Trichinella is an important parasitic nematode of animals worldwide. Heat shock proteins are ubiquitous in nature and allow organisms to quickly respond to environmental stress. A portion of the Tsdaf-21 gene, a Caenorhabditis elegans daf-21 homologue encoding heat-shock protein 90 (Hsp90) was cloned from Trichinella spiralis. The partial nucleotide sequence resided near the 5′-end of the gene and encoded a polypeptide of 254 amino acid residues harboring a HATPase-c superfamily domain and Hsp90 protein domain. Phylogenetic analysis revealed that Tsdaf-21 is highly conserved and formed a monophyletic clade with other nematodes. The partial Tsdaf-21 transcript was subcloned and expressed for antibody production. Results using PCR primers specific for the Tsdaf-21 transcript, and mouse polyclonal antisera specific for the recombinant protein showed that both the RNA transcript and the corresponding protein were ubiquitously and consistently expressed in newborn larvae, muscle larvae and both male and female adult worms in the absence of any external stress or stimulation

Cao, Lei

Qin, Weiwen

Tian, Guimei

Yang, Yurong

Zarlenga, Dante

English

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Publications from USDA-ARS / UNL Faculty U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska 2013 TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis Yurong Yang Xiamen University, yryang@xmu.edu.cn Weiwen Qin Xiamen University Dante Zarlenga ARS, Animal Parasitic Diseases Laboratory Lei Cao Xiamen University Guimei Tian Xiamen University Follow this and additional works at: https://digitalcommons.unl.edu/usdaarsfacpub Yang, Yurong; Qin, Weiwen; Zarlenga, Dante; Cao, Lei; and Tian, Guimei, "TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis" (2013). Publications from USDA-ARS / UNL Faculty. 2267. https://digitalcommons.unl.edu/usdaarsfacpub/2267 This Article is brought to you for free and open access by the U.S. Department of Agriculture: Agricultural Research Service, Lincoln, Nebraska at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Publications from USDA-ARS / UNL Faculty by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Veterinary Parasitology 194 (2013) 171– 174Contents lists available at SciVerse ScienceDirectVeterinary  Parasitologyjo u r nal homep age: www.elsev ier .com/ locate /vetparTsDAF-21/Hsp90  is  expressed  in  all  examined  stages  ofTrichinella  spiralisYurong  Yanga,∗, Weiwen  Qina,  Dante  Zarlengab,  Lei  Caoa, Guimei  Tianaa State Key Laboratory of Cellular Stress Biology, School of Life Science, Xiamen University, Xiamen, Fujian 361005, People’s Republic ofChinab ARS, Animal Parasitic Diseases Laboratory, Beltsville, MD  20705, USAa  r  t  i  c  l  e  i  n  f  oKeywords:Trichinella spiralisTsdaf-21/Hsp90Heat shock proteinsPhylogenetic analysisa  b  s  t  r  a  c  tTrichinella  is an  important  parasitic  nematode  of animals  worldwide.  Heat  shock  pro-teins are  ubiquitous  in  nature  and  allow  organisms  to  quickly  respond  to environmentalstress.  A portion  of the  Tsdaf-21  gene,  a  Caenorhabditis  elegans  daf-21  homologue  encodingheat-shock  protein  90 (Hsp90)  was  cloned  from  Trichinella  spiralis.  The  partial  nucleotidesequence  resided  near  the  5′-end  of the  gene  and  encoded  a  polypeptide  of 254  aminoacid  residues  harboring  a HATPase-c  superfamily  domain  and  Hsp90  protein  domain.  Phy-logenetic  analysis  revealed  that  Tsdaf-21  is highly  conserved  and  formed  a monophyleticclade  with other  nematodes.  The  partial  Tsdaf-21  transcript  was  subcloned  and expressedfor antibody  production.  Results  using  PCR  primers  specific  for the  Tsdaf-21  transcript,  andmouse polyclonal  antisera  specific  for  the  recombinant  protein  showed  that  both  the  RNAtranscript  and  the  corresponding  protein  were  ubiquitously  and  consistently  expressed  innewborn  larvae,  muscle  larvae  and  both  male and female  adult  worms  in the  absence  ofany external  stress  or stimulation.© 2013 Elsevier B.V. All rights reserved.1. IntroductionTrichinella is an important and global zoonotic parasite.Organisms belonging to this genus infect a wide variety ofanimals including humans. Infection is caused by ingest-ing meat contaminated with encysted muscle larvae (ML).The ML  can remain viable within the host tissue for longperiods of time even when stored at low temperatures (4 ◦Cin refrigerator).Heat shock proteins (Hsp) function as molecular chaper-ones and are involved in many cellular processes includingprotein trafficking, signal transduction, differentiation anddevelopment (Devaney, 2006). They play key roles in∗ Corresponding author at: Parasite Research Lab, Department of Biol-ogy, School of Life Science, Xiamen University, Xiamen, Fujian 361005,People’s Republic of China. Tel.: +86 592 2181792; fax: +86 592 2180038.E-mail address: yryang@xmu.edu.cn (Y. Yang).stress response, help denatured proteins to refold, pre-vent protein aggregation, and assist in targeting proteindegradation by proteasomes and lysosomes (Saunders andVerdin, 2009). This family of proteins also plays a rolein apoptosis and helps modulate signals involved in theimmune system and the inflammatory response (Shinyet al., 2011). Heat shock proteins are classified accordingto their molecular weights and grouped into many fam-ilies, such as small Hsps, Hsp60, Hsp70 and Hsp90. It isnow recognized that most of these proteins are induced inresponse to a wide variety of stresses, such as low or hightemperatures, heavy metal exposure or parasite infection.Heat shock proteins 60, 70 and 90 have been foundin Trichinella nativa,  Trichinella nelsoni, Trichinella pseu-dospiralis and Trichinella spiralis after larval exposureto hydrogen peroxide (H2O2) (Martinez et al., 2002a).Specifically, Hsp60 and Hsp70, but not Hsp90 were consti-tutively phosphorylated at their serine/threonine residues(Martinez et al., 2002a). Furthermore, the induction of0304-4017/$ – see front matter ©  2013 Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.vetpar.2013.01.048172 Y. Yang et al. / Veterinary Parasitology 194 (2013) 171– 174constitutive and higher molecular weight Hsp correlatedstrongly with the loss of larval viability and an increasein mortality following exposure to H2O2 (Martinez et al.,1999). After H2O2 shock, both Hsp90 and a 50 kDa Hsp(prot-50) were significantly elevated, whereas only Hsp90levels decreased after cold shock (Martinez et al., 2002b). Itwas also found that T. spiralis ML  stored in 199 medium at37 ◦C lost infectivity and this coincided with a concomitantreduction in Hsp70 and Hsp90. An inducible Hsp, Hsp90i,appeared as the expression of the more constitutive Hsp90decreased in stored larvae (Martinez et al., 2004).In Caenorhabditis elegans, daf-21 encodes an Hsp90 thatis primarily expressed in germline cells. After heat stress,daf-21 is expressed throughout the entire body of theworm (Inoue et al., 2003). Furthermore, daf-21 was  foundinvolved in egg hatching and motility; however, mutationsto the daf-21 gene had deleterious effects on animal fer-tility and induced early larval lethality. The daf-21 proteinindirectly regulates the meiotic prophase/metaphase tran-sition during oocyte development by ensuring the normalfunction of WEE-1.3 (Inoue et al., 2006). In contrast, daf-21 mutants show multiple chemosensory defects in taxisto volatile and non-volatile attractants, and inappropri-ate responses to the dauer-inducing pheromone (Birnbyet al., 2000). Except in response to stress, other functionsof Hsp90 in Trichinella spp. remain unclear. In this paper, wereport both gene and protein expression profiles of Hsp90in different stages of T. spiralis.2. Materials and methods2.1. Parasites and experimental infectionT. spiralis (ISS 534) was maintained by serial passagein mice. Muscle larvae were isolated 30 days post infec-tion (p.i.) from skeletal muscle by overnight digestion in1% pepsin: 3.6% HCl at 37 ◦C. To obtain adult worms fromthe intestine, mice infected with T. spiralis ML,  were euth-anized 6 days post infection. Newborn larvae (NBL) werecollected from female adult worms incubated for 24 h inRPMI 1640 medium (GIBCO). All worm preparations werewashed several times with physiological saline before DNAor RNA extraction.2.2. RNA extraction and cDNA cloningTotal RNA was prepared from NBL, ML, and maleand female adult worms using Trizol (MRC) accordingto the manufacturer; cDNA was synthesized using the3′-Full RACE Core set kit (Takara Biotechnology Co. Ltd.,Dalian). The resulting cDNA was used to amplify T. spi-ralis daf-21 cDNA by polymerase chain reaction (PCR) ina total volume of 50 L containing 1 L RT reaction, 2 L3′RACE Outer Primer, 7 L 1× cDNA dilution buffer II, 2 Lof Tsdaf21F primer (5′-CCCACGCGTCCGCGTTGTTTGTGCT;derived from the 5′ end of EST sequence ES566819), 5 L10 × ExTaq buffer, 0.25 L ExTaq polymerase, and 30.75 LddH2O. The PCR was performed as follows: 94 ◦C for 3 minfollowed by 36 cycles of 30 s at 94 ◦C, 30 s at 53.5 ◦C,1 min  at 72 ◦C; a final extension was performed at 72 ◦Cfor 10 min. The PCR product was gel purified, cloned intopMD18 T and used to transform Escherichia coli Dh5 cells.Positive colonies were identified by PCR and sequenced.One sequence was  subcloned into the expression vectorpET32 M (Invitrogen). The recombinant protein (rTsDAF-21) was  purified from E. coli BL21 (DE3) cells using Ni-NTAagarose (QIAGEN, Valencia, CA) according to the manu-facturer’s protocol. Antiserum was  obtained by injectingmice intraperitoneally with 100 g of recombinant protein,mixed with Freund’s complete adjuvant (Sigma, St. Louis,MO). The animals were boosted three times with 100 gprotein in Freund’s incomplete adjuvant and blood wascollected 5 weeks later.2.3. Sequence and phylogenetic analysisSequences were analyzed using NCBI utilities andBLAST searches (http://www.ncbi.nlm.nih.gov/BLAST).The putative amino acid sequence was  identi-fied using the ORF Finder program from the NCBI(http://www.ncbi.nlm.nih.gov/gorf/gorf.html). Amino acidsequences derived from the Tsdaf-21 were deposited intoGenBank (Accession No. JN836431). For tree reconstruc-tion, the predicted full-length amino acid sequence derivedfrom the T. spiralis genome (XM 003374508) (Mitreva et al.,2011) minus the 3′ terminal 46 amino acids was alignedusing Clustal W (Thompson et al., 1994) with thosefrom C. elegans (NP 506626.1), Drosophila melanogaster(NP 523899.1), Xenopus laevis (NP 001086624.1), Musmusculus (NP 034610.1), Rattus norvegicus (NP 786937.1),Homo sapiens (NP 031381.2), Danio rerio (AAC21566.1),Taeniopygia guttata (XP 002200608.1), Ascaris suum(ACO55134.1), Toxocara cati (ACO55135.1), Liriomyzasativae (AAW49253.2), Tigriopus japonicus (ACA03524.1),Heterodera glycines (ACR57215.1), Haemonchus contortus(ACU00668.1) and Brugia pahangi (O61998.2). Phyloge-netic analyses were inferred using the Neighbor-Joiningprogram in MEGA4 (Tamura et al., 2007). The evolutionarydistances were computed using the Poisson correctionwhere gaps and missing data were eliminated from thedataset; 684 amino acid positions were included in thefinal dataset.2.4. RT-PCR and protein analyses of Tsdaf-21Transcription levels of Tsdaf-21 were measured usingoligo (dT) primed cDNA (0.5 L) synthesized from NBL, MLand adult worms using superscript II reverse transcrip-tase (Invitrogen). Amplification of Tsdaf-21 was performedusing primers Thsp90F (5’-CTACATGGTGGATGCCATTG)and Thsp90R (5′-ACACGCTCGTACAACTGCTG) which gen-erate a 393 bp fragment that resides nearer the 3′ terminusof the sequence. The -actin control was  amplifiedusing forward (ActF: 5′-CGTGGTTACTCTTTCCCGCTG) andreverse (ActR: 5′-CATTTAGAAGCACTTGCGGTGAACGATGG)primers. Reactions were cycled 25–40 times (94 ◦C 30 s,53.5 ◦C 30 s, 72 ◦C 1 min) followed by a 72 ◦C, 8 min soak.Control samples without cDNA were included with eachPCR run. Amplified products were separated on a 1% (w/v)agarose gel, stained with ethidium bromide, and photo-graphed under UV light.Y. Yang et al. / Veterinary Parasitology 194 (2013) 171– 174 173Fig. 1. Phylogenetic tree (N–J) of DAF-21/Hsp90 proteins from 16 speciesrepresenting vertebrates, insects and nematodes. Evolutionary historywas inferred using the N–J method within MEGA4 (Tamura et al., 2007).The evolutionary distances were computed using a Poisson correction forthe  number of amino acid substitutions per site. The optimal tree with thesum of branch length = 1.00969711 is shown. Support numbers (bootstraptest; 1000 replicates) for each branch are also shown. Gaps and miss-ing  data were not considered in the final dataset which consisted of 684positions.For Western blots, worms  were homogenized in phos-phate buffered saline then centrifuged to remove debris.The protein content was determined using the Bradfordassay (Biorad). Worm lysates were separated on 12% SDS-PAGE gels, transferred to nitrocellulose, screened usingrTsDAF-21 polyclonal mouse anti-sera (1:500 dilution)then visualized using the ECL Western blotting system (Bio-rad) according to the manufacturer’s recommendations.Mouse anti--tubulin (1:500 dilution) was used as a con-trol screen to normalize protein loading.3. Results and discussion3.1. Sequence and phylogenetic analyses of Tsdaf-21The ML-derived Tsdaf-21 cDNA generated an ORFencoded a predicted protein of 254 amino acids. Thesequence contained HATPase c and Hsp90 domains(JN836431). It should be noted that the 3′ RACE primer didnot bind as anticipated to the poly A tail of the gene, butto an A rich region approximately 850 bp downstream ofthe translation start site. However, upon BLAST analysis,the partial sequence of Tsdaf-21 cloned herein demon-strated significant identity with sequences from a rangeof organisms, including plant parasitic nematodes, insectsand vertebrates. Phylogenetic analysis using the nearfull-length TsDAF-21 protein sequence (XM 003374508)derived from genomic data (Mitreva et al., 2011) displayedhigh similarity with 15 other known Hsp90 proteins wherethe conserved Hsp90 domain region was most highly con-served (data not shown). These Hsp90 proteins basicallyformed 2 clades (Fig. 1). Into one clade fell higher eukary-otes (human, mouse and frog) and insects (fruit fly and fly)whereas the Hsp90 from all nematodes formed a separateand monophyletic clade. The Tsdaf-21 protein sequenceFig. 2. Transcripts of the Tsdaf-21 in different stages of T. spiralis detectedby  RT-PCR (A) compared with an internal -actin control (B). (1) Negativecontrol; (2) NBL; (3) ML;  (4) female adult worms; (5) male adult worms;M  = molecular weight standards.predictably positioned within the clade comprised of othernematodes and consistent with a lineage that divergedearly in the evolution of the phylum Nematoda (Zarlengaet al., 2009). All nodes were well supported except for thesingle Hsp90 from T. japonicus, a marine gastropod inver-tebrate.There are multiple T. spiralis Hsp90 submissions in Gen-Bank derived from genes (2), mRNAs (5), proteins (5) andEST sequences (2). One sequence, XM 003374508, was pre-dicted from sequencing the T. spiralis genome (Mitrevaet al., 2011). Alignment provided information that theintegrity of the 3′-terminus is suspect. As such, 46 aminoacids of this putative full-length sequence were deletedprior to tree building. All additional sequences were over-lapping; two  are identical (XM003374508, EFV55915.1)and two EST sequences (ES570731 and ES562424) areincomplete but align well with Tsdaf-21. One sequence(XP003368967.1) was  found to be quite different; however,upon additional analyses this sequence conforms more toHsp75 than to Hsp90.3.2. Expression of Tsdaf-21 in developmental stages of T.spiralisThe transcript of Tsdaf-21 was detected by RT-PCR in allstages of T. spiralis. A specific band of 393 bp was  amplifiedfrom NBL, ML,  and adult male and female worms  (Fig. 2).The results suggest that Tsdaf-21 (Fig. 2A) is abundantlyand consistently expressed in all stages and in both sexes ofadult worms when referenced to actin as an internalcontrolFig. 3. Protein expression in different stages of T. spiralis detected byWestern blot. (A) Native TsDAF-21 (lane 1) in ML  crude worm extract andrTsDAF-21 (lane 2) were detected using mouse anti-rTsdaf-21 serum; M,pre-stained molecular weight markers. (B) Protein expression of TsDAF-21 in new born larvae (NBL), muscle larvae (ML), female adult worms (FA)and  male adult worms (MA) of T. spiralis detected using anti-rTsDAF-21serum (1:500 dilution; upper panel). Tubulin was  used to normalize pro-tein loading and was  detected with mouse anti--tubulin serum (1:500dilution; lower panel).174 Y. Yang et al. / Veterinary Parasitology 194 (2013) 171– 174(Fig. 2B). Similar conclusions were drawn using mouseanti-rTsDAF-21 serum to screen crude worm extracts inWestern blots. Data showed that the native TsDAF-21 wasapproximately 90 kDa in size and equally expressed in allthree stages examined including male and female adultworms (Fig. 3).In conclusion, our data suggest that the Tsdaf-21 andthe corresponding protein are ubiquitously and stronglyexpressed in all stages of Trichinella in the absence ofexternal stress; however, the function of the protein, itsregulation in response to external stress, and its role if any,in the infection process have yet to be investigated.Conflict of interestNo financial or personal relationships are maintainedwith other people or organizations that could inappropri-ately influence or bias this paper.AcknowledgementsThis work was supported in part by funding fromthe Natural Science Foundation of China (No. 30972181)and a grant from Xiamen Science Technology program(3502Z20061098).ReferencesBirnby, D.A., Link, E.M., Vowels, J.J., Tian, H., Colacurcio, P.L., Thomas,J.H., 2000. 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TsDAF-21/Hsp90 is expressed in all examined stages of
Trichinella spiralis

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TsDAF-21/Hsp90 is expressed in all examined stages of Trichinella spiralis

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