Acta Tropica 140 (2014) 34–40

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Opisthorchis viverrini: Analysis of the sperm-specific rhophilin associated tail protein 1-like Sitthichon Rattanachan a , Rudi Grams b , Smarn Tesana c , Peter M. Smooker d , Suksiri Vichasri Grams a,∗ a

Department of Biology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand Graduate Program in Biomedical Sciences, Faculty of Allied Health Sciences, Thammasat University, Pathumthani 12121, Thailand c Food-Borne Parasite Research Group, Department of Parasitology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand d School of Applied Sciences, RMIT University, Bundoora, Victoria 3083, Australia b

a r t i c l e

i n f o

Article history: Received 8 May 2014 Received in revised form 29 July 2014 Accepted 2 August 2014 Available online 11 August 2014 Keywords: Foodborne trematodiasis Opisthorchis viverrini ROPN1L Expression pattern Immune response

a b s t r a c t Concurrent deficiency of rhophilin associated tail protein (ROPN1) and ROPN1-like (ROPN1L) in mice causes structural abnormalities and immotility of sperm and thereby infertility. In the present research, ROPN1L of the human liver fluke Opisthorchis viverrini was molecularly characterized and showed unexpected potential as a diagnostic tool. ROPN1L transcripts were detected in 2-week-old juveniles by RT-PCR. Immunohistochemical analysis of the adult worm localized the protein in testis lobes, seminal vesicle and receptacle and immunoelectron microscopic analysis revealed its location on the tail of spermatozoa. Interestingly, sera of experimentally infected hamsters and sera of individuals suffering from opisthorchiasis showed reactivity to recombinant OvROPN1L (rOvROPN1L). The protein shows modest conservation to the human homolog at 47.2% sequence identity and a mouse anti-rOvROPN1L antiserum was not reactive with sperm protein extracts from hamsters, mice and rats. Unsurprisingly, conservation is higher in trematodes, e.g. 78.4% and 71.2% identity to Fasciola gigantica and Schistosoma haematobium, respectively and evaluation of diagnostic specificity is required using sera of individuals suffering from different trematodiases in Thailand. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Spermatogenesis, oogenesis and fertilization are important biological processes in the sexual reproduction of eukaryotic organisms. The life cycle of trematodes depends on the daily release of thousands of fertilized eggs during their reproductive stage and a decrease in fecundity would severely diminish their chances for survival. It is for this reason that gametogenesis and fertilization as well as the effects of anthelmintics on reproductive processes are important research areas in trematodiases. Ultimately, it is required to study the function of specific proteins involved in parasite reproduction, but surprisingly, there have been no reports on such proteins from trematodes as yet. In the important human fluke Opisthorchis viverrini gametogenesis and

∗ Corresponding author at: Department of Biology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok 10400, Thailand. Tel.: +66 2 201 5480; fax: +66 2 354 7161. E-mail address: [email protected] (S.V. Grams). http://dx.doi.org/10.1016/j.actatropica.2014.08.002 0001-706X/© 2014 Elsevier B.V. All rights reserved.

fertilization are essentially undescribed processes. We selected rhophilin associated tail protein 1-like (ROPN1L, or ropporin 1-like) of O. viverrini for application as a cell-type marker in the parasite due to its observed sperm-specific distribution in mammals and conservation in different animal phyla. In Mammalia, ROPN1L and the orthologous ROPN1 were located in the fibrous sheath of sperm and found to interact with A-kinase anchor protein 3 (AKAP3) through a N-terminal located domain also present in a few other proteins (Fujita et al., 2000; Carr et al., 2001; Brown et al., 2003). The properties of this domain are summarized under accession cd12084 in the National Center for Biotechnology Information (NCBI, National Library of Medicine, USA) Conserved Domain Database (CDD) as dimerization/docking domain of the regulatory subunit of cAMP-dependent protein kinase and similar domains (DD R PKA). Mice deficient in ROPN1L and ROPN1 produced immotile sperm with abnormal morphology and were infertile (Fiedler et al., 2013). While the exact biochemical mode of action of ROPN1L is at present unknown it has been suggested that it regulates the activity of AKAP3 which itself is important to target proteins, e.g. cAMP-dependent protein kinase, to specific

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subcellular areas (for a review of the role of AKAPs in mammalian reproduction see (Luconi et al., 2011)). In Thailand, O. viverrini has attracted intense scientific interest due to the connection of opisthorchiasis and cholangiocarcinoma, a bile duct cancer with a uniquely high prevalence rate in this country (Sripa et al., 2007). Among others, released antigens, tegumental surface antigens and egg antigens of the parasite have been analyzed for their application as diagnostic markers, targets for intervention or their role in tumorigenesis (Ruangsittichai et al., 2006; Pinlaor et al., 2009; Smout et al., 2009; Eursitthichai et al., 2010; Piratae et al., 2012; Sripa et al., 2012) and yet, as this research will demonstrate, antigens of the parasite sperm could also be involved in the host–parasite interaction and might warrant further attention.

2. Materials and methods 2.1. Parasites Metaceracariae of O. viverrini were collected from naturally infected cyprinoid fish and 2-, 4-, 6-, and 8-week-old parasites were collected from hamster (Mesocricetus auratus) experimentally infected by gastric intubation with 50 metacercariae each following the procedures as described in (Ruangsittichai et al., 2006). Blood samples were collected pre-infection and every 2 weeks postinfection from the orbital sinus and used to prepare sera which were stored at −20 ◦ C until used in western analysis and enzyme-linked immunosorbent assay (ELISA). Hamsters were euthanized using CO2 inhalation and had worm burdens ranging from 40% to 50%. The use of experimental animals in this study was approved by the Faculty of Science, Mahidol University Animal Care and Use Committee (22 April 2011, Project No. 204) and the animals were kept in compliance with the university guidelines. Metacercariae were mechanically excysted with disposable needles to obtain newly excysted juveniles (NEJ).

2.2. Isolation of a OvROPN1L cDNA and sequence analysis The 668 bp CDS of OvROPN1L was amplified from an adult stage cDNA library of O. viverrini (Eursitthichai et al., 2004) by standard polymerase chain reaction (PCR) with Taq polymerase and forward primer 5 -GCGGggatccATCGAGGGTCGCATGCCGCTTGTCAACGATCC-3 and reverse primer 5 -CCGCctgcagGCTCAGTCTAGTGGTTTGAG-3 . The primers were designed based on the sequence of an uncharacterized O. viverrini expressed sequence tag (EST) (GenBank: ES417466) containing the complete coding sequence (CDS) of OvROPN1L. The introduced BamHI and PstI restriction endonuclease sites (lower case) were used to subclone the cDNA fragment into the Escherichia coli expression vector pQE30 (QIAGEN, USA) by ligation using T4 DNA ligase (Promega, USA). Integrity of the cDNA was verified by sequencing and the deduced amino acid sequence was used in Basic Local Alignment Search Tool (BLAST)P searches to obtain the sequences of homologous proteins from Fasciola gigantica (contig 18487, http://research.vet.unimelb.edu.au/gasserlab/index.html) and Schistosoma haematobium (product of gene Sha 101854, http://www.schistodb.net/schisto/) databases. Human ROPN1L (UniProt: Q96C74) and ROPN1 (UniProt: Q9HAT0) sequences were directly received from the NCBI protein database. A multiple alignment of the sequences was calculated in Clustal Omega (Sievers et al., 2011), pairwise sequence identity and similarity values were calculated in European Molecular Biology Open Software Suite (EMBOSS) needle (Rice et al., 2000) with settings BLOSUM62 matrix, gap penalty 10, gap extension penalty 0.5; NCBI CDD was used to obtain information on conserved motifs in OvROPN1L.

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2.3. Reverse transcriptase (RT)-PCR with total RNA from developmental stages of O. viverrini Total RNA was extracted in TRIzol reagent (Invitrogen, USA) from metacercariae (N = 50), NEJ (N = 50), and 2-, 4-, 6-, and 8-weekold parasites (N = 50 each) following standard procedures. The extracted RNA was treated with DNase I (Fermentas Life Sciences, Lithuania) and 500 ng of each RNA sample was used as template for reverse transcription with the OvROPN1L-specific reverse primer 5 -GCTCAGTCTAGTGGTTTGAG-3 using a RevertAid reverse transcription kit (Fermentas Life Sciences, Lithuania). The RT-product was then used for standard PCR with Taq polymerase (Fermentas Life Sciences, Lithuania) and the OvROPN1L-specific forward primer 5 -ATGCCGCTTGTCAACGATCC-3 to yield the 669 bp CDS of OvROPN1L. A 298 bp cDNA fragment of OvGST was reverse transcribed and amplified in parallel as internal control as described in (Ruangsittichai et al., 2006). Densitometric analysis in ImageJ 1.49c (http://imagej.nih.gov/ij) was used for semiquantitative measurement of fragment abundance in the gel-resolved PCR products to calculate the ratios between the generated ROPN1L and GST cDNA fragments. 2.4. Expression of recombinant OvROPN1L in E. coli and production of anti-rOvROPN1L antisera E. coli M15 was transformed with pQE30-OvROPN1L, identified positive clones were cultured at 37 ◦ C in standard Luria–Bertani (LB) broth to an OD600 of 0.6 and induced with isopropyl beta-d-1thiogalactopyranoside (IPTG) at 1 mM final concentration. After 4 h the bacteria were pelleted and total bacterial protein was dissolved in 100 mM NaH2 PO4 , 10 mM Tris–HCl, pH 8.0, 6 M urea. Recombinant His-tagged OvROPN1L was then purified under denaturing conditions by nickel-nitrilotriacetic acid (Ni-NTA) chromatography following the supplied standard protocols (QIAGEN, USA). Concentration of purified protein was determined by the Lowry method using a DC Protein Assay Kit (Bio-Rad Laboratories, USA). Two female 5-week-old BALB/c mice were subcutaneously immunized 4 times with 20 ␮g rOvROPN1L each in 2-week intervals. The first immunization was carried out with rOvROPN1L mixed 1:1 in Freund’s complete adjuvant while the booster immunizations were carried out with incomplete Freund’s adjuvant. Final antisera were collected 7 days after the last immunization. 2.5. Preparation of parasite crude worm extract Adult parasites were transferred in lysis buffer (10 mM Tris–HCl, pH 7.2, 150 mM NaCl, 2% Triton-X 100, 1 mM ethylenediaminetetraacetic acid (EDTA) pH 7.2, 1 mM phenyl-methyl-sulphonylfluoride (PMSF), 4 M urea) and homogenized on ice in a glass rod homogenizer. The homogenate was rotated at 4 ◦ C, 1 h and then centrifuged at 12,000 × g, 15 min. The supernatant containing soluble crude worm protein was collected. Concentration of purified protein was determined by the Lowry method using a DC Protein Assay Kit (Bio-Rad Laboratories, USA). The protein extract was stored at −20 ◦ C until used in further experiments. 2.6. Preparation of mammalian sperm protein Sperm were first isolated from testes of Wistar rat, Syrian hamster, and ICR mouse using a testicular sperm extraction technique (Vera et al., 1984). Briefly, testes were first rinsed in normal saline and then immersed in 10 mM phosphate buffered saline (PBS), pH 7.2 and minced with scissors into pieces. Testicular fluid was obtained from the minced tissue by pressing it between two cleaned glass slides and the fluid was then filtered through sterile gauze to remove testicular tissue debris. Cleared testicular fluid was

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then resuspended in 10 mM PBS, pH 7.2 and centrifuged at 1500 × g, 4 ◦ C for 15 min. This was repeated 3 times to remove the seminal plasma and to obtain purified sperm. To obtain the sperm protein, the method of Fujita et al., 2000 (Fujita et al., 2000) was used with slight modifications. The sperm were homogenized in 100 ␮l of lysis buffer (10 mM Tris–HCl, pH 7.2, 150 mM NaCl, 2% Triton-X 100, 1 mM EDTA pH 7.2, 1 mM PMSF, 4 M urea) and then incubated on ice for 1 h with gentle shaking to promote the complete dissociation of intact sperm. Thereafter, the sperm lysates were centrifuged at 10,000 × g, 4 ◦ C for 15 min, and the cleared supernatants were collected and further analyzed for the presence of sperm protein by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE). 2.7. SDS-PAGE and Western analyses O. viverrini crude worm extract and sperm protein of hamster, mouse and rat were each dissolved at an amount of 10 ␮g in sample buffer (0.35 M Tris–Cl, pH 6.8, 0.35 M SDS, 30% glycerol, 0.6 mM dithiothreitol (DTT), 0.175 mM bromophenol blue) and resolved by 12.5% SDS-PAGE (Laemmli, 1970). The proteins were then transferred to a nitrocellulose membrane (Hybond–ECL, Amersham Biosciences, USA) using a wet blot apparatus (Hoefer TE 22, Amersham Biosciences, USA). Unspecific binding sites on the membrane were blocked by incubation in 3% skim milk in TBS (20 mM Tris–HCl, pH 7.5, 150 mM NaCl) containing 0.1% Tween 20 for 2 h at room temperature. The membrane was then incubated in mouse anti-rOvROPN1L antiserum, diluted 1:1000 and 0.5 ␮g/ml mouse monoclonal anti-␤ actin antibody (Sigma, USA) as internal control at room temperature for 2 h. Following washes the membrane was incubated in AP-conjugated goat anti-mouse IgG antibody (Abcam, UK) diluted 1:10,000 at room temperature for 1 h. For analysis of immune responses against native OvROPN1L in infected hamsters and humans 200 ng rOvROPN1L per lane was resolved by 12.5% SDS-PAGE and transferred to nitrocellulose membranes as described above. The membranes were cut in strips and each strip was first blocked and then detected with a specific serum. Pooled hamster sera (N = 12) collected pre-infection and 2, 4, 6, and 8 weeks postinfection were used at dilution 1:200 and the strips were incubated at room temperature for 2 h. AP-conjugated anti-Syrian hamster IgG antibody (Abcam, UK) was used as secondary antibody at dilution 1:10,000, room temperature for 1 h. Ten strips were detected with 1:2000 diluted sera of infected individuals at 4 ◦ C overnight, 1 strip with 1:2000 diluted pooled sera from 6 healthy individuals at 4 ◦ C overnight, AP-conjugated goat anti-human IgG antibody (Zymed Laboratories Inc., USA) at dilution 1:10,000 was used as secondary antibody at room temperature, 1 h. Finally, 1 strip was detected with mouse anti-rOvROPN1L antiserum (1:1000) at 4 ◦ C overnight as positive control and alkaline phosphatase (AP)-conjugated goat anti-mouse IgG antibody (Abcam, UK) diluted 1:10,000 at room temperature for 1 h. Colorimetric detection of all bound antibody complexes was obtained with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) substrates. The human sera used in this study were sampled in Khon Kaen Province, Thailand in a research project approved by the Khon Kaen University Ethics Committee (22 April 2005, project no. HE480315), further details can be found in (Eursitthichai et al., 2010). 2.8. Immunohistochemical detection of OvROPN1L in adult parasites The distribution of OvROPN1L in adult (6-week-old) parasites was analyzed in paraffin-embedded tissue as previously described (Chunchob et al., 2010). Mouse anti-rOvROPN1L antiserum was

used at dilution 1:200, mouse preimmune serum at the same dilution was used as negative control. 2.9. Subcellular detection of OvROPN1L by immunoelectron microscopy Adult (6-week-old) parasites were fixed in 4% paraformaldehyde, 0.5% glutaraldehyde in 10 mM PBS, pH 7.2, at 4 ◦ C for 2–4 h, dehydrated in a series of ethanol, and finally embedded in LR white (LR White embedding resin kit, Electron Microscopy Sciences, USA). Sections were cut at 60–90 nm thickness and subsequently placed on Formvar-coated nickel grids (100 meshes, Electron Microscopy Sciences, USA). The tissue was blocked in 0.1% glycine in 1× TBS, pH 7.4, 0.05% Tween 20 at room temperature for 10 min and then in 4% bovine serum albumin (BSA) in 1× TBS, pH 7.4, 0.05% Tween 20 at room temperature for 2 h. Following washes in 1% BSA in 1× TBS, pH 7.4, 0.05% Tween 20 the tissue was incubated in antiOvROPN1L antiserum (diluted 1:200 in 1% BSA in 1× TBS, pH 7.4) at room temperature for 2 h. After washes the grids were incubated in gold conjugated protein-G (1:20) at room temperature, 1 h. To enhance the contrast the washed sections were firstly incubated in 2% uranyl acetate in the dark, 30 s, touch washed in de-CO2 distilled water and incubated in de-CO2 1% lead citrate, 30 s. Finally, the tissue sections were touch washed in de-CO2 H2 O and dried. The sections were observed under a transmission electron microscope at 75 kV. 2.10. ELISA The wells of standard 96-well microplates were coated with 100 ng rOvROPN1L dissolved in 100 ␮l of 30 mM Na2 CO3 , 75 mM NaHCO3 , pH 9.6 at 4 ◦ C overnight. Following washes with H2 O and blocking in 3% BSA, 10 mM PBS, pH 7.4, 0.05% Tween 20 the sera from either infected hamsters (sampled 8 weeks postinfection, N = 12) or infected human individuals (N = 10) were added at a volume of 100 ␮l and a dilution of 1:200 in 1% BSA, 10 mM PBS, pH 7.4, 0.05% Tween 20 to each well. The same number of normal sera from the two species was used as negative control. The plates were incubated at 37 ◦ C for 2 h and washed before 100 ␮l of either AP-conjugated goat anti-human IgG antibody (Zymed Laboratories Inc., USA) or AP-conjugated goat anti-hamster IgG antibody (Abcam, UK) diluted 1:5000 in 1% BSA, 10 mM PBS, pH 7.4, 0.05% Tween 20 was added to each well. After incubation at 37 ◦ C for 1 h the wells were washed and colorimetric detection was performed with p-nitrophenyl phosphate (pNPP) substrate (Zymed Laboratories Inc., USA) diluted 1:100 in 0.1 M glycine, 1 mM MgCl2 , 1 mM ZnCl2 , pH 10.4. The obtained signals were measured in an ELISA reader at 405 nm wavelength. The ELISA was repeated three times with duplicate samples in each assay. Statistical analysis of the values was done in GraphPad Prism v6.0e by an unpaired t-test. 3. Results 3.1. Molecular cloning and sequence analysis of OvROPN1L A 879 bp cDNA encoding a protein homologous to mammalian ROPN1L was detected in a BLAST screen of O. viverrini sequence data at the NCBI databases for putative sperm-expressed proteins. The uncharacterized EST ES417466 was found to contain the complete 669 bp CDS for a putative O. viverrini ROPN1L. The 669 bp CDS (GenBank: KJ71930) was amplified by RT-PCR from total RNA of adult parasites, inserted into the vector pQE30 and sequenced. The deduced amino acid sequence of 222 residues (25.3 kDa calculated molecular weight) showed modest conservation to mammalian ROPN1L at below 50% identity (Fig. 1 and Table 1). Higher conservation was found with the homologous proteins of the trematodes

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OvROPN1L FgROPN1L ShROPN1L HsROPN1L HsROPN1

|----------- DD-R-PKA ------------| MPLVNDPYYCHEQIPIPPALPDILKQFTKAAIRTQPKDVLKWSYAYFRALANSEPPPVKD ..iSL..............F.......................h...k...hn......e ..iS.n.h......Q................................k.i.Q..I....e ...-P.TMf.Aq..H...E.................A...r..AG..S..sRGd.L.... ------MAQTDkPTC...E..Km..e.A.....V..q.liq.aAD..E..sRG.T...re

60 60 60 59 54

OvROPN1L FgROPN1L ShROPN1L HsROPN1L HsROPN1

RLEVPISTQKTDTGLTPGLLRVLNNQLAVLKTIPVTLIEEKWKDLSLPIDRFQELCRIGN ..................i..i.....sSm.Qv.lPv...r.......ve.......l.. ...l..............i..i......S..IvSlsv...........ve....i..... .m.m.Ta.........Q...k..hK.CHHKrYvEl.Dl.q...n.C..Kek.kA.LqlDP .S.RVALC--NRAE...E..ki.hs.v.GRLI.RAEElAqM..Vvn..T.L.NSvMNv.R

120 120 120 119 112

OvROPN1L FgROPN1L ShROPN1L HsROPN1L HsROPN1

FVGTCEWRHFLAIAASDLCATLEETLKLICELLTSDPEGGPARISFETWLDFYRYLGKLD ...s....K...l......KN.P.....v.....a.....A...Q.dQ..e.......i. .AN.....W...l.........S........i..a.....A..lP..Q..e.....A.i. CENKIk.In...lGC.M.GGs.NTA..Hl..i..D.........P.k.fSYV....Ar.. .TEEI..LK...l.C.A.GV.iTk...iv..v.sC.HN..SP..P.S.fQFL.T.iA.v.

180 180 180 179 172

OvROPN1L FgROPN1L ShROPN1L HsROPN1L HsROPN1

EISDA-HINHVMTYLTFD-IASQEGMIMPRNFMHPECPKLKPLD-------------..PeG-.....lA.....-..n.d..........Sd....N.R.-------------d.P.E-...K.lAh.s..-..t.N.A.A......KN....N.SeEVNLHAFIESTVED SDVSPLETESYlAS.KEnID.RkN...GLSd.FF.kRKL.eSieNSEDVGH------GEIS.S.vsRmlN.mEQe-vIGPd.i.TVNd.TQNPRVq.e-----------------

222 222 236 230 212

Fig. 1. Multiple alignment of ROPN1L sequences from O. viverrini, F. gigantica, S. haematobium, H. sapiens and the sequence of human ROPN1 (see Section 2 for details). Residues identical to those in OvROPN1L are indicated by a dot, similar residues (BLOSUM62) are shown in lower case characters, gaps are indicated by dashes.

Table 1 Pairwise alignment results for OvROPN1L against homologous proteins from F. gigantica, S. haematobium, and Homo sapiens. Compared sequences

Identical residues

Similar residues

Gaps

Score

OvROPN1L/FgROPN1L OvROPN1L/ShROPN1L OvROPN1L/HsROPN1L OvROPN1L/HsROPN1

174/222 (78.4%) 168/236 (71.2%) 109/231 (47.2%) 82/223 (36.8%)

203/222 (91.4%) 196/236 (83.1%) 149/231 (64.5%) 129/223 (57.8%)

0/222 (0.0%) 14/236 (5.9%) 10/231 (4.3%) 12/223 (5.4%)

993.0 941.0 519.0 389.5

F. gigantica (78.4% identity) and S. haematobium (71.2% identity) which are at present uncharacterized (Fig. 1 and Table 1). The functional significance of the N-terminal located dimerization/docking domain of 37 amino acid residues size (Fig. 1) is reflected in its higher regional sequence conservation (73.0%, 86.5%, 91.9% identity for Homo sapiens, F. gigantica and S. haematobium, respectively). This domain is present in other sperm-expressed proteins in mammals (NCBI CDD: cd12084) and we noticed putative homologs in trematodes, e.g. SP17 and cAMP-dependent protein kinase regulatory unit, but remarkably a trematode homolog of ROPN1 which is functionally partially redundant to ROPN1L could not be detected in the current molecular sequence databases at the NCBI, SchistoDB at http://schistodb.net/schisto/, and the Gasser laboratory at http://research.vet.unimelb.edu.au/gasserlab/index.html. 3.2. RT-PCR analysis of OvROPN1L transcripts during development

crude worm extract and sperm protein from hamster, mouse and rat (Fig. 4). A monoclonal antibody against beta-actin was used as a positive control and detected the expected 41 kDa protein in all extracts. A protein migrating at the expected 25.3 kDa molecular weight of ROPN1L was only detected in the parasite crude worm extract. 3.4. Light and electron microscopic detection of OvROPN1L in adult O. viverrini The immunohistochemical localization of OvROPN1L with antirOvROPN1L antiserum showed that it was concentrated in the male reproductive system of the adult parasite being clearly detected in testes, seminal vesicle and receptacle (Fig. 5). Seminal vesicle and receptacle act as sperm storage systems indicating that the protein is associated with spermatozoa as in mammals. Immunoelectron

RT-PCR analysis of total RNA from metacercariae, NEJ, and 2, 4-, 6- and 8-week-old parasites detected OvROPN1L transcripts starting from 2-week-old juveniles (Fig. 2) but compared to the internal control, glutathione S-transferase transcripts, the amount of OvROPN1L transcripts was higher in later developmental stages of the parasite as semiquantitatively measured with ratios of ROPNL1/GST at 0.31 in 2-week-old juveniles and 1.35 ± 0.03 in 4–8-week-old parasites, respectively. 3.3. Expression of recombinant OvROPN1L, preparation of mouse anti-rOvROPN1L antiserum, and western detection of parasite protein and mammalian sperm protein Recombinant, N-terminal His-tagged OvROPN1L was abundantly expressed in E. coli in insoluble form and purified by Ni-NTA affinity chromatography (Fig. 3). Purified rOvROPN1L was used to immunize two mice for the production of specific antisera. The antisera showed comparable reactivity against rOvROPN1L in western blots (data not shown) and were then used to probe parasite

Fig. 2. RT-PCR of total RNA extracted from metacercariae (MT), NEJ, 2-, 4-, 6-, and 8-week-old parasites with primers specific to O. viverrini glutathione S-transferase (GST, 298 bp) and OvROPN1L (669 bp). M: 100 bp DNA ladder (Fermentas). Negative control (nc): RT-PCR without template.

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Fig. 4. Western analysis of O. viverrini crude worm extract (Ov) and sperm protein from hamster (Ma), mouse (Mm), and rat (Rr) detected with mouse anti-rOvROPN1L antiserum and a monoclonal antibody against human beta-actin (positive control). The positions of the size standards are indicated on the left.

Fig. 3. Bacterial protein resolved by SDS-PAGE. (A) Expression of rOvROPN1L in E. coli M15 prior to induction (0) and 1–4 h after induction. The soluble protein fraction (Ps) does not contain rOvROPN1L, all of it is found in the insoluble protein fraction (Pi). (B) Purification of rOvROPN1L by Ni-NTA affinity chromatography. CL: cleared lysate, FT: flow through, W1, 2: wash fractions, D1, 4 and E1, 4: elution fractions in buffer D and E, respectively, M: size standards.

microscopy was then used to locate it in sections of testis lobes and gold particles were found on the tail regions of spermatozoa (Fig. 6). 3.5. Reactivity to OvROPN1L in sera of O. viverrini infected hamsters and humans Mammalian ROPN1L has been described as a fibrous sheath protein in spermatozoa and our results show a similar localization in parasite spermatozoa which suggests that it should be hidden from the host’s immune system. Yet, sera of infected hamster showed increasing reactivity against rOvROPN1L during the postinfection period of 8 weeks as detected by western analysis (Fig. 7A) suggesting exposure of the protein to the immune system and this result was supported by the reactivity of human infected sera to rOvROPN1L (Fig. 7B). Indirect ELISA was used to evaluate possible application of rOvROPN1L for routine diagnosis and the results confirmed the western analyses, infected sera of hamsters and humans were reactive to the recombinant parasite antigen (Fig. 8). The number of human sera was limited to 10 each for infected and uninfected samples in this preliminary analysis. The

Fig. 5. Immunohistochemical detection of OvROPN1L in a 6-week-old parasite. (A) Mouse preimmune serum showed no reactivity to any tissue; (B) mouse anti-rOvROPN1L antiserum detected OvROPN1L in the testis lobes (Te), seminal receptacle (Sr) and seminal vesicle (Sv). Vitellaria (Vi), intrauterine eggs (Eg), parenchyma (Pa), and intestinal tract (It) remained unstained. Size bar: 500 ␮m.

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Fig. 8. Scatter plots of absorbance values obtained by indirect ELISA against rOvROPN1L with 10 serum samples each collected from uninfected (uninf.) and naturally infected (inf.) individuals and 12 samples each from uninfected and experimentally infected hamsters. The hamster sera were collected 8 weeks postinfection with 50 metacercariae. Postmortem worm burden was 25–30 parasites/hamster.

4. Discussion

Fig. 6. Immunoelectron microscopic detection of OvROPN1L in sperm tails of the adult parasite. (A) Gold particles were absent in sections probed with preimmune serum. (B) Sections detected with anti-rOvROPN1L antiserum showed gold particles on the sperm tails (arrows). Size bar: 100 nm.

infected persons, 2 males and 8 females had an average age of 51.50 ± 11.72 years (range: 35–72 years) and similar fecal egg counts at 29 ± 4.67 eggs/sample (range: 24–36 eggs/sample). Reactivity of human and hamster infected and non-infected sera to rOvROPN1L were found to be statistically significant different at P-values of 0.0001. The uninfected persons had an average age of 34.3 ± 11.67 years (range: 25–58 years).

Molecular processes and their involved proteins that regulate spermatogenesis have not been investigated in trematodes as yet and the presented basic analysis of ROPN1L in O. viverrini is a first step to gather data on the male reproductive system at this level. In mammals ROPN1L and ROPN1, while having only modest sequence identity (39% in H. sapiens, (Carr et al., 2001)), were found to be partially functional redundant with only the double knockout showing the maximum phenotype of infertility (Fiedler et al., 2013). Based on our BLAST searches of trematode sequence data which is most complete in schistosomes (Berriman et al., 2009; Liu et al., 2009; Young et al., 2012) these parasites do not carry a ROPN1 gene in their genome and possibly, therefore, mammalian ROPN1 originated from ROPN1L by gene duplication and acquired additional functions in higher evolved animals. Loss of function of ROPN1L in trematodes could then be sufficient to cause sperm immotility and infertility if it is functional equivalent to the mammalian protein. Essentially, in O. viverrini ROPN1L was found located in spermatozoa as in mammals, but due to the rapid development of this parasite with intrauterine eggs already present in 2-week-old parasites (Kaewkes, 2003) maturity reached after 4 weeks, ROPN1L must be expressed early in development and transcripts could be detected as early as 2 weeks after metacercarial excystation. Unexpected was the immune response to OvROPN1L which was clearly detectable 6 weeks postinfection in hamster and subsequently confirmed in infected humans. Additional investigations are required to validate this result with a larger number of human serum samples and to conclude whether there are any correlations between age, fecal egg count, drug treatment and the ROPN1Lspecific immune response. ROPN1L is not a sperm surface protein but located in the fibrous sheath with a suggested function in regulation of AKAP3 activity in sperm (Fiedler et al., 2013). AKAP3 itself

Fig. 7. Western analyses of recombinant OvROPN1L detected with hamster and human sera. (A) Reactivity of pooled hamster sera (N = 12) collected prior to infection (NS) and 2, 4, 6, and 8 weeks postinfection with 50 O. viverrini metacercariae to rOvROPN1L. (B) Reactivity of 10 sera from infected individuals (1–10), mouse anti-rOvROPN1L antiserum (As), and pooled (N = 5) sera from uninfected individuals (NS). M indicates the lanes containing the size standards.

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binds other proteins and relocates them in the cell and the direct inhibition of AKAP3 with specific peptides caused equal arrest of sperm motility (Vijayaraghavan et al., 1997). It could be speculated that not all parasite spermatozoa that are (inadvertently) released in the bile are disposed into the host’s intestinal tract but that a sufficient number is either trapped in obstructed bile ducts or adhered to the bile duct wall through as yet not identified molecules. Their subsequent degradation would then allow uptake of the now exposed molecules by host cells and presentation to the immune system. If this is the case then other sperm-located proteins might cause similar immune responses and further inves´ tigation is indicated. Indeed, Gozdzik et al. (2012) have reported the application of a 17 kDa major sperm protein for diagnosis of infection with the lungworm Dictyocaulus viviparus in cattle. As spermatozoa will be produced in numbers much larger than eggs and continuously released they could present an unrecognized source of molecules with diagnostic potential. In conclusion, this analysis has demonstrated that OvROPN1L is a suitable marker for further research on spermatogenesis in the parasite, that it has potential as a diagnostic tool, and it suggests that other sperm proteins, eventually surface proteins, will be equally or better suited as diagnostic tools. Acknowledgments This research was supported by the Thailand Research Fund through a Royal Golden Jubilee Ph.D. scholarship (4.B.MU/49/P.1) to S. Ratanachan (student) and S. Vichasri Grams (advisor). We thank Sombat Singhakaew, Department of Biology, Faculty of Science, Mahidol University for technical advice and acknowledge Todsamon Narizp, Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University for electron microscopic services. References Berriman, M., Haas, B.J., Loverde, P.T., Wilson, R.A., Dillon, G.P., Cerqueira, G.C., Mashiyama, S.T., Al-Lazikani, B., Andrade, L.F., Ashton, P.D., Aslett, M.A., Bartholomeu, D.C., Blandin, G., Caffrey, C.R., Coghlan, A., Coulson, R., Day, T.A., Delcher, A., Demarco, R., Djikeng, A., Eyre, T., Gamble, J.A., Ghedin, E., Gu, Y., Hertz-Fowler, C., Hirai, H., Hirai, Y., Houston, R., Ivens, A., Johnston, D.A., Lacerda, D., Macedo, C.D., McVeigh, P., Ning, Z., Oliveira, G., Overington, J.P., Parkhill, J., Pertea, M., Pierce, R.J., Protasio, A.V., Quail, M.A., Rajandream, M.A., Rogers, J., Sajid, M., Salzberg, S.L., Stanke, M., Tivey, A.R., White, O., Williams, D.L., Wortman, J., Wu, W., Zamanian, M., Zerlotini, A., Fraser-Liggett, C.M., Barrell, B.G., El-Sayed, N.M., 2009. The genome of the blood fluke Schistosoma mansoni. Nature 460, 352–358. Brown, P.R., Miki, K., Harper, D.B., Eddy, E.M., 2003. A-kinase anchoring protein 4 binding proteins in the fibrous sheath of the sperm flagellum. Biol. Reprod. 68, 2241–2248. Carr, D.W., Fujita, A., Stentz, C.L., Liberty, G.A., Olson, G.E., Narumiya, S., 2001. Identification of sperm-specific proteins that interact with A-kinase anchoring proteins in a manner similar to the type II regulatory subunit of PKA. J. Biol. Chem. 276, 17332–17338. Chunchob, S., Grams, R., Viyanant, V., Smooker, P.M., Vichasri-Grams, S., 2010. Comparative analysis of two fatty acid binding proteins from Fasciola gigantica. Parasitology 137, 1805–1817.

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Opisthorchis viverrini: analysis of the sperm-specific rhophilin associated tail protein 1-like.

Concurrent deficiency of rhophilin associated tail protein (ROPN1) and ROPN1-like (ROPN1L) in mice causes structural abnormalities and immotility of s...
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