAgB3b/c). Delipidation of bacterial fatty acids was done through Octyl-Sepharose 4 reversephase hydrophobic column (C8) or Sephadex-LH beads (C3). Each extract (2 µg/µl protein) was serially incubated in 96-well microtiter plate (0-10 µl) as described in the Materials and Methods (n = 3, mean ± S.D.). Closed circle, mock E. coli extract; open circle, rEmAgB3b/c induced E. coli extract; open triangle, GST-tag cleaved rEmAgB3b/c (precleared rEmAgB3b/c); closed rectangle, rEmAgB3b/c treated with Sephadex-LH beads; open rectangle, rEmAgB3b/c delipidated with Octyl-Sepharose 4 column. *P < 0.05; **P < 0.01. (Inset) Palmitic acid (1 nM/µl) control was used to establish standard curve. 90x141mm (300 x 300 DPI)


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Figure 10. In vitro hydrophobic-ligand-binding activity and displacement of protein-ligand binary complex with oleic acid (OA). (A-D) Increasing doses of rEmAgB3b/c or rEgAgB3 were incubated each with 10 µM 1anilinonapthalene 8-sulfonic acid (1,8-ANS), cis-parinaric acid (cPnA), or 16-(9-anthroyloxy) palmitic acid (16-AP) for 2 min. Fluorescence emission spectra were recorded at 25°C using black 96-well plates (Exmax/Emmax = 370/460 nm for 1,8-ANS, Exmax/Emmax = 315/420 nm for cPnA, and Exmax/Emmax = 360/460 nm for 16-AP). TsM 150 kDa HLBP was used as a positive control. (E-G) Displacement of 1,8-ANS and 16-AP bound to rEmAgB3b/c or rEgAgB3 by competitive binding of OA (2-8 µM). The displacement by OA is shown in histogram (n = 3-5, mean ± S.D.). 263x163mm (300 x 300 DPI)



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Figure 11. Steady-state kinetics of 1,8-ANS (A), cPnA (B), and 16-AP (C) bound to rEmAgB3b/c and 16-AP bound to rEgAgB3 (D). Changes in fluorescence intensity of reaction mixtures containing rEmAgB3b/c (2 µg) and increasing doses of 1-anilinonapthalene 8-sulfonic acid (1,8-ANS; 0.1-80 µM), cis-parinaric acid (cPnA; 0.05-20 µM), or 16-(9-anthroyloxy) palmitic acid (16-AP; 0.1-80 µM) were monitored. The curve was used to derive the equilibrium dissociation constant (Kd) for 1,8-ANS:rEmAgB3b/c, cPnA:rEmAgB3b/c, 16AP:rEmAgB3b/c, and 16-AP:rEgAgB3 interactions. The curved lines represent theoretical binding with Kd by fitting experimental curves to a single-site binding model. The number of binding sites (n) per monomer is also shown. 142x149mm (300 x 300 DPI)


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