Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion

Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion Cong Li, Qiping Zhao, Shunhai Zhu, Qingjie Wang, Haixia Wang, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.2.17982/v3 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Jul, 2020 Read the published version in Parasites & Vectors → Version 3 posted 3 You are reading this latest preprint version Show more versions Abstract Background: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several species of the protozoan parasite Eimeria . Among these species, E. tenella causes hemorrhagic pathologies and high mortality. These parasites have complex and diverse lifestyles that require the invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative Et AMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative Et AMA1-interacting protein, E. tenella Eimeria -specific protein ( Et Esp). Methods : Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between Et AMA1 and Et Esp in vivo and in vitro. The expression of Et Esp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of Et Esp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C.The localization of Et Esp was analyzed with an immunofluorescence assay. An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against Et Esp to inhibit cell invasion by E. tenella sporozoites. Results: The interaction between Et AMA1 and Et Esp was confirmed with BiFC in vivo and by GST pull-down in vitro . Our results show that Et Esp is differentially expressed during distinct phases of the parasite life cycle. An immunofluorescence analysis showed that the Et Esp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine, we showed that Et Esp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-r Et Esp antibody attenuated the capacity of E. tenella to invade host cells in vitro . Conclusion: In this study, we show that Et Esp interacts with Et AMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in the sporozoite invasion of host cells and maybe involved in the growth of the parasite in the host. These data have implications for the use of Et AMA1 or Et AMA1-interacting proteins as targets in intervention strategies against avian coccidiosis. Parasitology Eimeria tenella Apical membrane antigen 1 Eimeria -specific protein Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Background Avian coccidiosis is a widespread, economically significant disease of poultry that results in annual global economic losses of approximately $2.4 billion, including both production losses and disease prevention and treatment costs [1]. It is an enteric disease caused by several species of the protozoan parasite Eimeria , predominantly E. brunetti , E. necatrix , E. tenella , E. acervulina , E. maxima , E. mitis , and E. praecox [2]. Of these, E. tenella is one of the species that causes hemorrhagic pathologies and high mortality. Eimeria belongs to the phylum Apicomplexa, which includes important pathogens of humans and domestic animals, such as the causative agents of malaria ( Plasmodium spp.), toxoplasmosis ( Toxoplasma gondii ), babesiosis ( Babesia spp.), and coccidiosis ( Eimeria spp.). Most apicomplexans are obligate intracellular parasites and are characterized by their apical complexes of specialized secretory organelles (micronemes, rhoptries, and dense granules) [3]. They use actin-based motility coupled to regulated protein secretion from their apical organelles to actively invade host cells [4]. These parasites have complex and diverse lifestyles that involve the invasion of many different cell types, including erythrocytes, lymphocytes, macrophages, and digestive-tract cells. Despite the diversity of their target host cells, they maintain a highly conserved mechanism for this active invasion process [5]. The host-cell invasion mechanism involves the following steps: attachment, apical reorientation, moving junction formation, and the formation of a protective parasitophorous vacuole. Each invasion step is mediated by various proteins, which are secreted from apical secretory organelles [6]. Apical membrane antigen 1 (AMA1), a type I transmembrane protein, is one of a number of proteins released from micronemes that are conserved across all apicomplexans. It is known to play several important roles during host-cell penetration [7]. For instance, previous reports have shown that antibodies against AMA1 or small specific AMA1-binding peptides inhibit the invasion of host cells by Toxoplasma , E. tenella, Babesia, Neospora , and Plasmodium [3,8–11]. AMA1 is also a long-standing effective candidate vaccine for some apicomplexans, including N. caninum , T. gondii and Plasmodium [11–13]. In Toxoplasma and Plasmodium , AMA1 is reportedly involved in apical reorientation [14], host-cell attachment [7, 15], invasion and establishment of the moving-junction[16], , and the provision of a signal that initiates intracellular replication [17]. In contrast to the functions of AMA1 in other apicomplexan parasites, there are only a few reports of this conserved protein in Eimeria . In a previous in vitro study, AMA1 antibodies or specific Et AMA1-binding peptides inhibited the invasion of host cells by E. tenella sporozoites [10, 18]. Et AMA1 also partially protected host cells against homologous challenge with E. tenella when used as a recombinant protein vaccine and against heterologous challenge with E. maxima when the AMA1 protein from E. maxima was expressed as a live vectored vaccine [19]. Although AMA1 plays an important role in host-cell invasion by E. tenella sporozoites, its precise functions are unknown. Proteins perform a vast number of cellular functions when they interact with one or multiple binding partners. Protein–protein interactions are essential in the mediation of almost all cellular processes, including replication, transcription, translation, and signal transduction [20]. The biochemical analysis of protein complexes and the identification of their components have been fundamental to our understanding of their biological functions in cells [21]. To understand the precise functions of Et AMA1 during host-cell invasion, we screened Et AMA1-interacting proteins with a yeast two-hybrid system and identified 14 putative Et AMA1-interacting proteins in a previous study [22]. E. tenella Eimeria -specific protein ( Et Esp) (GenBank accession number: JZ905773) is one of these putative interacting proteins. In this study, we cloned and characterized Et Esp. We systematically analyzed its interaction with Et AMA1 using bimolecular fluorescence complementation (BiFC) in vivo and a glutathione S-transferase (GST) pull-down assay in vitro . Our results show that the Et Esp is secreted protein, but not from micronemes, interacts with Et AMA1, and is involved in the invasion of host cells by E. tenella sporozoites. Methods Parasite collection E ． tenella was obtained from the Key Laboratory of Animal Parasitology of the Ministry of Agriculture, Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences，Shanghai, China. The parasites were maintained and propagated by passage through coccidia-free, 2-week-old chickens, as previously described [23]. Coccidia-free 14-day-old chickens were inoculated with 1 × 10 4 sporulated oocysts of E. tenella . Unsporulated oocysts (UO) were collected from infected chicken ceca at 7 days postinfection. Sporulated oocysts (SO) were derived from UO that had undergone sporulation in 2% potassium dichromate at a temperature 28–30 °C for 72–120 h, under forced aeration with a suitable pump. When more than 90% of the oocysts had sporulated, the oocysts were collected and purified. The sporozoites (Spz) were purified from cleaned SO with in vitro excystation [24]. Second-generation merozoites (sMrz) were isolated from infected chicken ceca at 115 h postinoculation, as described previously [25]. All parasites were collected and frozen in liquid nitrogen. Chickens and rabbits were fed and used according to a protocol approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. The chicken embryo fibroblast cell line, DF-1, a derivative of the East Lansing Line (ELL-0) (Jiang et al. 2012), was used for BiFC and in vitro infection experiments. Molecular cloning and sequence analysis of E . tenella -specific protein Total RNA was extracted from E. tenella sporozoites with TRIzol Reagent (Invitrogen, USA). GeneRacer™ primers (GR5P and GR5N) were provided for the random amplification of PCR ends (RACE) in the GeneRacer™ Kit (Invitrogen) and gene-specific primers (GS5P and GS5N) were designed based on the expressed sequence tag (EST) sequence (GenBank accession number: JZ905773) which is 790 bp in length and contains a poly(A) at the 3¢ end (Table S). The 5¢ end of this gene was determined according to the manufacturer’s protocol. The PCR-amplified fragment was then ligated into the pGEM-T Easy Vector (Promega, USA) and used to transform competent Escherichia coli TOP10 cells. After PCR identification, the plasmid DNA was sequenced. After the resulting sequence was assembled and aligned with the original EST sequence, the full-length cDNA sequence of the gene was determined and submitted to the National Center for Biotechnology Information (NCBI) GenBank (accession number: MN161778). The full-length Et Esp cDNA sequence was used in a BLAST search of GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and the E. tenella genome database (http://www.genedb.org/Homepage/Etenella). The deduced amino acid sequence was obtained with the ORF Finder tool at NCBI. The molecular mass and theoretical isoelectric point were calculated with ProtParam tools ( http://web.expasy.org/protparam/ ). The signal peptide sequence was identified with the SignalP 4.1 server ( http://www.cbs.dtu.dk/services/SignalP/ ), and transmembrane regions were predicted with the TMHMM server v. 2.0 ( http://www.cbs.dtu.dk/services/TMHMM/ ). Protein motifs were scanned with Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan). Recombinant protein expression and polyclonal anti-r Et Esp serum The Et Esp open reading frame (ORF) cDNA was amplified with PCR using primers Et Esp-UP and Et Esp-LP (Table S), which contained Bam HI and Xho I restriction sites, respectively. The PCR fragment was then ligated into the prokaryotic expression vector pET28a(+) digested with the same restriction endonucleases, to construct the recombinant expression plasmid pET– Et Esp. The recombinant protein His– Et Esp (r Et Esp) was expressed in Escherichia coli BL21 cells at 37 °C with 1 mM isopropyl-thio-α-d-galactoside. The cell pellet was lysed with sonication and digested with 10 µg/mL lysozyme (Sigma-Aldrich, USA). The lysate was then analyzed with 12% SDS-PAGE to confirm that the recombinant protein was present as a soluble protein or inclusion bodies. r Et Esp was purified with His·Bind® Resin (Merck, USA) and its concentration measured with a BCA Protein Assay Kit (Beyotime, China). Two 2-month-old male rabbits were inoculated with 200 μg of purified r Et Esp emulsified in Freund’s complete adjuvant (Sigma-Aldrich). After 14 days, a booster of 200 μg of purified r Et Esp in Freund’s incomplete adjuvant (Sigma-Aldrich) was administered, followed by a second and third booster on days 28 and 42. One week after the final booster, the rabbit serum was collected and stored at −20 °C until use. Analysis of Et Esp transcript levels with real-time quantitative PCR (qPCR) The expression profiles of Et Esp mRNA were examined in four developmental stages of E. tenella (UO, SO, Spz, and sMrz) with qPCR. cDNA samples were synthesized from DNaseI-treated total RNAs of the E. tenella developmental stages using SuperScript™ II Reverse Transcriptase (Invitrogen) and random pd(N)6 primer. The housekeeping gene 18S rRNA was used as the internal control. The primers used to amplify the Et Esp cDNA ( Et Esp-SP and Et Esp-AP) and the 18S rRNA gene (18S-SP and 18S-AP) were designed with Primer3 v. 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) (Table S). qPCR was performed with the StepOnePlus™ Real-Time PCR System using the SYBR® Premix Ex Taq™ II kit (Takara, Japan). All experiments were performed twice, with separate biological replicates. In each experiment, the reactions were performed in triplicate. A dilution series of cDNA templates of the sporozoites was used to establish standard curves, and all standard curves had correlation coefficients of R 2 > 0.99. The comparative 2 −ΔΔCt method was used to analyze the relative levels of gene expression. SDS-PAGE and western blotting Protein samples were prepared from the four E. tenella developmental stages (UO, SO, Spz, and sMrz), and from DF-1 cells transfected with the recombinant plasmids, for western blotting. The protein concentrations were determined with a BCA Protein Assay Kit (Beyotime,China). The purified r Et Esp and protein lysates were separated with SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes. Rabbit antiserum (1:100) against sporozoite proteins, previously produced in our laboratory [26], a rabbit anti-r Et Esp antibody (1:100), a mouse monoclonal anti-α-tubulin antibody (1:1000) (Sigma-Aldrich), and a monoclonal anti-His antibody (1:1000) were used as the primary antibodies to detect r Et Esp or native Et Esp. Naïve rabbit serum (1:100) was used as the negative control. IRDye-800CW-labelled goat anti-rabbit IgG antibody (1:25,000) and IRDye-680RD-labeled donkey anti-mouse IgG antibody (1:25,000; LI-Cor, Lincoln, NE, USA) were used as the secondary antibodies. The IRDyes were detected with the Odyssey Infrared Imaging System (LI-Cor). BiFC assay The ORF fragments of Et Esp and the Et AMA1 ectodomain, with no stop codon, were amplified from the first-strand cDNA with two pairs of primers (Bf Et Esp-UP/Bf Et Esp-LP and Bf Et AMA1-UP/Bf Et AMA1-LP, respectively), which contained Eco RI and Bgl II restriction sites ( Et Esp) or Eco RI and Xho II restriction sites ( Et AMA1). The fragments were then digested with the appropriate restriction enzymes and ligated into the pBiFC-VN155 and pBiFC-VC155 vectors digested with the same enzymes, respectively, to construct the recombinant plasmids pBiFC-VN155– Et Esp and pBiFC-VC155– Et AMA1, respectively. Before the BiFC assay, the uptake of the expression vectors by the cells was confirmed. DF-1 cells were transfected with the recombinant plasmid pBiFC-VN155– Et Esp or pBiFC-VC155– Et AMA1 using Lipofectamine™ 2000 Transfection Reagent (Invitrogen, USA), according to the manufacturer’s instructions. At 48 h after transfection, the cells were harvested and the proteins were extracted with RIPA Lysis Buffer (Beyotime). Western blots were probed with rabbit anti-r Et Esp antibody and rabbit anti-r Et AMA1 antibody, which were previously produced in our laboratory [10]. After confirmation that the cells had expressed the two constructs, DF-1 cells were cotransfected with pBiFC-VN155– Et Esp and pBiFC-VC155– Et AMA1. DF-1 cells were also cotransfected with pBiFC-bJunVN55 (I152L) and pBiFC-bFosVC155 or pBiFC-VC155– Et AMA1 and pBiFC-VN155 empty vector, or pBiFC-VN155– Et Esp and pBiFC-VC155 empty vector as the positive or negative control, respectively. The DF-1 cells were observed with fluorescence microscopy 24 h after transfection with the different constructs. GST pull-down To confirm the interaction between Et AMA1 and Et Esp317 in vitro , a GST pull-down assay was performed with the Pierce™ GST Protein Interaction Pull-Down Kit (Thermo Scientific, USA), according to the manufacturer’s instructions. The recombinant plasmid pGEX-6P– Et AMA1 was previously constructed in our laboratory [10]. The expression of the recombinant protein GST– Et AMA1 was induced and the protein purified with GST resin for use as the bait protein. The ORF of Et Esp was inserted into the pET-28a vector to express the recombinant protein His– Et Esp (r Et Esp) as the prey protein. GST– Et AMA1 was incubated with equilibrated glutathione-agarose to immobilize the bait protein. r Et Esp was then added to the glutathione-agarose and incubated with the bait protein. The bait and prey proteins were eluted from the glutathione-agarose. E. coli BL21 cells were transformed with recombinant plasmid pET– Et MIC2, constructed previously in our laboratory [27], to express the recombinant protein His– Et MIC2 as the negative control. Another, r Et Esp was loaded in an empty glutathione-agarose column as the negative. All the proteins were then resolved with 12% SDS-PAGE and detected with western blotting using the appropriate antibodies, as described above. Assay of Et Esp secretion Freshly excysted sporozoites (4 × 10 6 ) were incubated in 100 μL of complete medium (CM; Dulbecco’s modified Eagle’s medium [DMEM] supplemented with 10% fetal bovine serum [FBS], 100 U/mL penicillin/streptomycin, 2 mM l-glutamine) for 2 h at 41 °C under 5% CO 2 for the secretion experiments. They were then incubated with 5, 10, or 20 μM staurosporine (Sigma; dissolved in dimethylsulfoxide [DMSO]) or an appropriate volume of carrier DMSO, as described previously [28]. The secretion of Et MIC2 and Et GRA( Tg GRA7 homologous protein) was used as the control. The sporozoites were then pelleted by centrifugation for 10 min at 6000 × g. The supernatants and sporozoites were recovered and analyzed with western blotting using a rabbit anti-r Et Esp antibody and rabbit anti-r Et MIC2 antibody generated previously in our laboratory [27] and mouse anti- Tg GAR7 antibody which generated previous in other laboratory of our Institute . Immunofluorescence staining of parasites Purified differentially developed parasites (Spz, sporocysts [Sporo], and sMrz) were transferred to glass slides and air-dried, as previously described [10, 29]. Purified freshly sporozoites were infected DF-1 cells after incubation in CM for 2 h at 41°C. At different time points after infection, the DF-1 cells were collected, washed, transferred to glass slides, and air-dried. The slides were then fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) and placed in 1% Triton X-100 in PBS for 15 min to increase their permeability. Non permeabilized sporozoites and sporocysts were used as control. The slides were blocked with PBS containing 2% (w/v) bovine serum albumin for overnight at 4 °C. A rabbit anti-r Et Esp antibody (1:100) was added and the cells were incubated for 1 h at 37 °C. A 1:500 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) was then added and the cells incubated for 1 h at 37 °C. The cell nuclei were stained by incubation in 10 μg/mL 4¢,6-diamidino-2-phenylindole (Beyotime) at room temperature for 10 min. After each step, the slides were washed three times for 10 min each with PBS containing 0.05% Tween 20. The slides were finally mounted with 50 μL of Fluoromount Aqueous Mounting Medium (Sigma-Aldrich) before observation with a fluorescence microscope (Olympus, Tokyo, Japan). At the same time, we performed the co-localization of Et Esp and Et AMA1 in sporozoites. Purified sporozoites were treated with mouse anti-r Et AMA1 antibody (1:100) and rabbit anti-r Et Esp antibody(1:100), then goat anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated antibody(1:500) and goat anti-mouse IgG cyanine (Cy3)-conjugated antibody (Sigma-Aldrich)(1:500) were secondary antibody. Invasion inhibition assay in vitro The invasion inhibition assay was based on previous reports the invasion of DF-1 cells by E. tenella sporozoites. Antibodies were purified with Protein A+G Agarose (Beyotime). DF-1 cells (2 × 10 5 cells per well) were cultured in 24-well plates (Corning) in CM for 24 h at 37 °C under 5% CO 2 . The freshly purified sporozoites were counted and labeled with carboxyfluorescein diacetate succinimidyl ester (Beyotime). The labeled sporozoites were incubated at 37 °C with 50, 100, 200, 300 or 400 μg/mL purified IgG directed against r Et Esp for 2 h. The same quantity of IgG from naïve rabbit serum (Sigma-Aldrich) was used as the negative control, and an equivalent volume of PBS as the normal control. After they were washed twice with sterile PBS, DF-1 cells (10 5 /well) were infected with the labeled sporozoites (10 5 /well) in 24-well plates and cultured for 16 h at 41 °C under 5% CO 2 . The cells were then collected and analyzed with flow cytometry on a Cytomics™ FC 500 (Beckman Coulter, Indianapolis, IN, USA). The controls were uninfected DF-1 cells. The infected cells, uninfected cells, and free sporozoites were gated with the CXP software to count the infected (labeled sporozoites) and uninfected (fluorescence-free) cells. All assays were performed in triplicate. The percentages of infected cells in the presence or absence of an anti-r Et Esp polyclonal antibody were used to calculate the inhibition rates, as previously described [10]. Results Cloning and sequence analysis of full-length Et Esp cDNA The 1108-bp full-length cDNA of Et Esp was obtained with RACE. A sequence analysis showed that the full-length cDNA included a 5¢-untranslated region (UTR) of 70 bp, a 3¢-UTR of 542 bp with a poly(A) tail, and an ORF of 501 bp, which encoded 166 amino acids with a calculated molecular weight of 18.1 kDa and a theoretical isoelectric point of 4.2 (Fig. 1). Analysis of the amino acid sequence showed a signal peptide of 19 amino acids at the N-terminus and no transmembrane region. Searches in the Motif Database and the Conserved Domain Database revealed the presence of one N-glycosylation site, five casein kinase II phosphorylation sites, six N-myristoylation sites, one tyrosine kinase phosphorylation site, one intein DOD-type homing endonuclease domain, and no conserved domains (Fig. 1). A BLAST search of the E. tenella genome database showed that the ORF sequence shared 100% sequence identity with ETH_00016590, which encodes an Eimeria -specific protein, on supercontig Eth_scaff124: 9216–10141. The amino acid sequence shared 100% homology with the E. tenella Eimeria -specific protein (XP_013228647.1) and 92% (152/170) identity with the E. necatrix Eimeria -specific protein (XP_013435139.1) in NCBI. Therefore, this gene was designated Et Esp and submitted to NCBI GenBank (GenBank accession no. MN161778). It also shared 68% (106/157) amino acid identity with E. brunetti conserved hypothetical protein (CDJ53027.1), 63% (108/172) identity with E. praecox conserved hypothetical protein (CDI81636.1), 74% (97/131) identity with E. maxima conserved hypothetical protein (XP_013336310.1), and 73% (91/124) identity with E. acervulina conserved hypothetical protein (XP_013248166.1). Also, this protein is not found in other apicomplexa parasites. These results show that the protein is conserved in Eimeria spp. Expression and characterization of recombinant Et Esp r Et Esp was expressed as a His6-tagged fusion protein. SDS-PAGE showed that r Et Esp was mainly present in the soluble fraction of the bacterial lysate. After the purification of r Et Esp with Ni-NTA chromatography, a protein of approximately 21 kDa was observed with SDS-PAGE. Because 3 kDa of the fusion protein was derived from the vector, the predicted molecular mass of Et Esp was about 18.1 kDa. Western blotting showed that purified r Et Esp was recognized by rabbit serum directed against sporozoites and by a monoclonal anti-His6 antibody. Naïve rabbit serum failed to recognize any protein corresponding to the expected size of r Et Esp (Fig. 2). These results indicate that r Et Esp was recognized specifically by rabbit serum directed against a soluble sporozoite protein and by a monoclonal anti-His antibody. Et Esp mRNA and protein expression at different developmental stages of E. tenella qPCR was used to analyze the UO, SO, Spz, and sMrz stages of E. tenella for the presence of Et Esp mRNA. The levels of Et Esp mRNA were much higher in the sMrz stage than in the other three stages, and Et Esp mRNA was almost undetectable in UO (Fig. 3a). The expression of Et Esp in the four developmental stages was also determined with immunoblotting using rabbit antiserum against r Et Esp. A monoclonal anti-α-tubulin antibody was used as the control. Western blotting showed that the anti-r Et Esp antibody reacted with a band of approximately 18 kDa in the parasite lysates prepared from the four different developmental stages of E. tenella The expression levels of Et Esp were higher in sporozoites than in other three stages(Fig. 3b,c). Confirmation of the interaction between Et AMA1 and Et Esp To characterize the interaction between Et AMA1 and Et Esp in vivo , a BiFC assay was performed. For the BiFC assay, fragments of the Et Esp ORF and the Et AMA1 ectodomain sequence were cloned into the plasmids pBiFC-VN155 and pBiFC-VC155, respectively, to generate the constructs pBiFC-VN155– Et Esp and pBiFC-VC155– Et AMA1, respectively. The total proteins were extracted from DF-1 cells transfected separately with one or other construct. Western blotting showed that the two constructs were expressed individually in the DF-1 cells at 48 h after transfection (Fig. 4a). Strong green fluorescence was observed in DF-1 cells 48 h after they were cotransfected with both constructs. Green fluorescence was also observed in the positive control. However, there was no visible fluorescence in the DF-1 cells cotransfected with pBiFC-VC155– Et AMA1 and pBiFC-VN155 empty vector or pBiFC-VN155– Et Esp and pBiFC-VC155 empty vector. These results indicate that Et Esp interacts with Et AMA1 in cells (Fig. 4b). GST pull-down To confirm the interaction between Et AMA1 and Et Esp in vitro , a GST pull-down assay was performed. GST– Et AMA1 and His– Et Esp were expressed individually in E. coli and purified. GST– Et AMA1 was bound to an equilibrated glutathione–agarose column, and then His– Et ESP was added to the column. The proteins bound to the glutathione–agarose, and any nonspecifically bound proteins were removed by elution with buffer. The proteins retained on the column were then eluted and detected with immunoblotting using anti-r Et AMA1 and anti-r Et Esp antibodies (Fig. 5). The results clearly indicated a direct interaction between the Et AMA1 and Et Esp proteins. Et Esp is not secreted from the microneme To examine the secretion of Et Esp, sporozoites were incubated in CM at 41 °C. The supernatant containing the excretory–secretory antigens (ESA) from the incubated sporozoites and sporozoites pellets were analyzed with western blotting. Immunoblots of the ESA samples and sporozoites were probed with an anti-r Et Esp antibody and showed that Et Esp was secreted when the sporozoites were incubated at 41 °C under 5% CO 2 in CM. Rabbit serum raised against the micronemal protein Et MIC2 was used as the experimental control [27]. To demonstrate whether Et Esp secretion is dependent on the micronemal pathway, we added staurosporine to the CM because staurosporine is a protein kinase inhibitor known to specifically inhibit microneme secretion [28]. In the parasites treated with 5, 10, or 20 μM staurosporine, the secretion of Et Esp and Et GRA into the supernatant was not affected, but the secretion of Et MIC2 in supernatant was significant reduced compared with their secretion in the presence of the DMSO solvent only (Fig. 6a,b). These results show that Et Esp is a secreted protein, but not a micronemal protein. Immunolocalization of Et Esp at different developmental stages of E. tenella To investigate the localization and distribution of the Et Esp protein in different development stages of E. tenella , including sporozoites, second-generation merozoites, immature schizonts, and mature schizonts, it was localized with immunofluorescence in vitro using an antibody against r Et Esp. The Et Esp protein was mainly distributed on the surfaces of the permeabilized parasite sporozoites, sporocysts, and second-generation merozoites (Fig. 7a-a, b-a, k,). The protein were also mainly located on the surface of non permeabilized sporozoites and sporocysts (Fig. 7a-b, b-b). After incubation in CM for 2 h, the fluorescence increased and mainly localized to the anterior and surface of parasites (Fig. 7c). Et Esp protein was also mainly located on the surfaces of parasites 2 h after their invasion of DF-1 cells (Fig. 7d). At 12 h after the sporozoites were added to DF-1 cells, Et Esp also localized to the cytoplasm of the sporozoites, except for the refractile body in the posterior section of the parasites, and the intensity of Et Esp staining had increased (Fig. 7e). At 24–72 h postinfection, the Et Esp protein was uniformly distributed in trophozoites, immature schizonts, and mature schizonts, and the protein’s expression had increased (Fig. 7e–j). Co-localization of Et AMA1 and Et Esp IFAs were performed to determine the location of Et AMA1 and Et Esp. Purified sporozoites were treated with mouse anti-r Et AMA1 antibody and rabbit anti-r Et Esp antibody. The results showed Et Esp was mainly located on the surface of sporozoites, Et AMA1 was distributed throughout the cytoplasm and the membrane of sporozoites with the exception of refractile bodies (Fig 8). Anti-r Et Esp antibodies inhibit DF-1 cell invasion To evaluate the effect of the Et Esp protein on the invasion of DF-1 cells by E. tenella sporozoites, an invasion inhibition assay of sporozoites was performed in vitro . When the sporozoites were incubated with purified anti-r Et Esp antibody before infection, their capacity to invade the DF-1 cells was significantly reduced. After pretreatment with 50, 100, 200, 300, or 400 μg/mL anti- rEt Esp IgG antibody, their invasion of cells was highly significantly reduced compared with that of sporozoites treated with naïve rabbit IgG (negative control) (P < 0.01). Under these experimental conditions, an inhibition plateau of 62.9% was reached at an antibody concentration of 300 μg/mL. In a comparative analysis, the same dose of the naïve rabbit serum IgG antibody did not significantly affect invasion (Fig. 9). Discussion In this study, we cloned and characterized the E. tenella Eimeria -specific protein, a putative Et AMA1-interacting protein, using a yeast two-hybrid system in our laboratory [22]. Although the yeast two-hybrid system is a widely used and powerful method for identifying the partners of proteins in regulatory complexes and in the analysis of protein–protein interactions [30], the system has several limitations, including the possibility of isolating very large numbers of clones with no biological relevance [31]. Therefore, the interaction between Et AMA1 and Et Esp required validation with an alternative technique, such as a BiFC assay or GST pull-down assay. The BiFC assay is a versatile technique for investigating protein–protein interactions in living systems, and is based on the reconstitution of a fluorescent protein in vivo [32]. GST pull-down is amenable to more specific investigations of protein–protein interactions in vitro , but relies on purified proteins that may not fully mimic the protein’s native conformation or posttranslational modification, which mediate its interactions [33]. Although these assays have some advantages in identifying protein–protein interactions, each also has its drawbacks. Therefore, in many research fields, these methods are often combined to identify the interactions between two proteins [33–35]. In this study, the interaction between Et AMA1 and Et Esp was confirmed with a GST pull-down assay in vitro and a BiFC assay in vivo . These results indicated that Et Esp interacts with Et AMA1. Proteins perform a vast number of cellular functions through their interactions with one or multiple binding partners. Moreover, many protein–protein interactions are regulated by posttranscriptional modification (e.g., phosphorylation) of the protein of interest, and these modifications are induced by exposure to certain circumstances [36]. In this study, an amino acid sequence analysis predicted that Et Esp contains one N-glycosylation site, five casein kinase II phosphorylation sites, six N-myristoylation sites, one tyrosine kinase phosphorylation site and one intein DOD-type homing endonuclease domain. Inteins, also called protein introns, are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins[37]. These data suggest that its functions may be regulated by posttranslational modification. We supposed that the interaction of Et AMA1 with Et Esp may be regulated by posttranslational modification. To understand the expression of Et Esp in the different developmental stages of parasite, we examined its expression patterns with qPCR and western blotting. Our results indicated that Et Esp mRNA levels were higher in second-generation merozoites and sporozoites than in sporulated oocysts or unsporulated oocysts. But western blotting showed that the expression of Et Esp was higher in sporozoites than other developmental stages of E.tenella. Immunofluorescent localization showed that the expression of the protein increased with the development of the parasites in DF-1 cells. Previous proteomic and transcriptomic data confirm that Et AMA paralogues are tightly stage-regulated [38, 39]. Et AMA1 is a sporozoite-specific protein involved in the invasion process of sporozoites [10, 19, 38]. While another Et AMA1 paralogues, Et AMA2 is a merozoites-specific protein not involved in the parasite invasion. All these finding indicate that E. tenella parasites harbour stage-specific AMA proteins that could be relevant during specific phases of the parasite cycle [19]. In this study, Et Esp is differentially expressed during the distinct phases of the parasite life cycle, and may be very important in the invasion and development of the parasite life cycle. Immunofluorescent localization showed that Et Esp was located on the surface of the sporozoite and concentrated around the anterior of the parasite during its incubation in CM. However, the protein has no transmembrane region or glycophosphatidyl inositol (GPI)-anchor sequence, but has a signal peptide, and six N-myristoylation sites. The presence of a signal peptide is necessary for the translocation of proteins from their ribosomal sites of translation into the lumen of the endoplasmic reticulum, from where they are trafficked in the endomembrane system to their final locations within the cell or beyond [40]. We speculated that Et Esp also undergoes posttranslational modification according to the amino acid sequence analysis, including phosphorylation, myristoylation, and glycosylation. Among these modifications, myristoylation is the key factor in the membrane localization of signal-transducing proteins [41].. Most surface antigens are involved in the invasion, pathogenesis, and immune evasion of parasites. For example, in Plasmodium , merozoite surface proteins are critical for parasite invasion, and represent attractive targets for antibody-based therapies against clinical malaria [42]. We also found that the expression of Et Esp increased and that the protein mainly localized on the anterior and surface of the parasites after incubation in CM for 2 h. This suggests that the protein is involved in the sporozoite invasion of host cells. To investigate the function of Et Esp in the invasion process, we performed an invasion test in vitro and found that polyclonal rabbit anti-r Et Esp serum efficiently reduced the sporozoite invasion of cultured DF-1 cells. Et AMA1 also localized to the anterior of the sporozoites after their invasion of DF-1 cells [10]. Previous reports have shown that monospecific mouse anti-r Et AMA1 serum or polyclonal rabbit antiserum against r Et AMA1 also blocked the invasion of host cells in vitro [10,19]. . In this study, Et Esp is involved in invasion as demonstrated by using antibodies raised against Et Esp in vitro. We tested that Et Esp is an interacting protein with Et AMA1 by using BiFC, GST-pull down and yeast two-hybrid system.. Therefore, we speculated that Et Esp mediates sporozoites invasion in host cells by interacting with Et AMA1. The exact function of Et Esp needs further study. Previous studies have shown that in T. gondii and Plasmodium , AMA1 interacts directly with rhoptry neck protein 2 (RON2), which is secreted from the parasite rhoptries and specifically localizes at the moving junction. The RON2–AMA1 interaction is a critical step in the moving-junction-dependent invasion of host cells by apicomplexan parasites [43, 44]. Although the interaction between AMA1 and RON2 has not been reported in Eimeria spp., E. tenella is an apicomplexan and AMA1 is conserved in this phylum. Therefore, we inferred that Et AMA1 may also interact with Et RON2 and specifically localize to the moving junction during the invasion of host cells by E. tenella . In this study, we have shown that Et Esp is an Et AMA1-interacting protein, but whether it localizes to the moving junction during invasion requires further study. Conclusions In this study, we have shown that interacts with Et AMA1 using a BIFC assay in vivo and a GST pull-down assay in vitro . Using staurosporine, we showed that Et Esp is a secreted protein of sporozoites but not from micronemes. An invasion inhibition assay revealed that an antibody against r Et Esp also blocked the parasite invasion of its host cells by more than 62%. These data have implications for the use of Et AMA1 or Et AMA1-interacting proteins as targets in therapeutic intervention strategies against avian coccidiosis. Supplementary Information Additional file 1 : Table S. Primers sequence used in this study. Abbreviations Et AMA1: Eimeria tenella apical membrane antigen 1; Et Esp: E. tenella Eimeria -specific protein; BiFC: bimolecular fluorescence complementation; GST pull-down: glutathione S-transferase pull-down; RACE: random amplification of PCR ends; UO: unsporulated oocysts; SO: sporulated oocysts; Spz: sporozoites; sMrz: second-generation merozoites; NCBI: the National Center for Biotechnology Information; ORF: open reading frame; CM: complete medium. Declarations Acknowledgments We would like to thank all organizations which funded this work and all the teachers who cooperated in technical assistance and Dr Wang quan who provided mouse anti-TgGRA7 antibody of T.gondii . Declarations Ethics approval and consent to participate The protocol was approved and authorized by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences. Consent for publication Not applicable. Availability of data and materials Not applicable. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the National Natural Science Foundation of China (Grant Nos. 31572266 and 31672551) and National Key Research and Development Program of China (2018YFD0500302) and National Sharing Service Platform for Parasite Resources (No. TDRC-22) and Shanghai Minhang District talent development special funds. 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Shen B, Sibley The moving junction, a key portal to host cell invasion by apicomplexan parasites. Curr Opin Microbiol. 2012; 15:449-55. Tables Table 1. Primers sequence used in this study Primer name Primer sequence GS5P (5' Primer) 5'-ATGGTCTCGGGCCAGTTCTCGTTCA -3' GS5N (5' Nested Primer) 5'-GGAGATGAGACCCAGGCGGATGAAA -3' Et Esp-UP 5'-GCGGATCCATGAAGGGCCTGTTCTTCACCGTCG-3' Et Esp-LP 5'-GCCTCGAGCGAATCTACTTCAAGAAAAGCCACG-3' Et Esp -SP 5'-CCCCGACTACCTCAAGTTCCTCAGC -3' Et Esp -AP 5'-TGGGTCCGTCTCCCCCTCCTTGGTG -3' 18S-SP 5'-TGTAGTGGAGTCTTGGTGATTC-3' 18S-AP Bf Et ESp-UP Bf Et ESp-LP Bf Et AMA1-UP Bf Et AMA1-LP 5'-CCTGCTGCCTTCCTTAGATG-3' 5'-GCGAATTCGGGCCACCATGAAGGGCCTGTTCTT-3' 5'-GCAGATCTGCTGCTCGCGTTGCCAGCAGAT -3' 5'-GCGAATTCGGGCCACCATGCAGCCGCCCTAT-3' 5'-GCCTCGAGGGTATTCCTGGTCCAG-3' Supplementary Files supplement1.doc Cite Share Download PDF Status: Published Journal Publication published 25 Jul, 2020 Read the published version in Parasites & Vectors → Version 3 posted Editor assigned by journal 09 Jun, 2020 Submission checks completed at journal 08 Jun, 2020 Editor invited by journal 08 Jun, 2020 You are reading this latest preprint version Show more versions Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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11:43:50","currentVersionCode":3,"declarations":"","doi":"10.21203/rs.2.17982/v3","doiUrl":"https://doi.org/10.21203/rs.2.17982/v3","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13071-020-04229-5","type":"published","date":"2020-07-25T12:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":1324378,"identity":"27beb894-6cca-48d2-a36d-29c2443c7d41","added_by":"auto","created_at":"2020-06-13 20:13:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1086190,"visible":true,"origin":"","legend":"Nucleotide sequence of the full-length cDNA of EtEsp and the deduced amino acid sequence. Start and stop codons are underlined. One putative intein DOD-type homing endonuclease domain is shown with wavy underlining. A putative N-glycosylation site has a double line. Five putative casein kinase II phosphorylation sites are shown in yellow. Six putative N-myristoylation sites are shown in gray.","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig1.jpg"},{"id":1324379,"identity":"e85da864-90e8-4210-98c4-db8456f68761","added_by":"auto","created_at":"2020-06-13 20:13:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29853,"visible":true,"origin":"","legend":"Immunogenicity of rEtEsp. rEtEsp protein was subjected to western blotting. Lane M: protein marker; lane 1: anti-His-tag monoclonal antibody as the primary antibody; lane 2: rabbit serum against sporozoites as the primary antibody; lane 3: naïve rabbit IgG as the primary antibody.","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig2.jpg"},{"id":1324380,"identity":"0daaf364-476e-4093-a6ba-e99acde6e626","added_by":"auto","created_at":"2020-06-13 20:13:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":63622,"visible":true,"origin":"","legend":"EtEsp expression at different developmental stages of E. tenella. UO, unsporulated oocysts; SO, sporulated oocysts; Spz, sporozoites; sMrz, second-generation merozoites. a. qPCR of EtEsp at different developmental stages of E. tenella. b. Western blot showing EtEsp at different developmental stages, probed with rabbit anti-rEtEsp serum or mouse monoclonal anti-α-tubulin antibody. c. The densitometric intensity of western blot images was analyzed using ImageJ software.\n\n","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig3.jpg"},{"id":1324381,"identity":"ca62b19e-ea74-400c-8c9c-068eaa8e25c5","added_by":"auto","created_at":"2020-06-13 20:13:49","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":68662,"visible":true,"origin":"","legend":"Interaction between EtAMA1 and EtEsp in DF-1 cells assessed with BiFC. a. DF-1 cells were transfected with VC155–EtAMA1 and VN155–EtEsp and the cellular lysates were analyzed with immunoblotting using antisera against EtAMA1 and EtEsp, 1. anti-r EtAMA1 antibody;2. anti-r EtEsp antibody. b. BiFC was performed. a, DF-1 cells were cotransfected with VC155–EtAMA1 and VN155–EtEsp. b, DF-1 cells were cotransfected with positive controls bFos and bJun. c, DF-1 cells were cotransfected with pBiFC-VC155–EtAMA1 and pBiFC-VN155 empty vector. . d, DF-1 cells were cotransfected with pBiFC-VN155–EtEsp and pBiFC-VC155 empty vector. ","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig4.jpg"},{"id":1324382,"identity":"10f93468-873c-415f-8d87-3e2af84aba06","added_by":"auto","created_at":"2020-06-13 20:13:49","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":43930,"visible":true,"origin":"","legend":"In vitro pull-down assay between EtAMA1 and EtEsp. Lane M: protein marker; lane 1:rEtMIC2 protein incubated with rEtEsp，detected with anti-rEtAMA1 and anti-rEtMIC2 as negative control; lane 2: rEtAMA1 protein incubated with rEtEsp，detected with anti-rEtAMA1 and anti-rEtEsp; lane 3: rEtEsp loaded in an empty glutathione-agarose column, detected with anti-rEtEsp as negative control.\n\n","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig5.jpg"},{"id":1324383,"identity":"9986abaa-2277-4eca-a52f-e59c065013d9","added_by":"auto","created_at":"2020-06-13 20:13:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":76640,"visible":true,"origin":"","legend":"Western blotting analysis of secretion assays (supernatants and sporozoites pellet). (a): the supernants(Sup); (b): sporozoites pellet(Spz). Lane M: protein marker; lanes 1–3: 5, 10, and 20 μM staurosporine dissolved in DMSO.; lanes 4–6: volumes of DMSO solvent corresponding to 5, 10, or 20 μM staurosporine ","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig6.jpg"},{"id":1324384,"identity":"245bca10-ed7a-411e-b3d1-20b8b325db32","added_by":"auto","created_at":"2020-06-13 20:13:49","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":138542,"visible":true,"origin":"","legend":"Immunofluorescent localization of EtEsp at different developmental stages of E. tenella. Parasites were immunostained with anti-rEtEsp antibody. a, Sporozoites (Spz) were incubated in PBS, a-a permeabilized sporozoites, a-b: non permeabilized sporozoites; b, sporocysts (Sporo) were incubated in PBS, b-a: permeabilized sporocysts, b-b: non permeabilized sporocysts; c, sporozoites (Spz) were incubated in complete medium (CM) for 2 h at 41 °C; d, e, intracellular sporozoites (iSpz) at 2 h and 12 h postinfection, respectively; f, trophozoites (iTropho) at 24 h postinfection; g, h, immature schizonts (iSc) at 48 and 60 h postinfection, respectively; i, mature schizonts (mSc) at 68 h postinfection; j, first-generation merozoites (fMrz) at 72 h postinfection; k, second-generation merozoites (sMrz) in PBS.","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig7.jpg"},{"id":1324385,"identity":"a5a66ab5-4bb4-45f8-a76a-5f69b4f20bf0","added_by":"auto","created_at":"2020-06-13 20:13:50","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":147638,"visible":true,"origin":"","legend":"Co-localization of EtEsp and EtAMA1 in sporozoites using mouse anti-rEtAMA1 antibody and rabbit anti-rEtEsp antibody by IFA.","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig8.jpg"},{"id":1324386,"identity":"0a3afc3a-94a0-47fb-98ef-8ab80521fbe9","added_by":"auto","created_at":"2020-06-13 20:13:50","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":142625,"visible":true,"origin":"","legend":"Inhibition of sporozoite invasion in vitro. All the assays were performed in triplicate (anti-rEtEsp, rabbit antiserum generated against recombinant EtESP protein; NI, IgG from naïve rabbit serum). **P \u003c 0.01 for differences between treatment with antibody against rEtEsp or with naïve rabbit serum with the same IgG concentration.","description":"","filename":"Fig9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/Fig9.jpg"},{"id":15667426,"identity":"cb2f3719-b5d2-48b3-9f72-26d4c844725a","added_by":"auto","created_at":"2021-11-18 13:42:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1071642,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/b0e7a03e-96ab-44e0-a577-4210cb106003.pdf"},{"id":2626073,"identity":"f28f9441-50a9-4954-b182-5b69fee3d598","added_by":"d7e98867-8a6f-41f6-a6c5-81a52a808679","created_at":"2020-09-25 21:02:30","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":722944,"visible":true,"origin":"","legend":"","description":"","filename":"supplement1.doc","url":"https://assets-eu.researchsquare.com/files/rs-8611/v3/supplement_1.doc"}],"financialInterests":"","formattedTitle":"\u003cp\u003e\t\u003cem\u003eEimeria tenella Eimeria\u003c/em\u003e-specific protein that interacts with apical membrane antigen 1 (\u003cem\u003eEt\u003c/em\u003eAMA1) is involved in host cell invasion\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eAvian coccidiosis is a widespread, economically significant disease of poultry that results in annual global economic losses of approximately $2.4 billion, including both production losses and disease prevention and treatment costs [1]. It is an enteric disease caused by several species of the protozoan parasite \u003cem\u003eEimeria\u003c/em\u003e, predominantly \u003cem\u003eE. brunetti\u003c/em\u003e, \u003cem\u003eE. necatrix\u003c/em\u003e, \u003cem\u003eE. tenella\u003c/em\u003e, \u003cem\u003eE. acervulina\u003c/em\u003e, \u003cem\u003eE. maxima\u003c/em\u003e,\u003cem\u003e E. mitis\u003c/em\u003e, and \u003cem\u003eE. praecox \u003c/em\u003e[2]. Of these, \u003cem\u003eE. tenella\u003c/em\u003e is one of the species that causes hemorrhagic pathologies and high mortality. \u003cem\u003eEimeria \u003c/em\u003ebelongs to the phylum Apicomplexa, which includes important pathogens of humans and domestic animals, such as the causative agents of malaria (\u003cem\u003ePlasmodium \u003c/em\u003espp.), toxoplasmosis (\u003cem\u003eToxoplasma gondii\u003c/em\u003e), babesiosis (\u003cem\u003eBabesia\u003c/em\u003e spp.), and coccidiosis (\u003cem\u003eEimeria \u003c/em\u003espp.). Most apicomplexans are obligate intracellular parasites and are characterized by their apical complexes of specialized secretory organelles (micronemes, rhoptries, and dense granules) [3]. They use actin-based motility coupled to regulated protein secretion from their apical organelles to actively invade host cells [4]. These parasites have complex and diverse lifestyles that involve the invasion of many different cell types, including erythrocytes, lymphocytes, macrophages, and digestive-tract cells. Despite the diversity of their target host cells, they maintain a highly conserved mechanism for this active invasion process [5].\u003c/p\u003e\n\u003cp\u003eThe host-cell invasion mechanism involves the following steps: attachment, apical reorientation, moving junction formation, and the formation of a protective parasitophorous vacuole. Each invasion step is mediated by various proteins, which are secreted from apical secretory organelles [6]. Apical membrane antigen 1 (AMA1), a type I transmembrane protein, is one of a number of proteins released from micronemes that are conserved across all apicomplexans. It is known to play several important roles during host-cell penetration [7]. For instance, previous reports have shown that antibodies against AMA1 or small specific AMA1-binding peptides inhibit the invasion of host cells by \u003cem\u003eToxoplasma\u003c/em\u003e, \u003cem\u003eE. tenella,\u003c/em\u003e \u003cem\u003eBabesia, Neospora\u003c/em\u003e, and \u003cem\u003ePlasmodium\u003c/em\u003e [3,8\u0026ndash;11]. AMA1 is also a long-standing effective candidate vaccine for some apicomplexans, including \u003cem\u003eN. caninum\u003c/em\u003e, \u003cem\u003eT. gondii\u003c/em\u003e and \u003cem\u003ePlasmodium\u003c/em\u003e [11\u0026ndash;13]. In \u003cem\u003eToxoplasma\u003c/em\u003e and \u003cem\u003ePlasmodium\u003c/em\u003e, AMA1 is reportedly involved in apical reorientation [14], host-cell attachment [7, 15], invasion and establishment of the moving-junction[16], , and the provision of a signal that initiates intracellular replication [17].\u003c/p\u003e\n\u003cp\u003eIn contrast to the functions of AMA1 in other apicomplexan parasites, there are only a few reports of this conserved protein in \u003cem\u003eEimeria\u003c/em\u003e. In a previous \u003cem\u003ein vitro\u003c/em\u003e study, AMA1 antibodies or specific \u003cem\u003eEt\u003c/em\u003eAMA1-binding peptides inhibited the invasion of host cells by \u003cem\u003eE. tenella\u003c/em\u003e sporozoites [10, 18]. \u003cem\u003eEt\u003c/em\u003eAMA1 also partially protected host cells against homologous challenge with \u003cem\u003eE. tenella\u003c/em\u003e when used as a recombinant protein vaccine and against heterologous challenge with \u003cem\u003eE. maxima\u003c/em\u003e when the AMA1 protein from \u003cem\u003eE. maxima \u003c/em\u003ewas expressed as a live vectored vaccine [19]. Although AMA1 plays an important role in host-cell invasion by \u003cem\u003eE. tenella\u003c/em\u003e sporozoites, its precise functions are unknown.\u003c/p\u003e\n\u003cp\u003eProteins perform a vast number of cellular functions when they interact with one or multiple binding partners. Protein\u0026ndash;protein interactions are essential in the mediation of almost all cellular processes, including replication, transcription, translation, and signal transduction [20]. The biochemical analysis of protein complexes and the identification of their components have been fundamental to our understanding of their biological functions in cells [21].\u003c/p\u003e\n\u003cp\u003eTo understand the precise functions of \u003cem\u003eEt\u003c/em\u003eAMA1 during host-cell invasion, we screened \u003cem\u003eEt\u003c/em\u003eAMA1-interacting proteins with a yeast two-hybrid system and identified 14 putative \u003cem\u003eEt\u003c/em\u003eAMA1-interacting proteins in a previous study [22]. \u003cem\u003eE. tenella \u003c/em\u003e\u003cem\u003eEimeria\u003c/em\u003e -specific protein (\u003cem\u003eEt\u003c/em\u003eEsp) (GenBank accession number: JZ905773) is one of these putative interacting proteins. In this study, we cloned and characterized \u003cem\u003eEt\u003c/em\u003eEsp. We systematically analyzed its interaction with\u003cem\u003e Et\u003c/em\u003eAMA1 using bimolecular fluorescence complementation (BiFC) \u003cem\u003ein vivo \u003c/em\u003eand a glutathione S-transferase (GST) pull-down assay \u003cem\u003ein vitro\u003c/em\u003e. Our results show that the \u003cem\u003eEt\u003c/em\u003eEsp is secreted protein, but not from micronemes, interacts with \u003cem\u003eEt\u003c/em\u003eAMA1, and is involved in the invasion of host cells by \u003cem\u003eE. tenella\u003c/em\u003e sporozoites.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eParasite collection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eE\u003c/em\u003e\u003cem\u003e．\u003c/em\u003e\u003cem\u003etenella \u003c/em\u003ewas obtained from the Key Laboratory of Animal Parasitology of the Ministry of Agriculture, Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences，Shanghai, China. The parasites were maintained and propagated by passage through coccidia-free, 2-week-old chickens, as previously described [23]. Coccidia-free 14-day-old chickens were inoculated with 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e sporulated oocysts of\u003cem\u003e E. tenella\u003c/em\u003e. Unsporulated oocysts (UO) were collected from infected chicken ceca at 7 days postinfection. Sporulated oocysts (SO) were derived from UO that had undergone sporulation in 2% potassium dichromate at a temperature 28\u0026ndash;30 \u0026deg;C for 72\u0026ndash;120 h, under forced aeration with a suitable pump. When more than 90% of the oocysts had sporulated, the oocysts were collected and purified. The sporozoites (Spz) were purified from cleaned SO with \u003cem\u003ein vitro\u003c/em\u003e excystation [24]. Second-generation merozoites (sMrz) were isolated from infected chicken ceca at 115 h postinoculation, as described previously [25]. All parasites were collected and frozen in liquid nitrogen.\u003c/p\u003e\n\u003cp\u003eChickens and rabbits were fed and used according to a protocol approved by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.\u003c/p\u003e\n\u003cp\u003eThe chicken embryo fibroblast cell line, DF-1, a derivative of the East Lansing Line (ELL-0) (Jiang et al. 2012), was used for BiFC and \u003cem\u003ein vitro\u003c/em\u003e infection experiments.\u003c/p\u003e\n\u003ch1\u003eMolecular cloning and sequence analysis of \u003cem\u003eE\u003c/em\u003e\u003cem\u003e.\u003c/em\u003e\u003cem\u003e tenella\u003c/em\u003e-specific protein\u003c/h1\u003e\n\u003cp\u003eTotal RNA was extracted from \u003cem\u003eE. tenella\u003c/em\u003e sporozoites with TRIzol Reagent (Invitrogen, USA). GeneRacer\u0026trade; primers (GR5P and GR5N) were provided for the random amplification of PCR ends (RACE) in the GeneRacer\u0026trade; Kit (Invitrogen) and gene-specific primers (GS5P and GS5N) were designed based on the expressed sequence tag (EST) sequence (GenBank accession number: JZ905773) which is 790 bp in length and contains a poly(A) at the 3\u0026cent; end (Table S). The 5\u0026cent; end of this gene was determined according to the manufacturer\u0026rsquo;s protocol. The PCR-amplified fragment was then ligated into the pGEM-T Easy Vector (Promega, USA) and used to transform competent\u003cem\u003e Escherichia coli\u003c/em\u003e TOP10 cells. After PCR identification, the plasmid DNA was sequenced. After the resulting sequence was assembled and aligned with the original EST sequence, the full-length cDNA sequence of the gene was determined and submitted to the National Center for Biotechnology Information (NCBI) GenBank (accession number: MN161778). The full-length \u003cem\u003eEt\u003c/em\u003eEsp cDNA sequence was used in a BLAST search of GenBank (http://www.ncbi.nlm.nih.gov/BLAST/) and the \u003cem\u003eE. tenella \u003c/em\u003egenome database (http://www.genedb.org/Homepage/Etenella). The deduced amino acid sequence was obtained with the ORF Finder tool at NCBI. The molecular mass and theoretical isoelectric point were calculated with ProtParam tools (\u003ca href=\"http://web.expasy.org/protparam/\"\u003ehttp://web.expasy.org/protparam/\u003c/a\u003e). The signal peptide sequence was identified with the SignalP 4.1 server (\u003ca href=\"http://www.cbs.dtu.dk/services/SignalP/\"\u003ehttp://www.cbs.dtu.dk/services/SignalP/\u003c/a\u003e), and transmembrane regions were predicted with the TMHMM server v. 2.0 (\u003ca href=\"http://www.cbs.dtu.dk/services/TMHMM/\"\u003ehttp://www.cbs.dtu.dk/services/TMHMM/\u003c/a\u003e). Protein motifs were scanned with Motif Scan (http://myhits.isb-sib.ch/cgi-bin/motif_scan).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRecombinant protein expression and polyclonal anti-r\u003cem\u003eEt\u003c/em\u003eEsp serum\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eEt\u003c/em\u003eEsp open reading frame (ORF) cDNA was amplified with PCR using primers \u003cem\u003eEt\u003c/em\u003eEsp-UP and \u003cem\u003eEt\u003c/em\u003eEsp-LP (Table S), which contained \u003cem\u003eBam\u003c/em\u003eHI and \u003cem\u003eXho\u003c/em\u003eI restriction sites, respectively. The PCR fragment was then ligated into the prokaryotic expression vector pET28a(+) digested with the same restriction endonucleases, to construct the recombinant expression plasmid pET\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp. The recombinant protein His\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp (r\u003cem\u003eEt\u003c/em\u003eEsp) was expressed in \u003cem\u003eEscherichia coli\u003c/em\u003e BL21 cells at 37 \u0026deg;C with 1 mM isopropyl-thio-\u0026alpha;-d-galactoside. The cell pellet was lysed with sonication and digested with 10 \u0026micro;g/mL lysozyme (Sigma-Aldrich, USA). The lysate was then analyzed with 12% SDS-PAGE to confirm that the recombinant protein was present as a soluble protein or inclusion bodies. r\u003cem\u003eEt\u003c/em\u003eEsp was purified with His\u0026middot;Bind\u0026reg; Resin (Merck, USA) and its concentration measured with a BCA Protein Assay Kit (Beyotime, China).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; Two 2-month-old male rabbits were inoculated with 200 \u0026mu;g of purified r\u003cem\u003eEt\u003c/em\u003eEsp emulsified in Freund\u0026rsquo;s complete adjuvant (Sigma-Aldrich). After 14 days, a booster of 200 \u0026mu;g of purified r\u003cem\u003eEt\u003c/em\u003eEsp in Freund\u0026rsquo;s incomplete adjuvant (Sigma-Aldrich) was administered, followed by a second and third booster on days 28 and 42. One week after the final booster, the rabbit serum was collected and stored at \u0026minus;20 \u0026deg;C until use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnalysis of \u003cem\u003eEt\u003c/em\u003eEsp transcript levels with real-time quantitative PCR (qPCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression profiles of \u003cem\u003eEt\u003c/em\u003eEsp mRNA were examined in four developmental stages of \u003cem\u003eE. tenella\u003c/em\u003e (UO, SO, Spz, and sMrz) with qPCR. cDNA samples were synthesized from DNaseI-treated total RNAs of the \u003cem\u003eE. tenella\u003c/em\u003e developmental stages using SuperScript\u0026trade; II Reverse Transcriptase (Invitrogen) and random pd(N)6 primer. The housekeeping gene 18S rRNA was used as the internal control. The primers used to amplify the \u003cem\u003eEt\u003c/em\u003eEsp cDNA (\u003cem\u003eEt\u003c/em\u003eEsp-SP and \u003cem\u003eEt\u003c/em\u003eEsp-AP) and the 18S rRNA gene (18S-SP and 18S-AP) were designed with Primer3 v. 0.4.0 (http://bioinfo.ut.ee/primer3-0.4.0/) (Table S). qPCR was performed with the StepOnePlus\u0026trade; Real-Time PCR System using the SYBR\u0026reg; Premix Ex Taq\u0026trade; II kit (Takara, Japan). All experiments were performed twice, with separate biological replicates. In each experiment, the reactions were performed in triplicate. A dilution series of cDNA templates of the sporozoites was used to establish standard curves, and all standard curves had correlation coefficients of R\u003csup\u003e2\u003c/sup\u003e \u0026gt; 0.99. The comparative 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method was used to analyze the relative levels of gene expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSDS-PAGE and western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProtein samples were prepared from the four \u003cem\u003eE. tenella\u003c/em\u003e developmental stages (UO, SO, Spz, and sMrz), and from DF-1 cells transfected with the recombinant plasmids, for western blotting. The protein concentrations were determined with a BCA Protein Assay Kit (Beyotime,China). The purified r\u003cem\u003eEt\u003c/em\u003eEsp and protein lysates were separated with SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes. Rabbit antiserum (1:100) against sporozoite proteins, previously produced in our laboratory [26], a rabbit anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody (1:100), a mouse monoclonal anti-\u0026alpha;-tubulin antibody (1:1000) (Sigma-Aldrich), and a monoclonal anti-His antibody (1:1000) were used as the primary antibodies to detect r\u003cem\u003eEt\u003c/em\u003eEsp or native \u003cem\u003eEt\u003c/em\u003eEsp. Na\u0026iuml;ve rabbit serum (1:100) was used as the negative control. IRDye-800CW-labelled goat anti-rabbit IgG antibody (1:25,000) and IRDye-680RD-labeled donkey anti-mouse IgG antibody (1:25,000; LI-Cor, Lincoln, NE, USA) were used as the secondary antibodies. The IRDyes were detected with the Odyssey Infrared Imaging System (LI-Cor).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBiFC assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe ORF fragments of \u003cem\u003eEt\u003c/em\u003eEsp and the \u003cem\u003eEt\u003c/em\u003eAMA1 ectodomain, with no stop codon, were amplified from the first-strand cDNA with two pairs of primers (Bf\u003cem\u003eEt\u003c/em\u003eEsp-UP/Bf\u003cem\u003eEt\u003c/em\u003eEsp-LP and Bf\u003cem\u003eEt\u003c/em\u003eAMA1-UP/Bf\u003cem\u003eEt\u003c/em\u003eAMA1-LP, respectively), which contained\u003cem\u003e Eco\u003c/em\u003eRI and \u003cem\u003eBgl\u003c/em\u003eII restriction sites (\u003cem\u003eEt\u003c/em\u003eEsp) or \u003cem\u003eEco\u003c/em\u003eRI and \u003cem\u003eXho\u003c/em\u003eII restriction sites (\u003cem\u003eEt\u003c/em\u003eAMA1). The fragments were then digested with the appropriate restriction enzymes and ligated into the pBiFC-VN155 and pBiFC-VC155 vectors digested with the same enzymes, respectively, to construct the recombinant plasmids pBiFC-VN155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp and pBiFC-VC155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1, respectively. Before the BiFC assay, the uptake of the expression vectors by the cells was confirmed. DF-1 cells were transfected with the recombinant plasmid pBiFC-VN155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp or pBiFC-VC155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 using Lipofectamine\u0026trade; 2000 Transfection Reagent (Invitrogen, USA), according to the manufacturer\u0026rsquo;s instructions. At 48 h after transfection, the cells were harvested and the proteins were extracted with RIPA Lysis Buffer (Beyotime). Western blots were probed with rabbit anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody and rabbit anti-r\u003cem\u003eEt\u003c/em\u003eAMA1 antibody, which were previously produced in our laboratory [10]. After confirmation that the cells had expressed the two constructs, DF-1 cells were cotransfected with pBiFC-VN155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp and pBiFC-VC155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1. DF-1 cells were also cotransfected with pBiFC-bJunVN55 (I152L) and pBiFC-bFosVC155 or pBiFC-VC155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 and pBiFC-VN155 empty vector, or pBiFC-VN155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp and pBiFC-VC155 empty vector as the positive or negative control, respectively. The DF-1 cells were observed with fluorescence microscopy 24 h after transfection with the different constructs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGST pull-down\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp317 \u003cem\u003ein vitro\u003c/em\u003e, a GST pull-down assay was performed with the Pierce\u0026trade; GST Protein Interaction Pull-Down Kit (Thermo Scientific, USA), according to the manufacturer\u0026rsquo;s instructions. The recombinant plasmid pGEX-6P\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 was previously constructed in our laboratory [10]. The expression of the recombinant protein GST\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 was induced and the protein purified with GST resin for use as the bait protein. The ORF of \u003cem\u003eEt\u003c/em\u003eEsp was inserted into the pET-28a vector to express the recombinant protein His\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp (r\u003cem\u003eEt\u003c/em\u003eEsp) as the prey protein. GST\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 was incubated with equilibrated glutathione-agarose to immobilize the bait protein. r\u003cem\u003eEt\u003c/em\u003eEsp was then added to the glutathione-agarose and incubated with the bait protein. The bait and prey proteins were eluted from the glutathione-agarose. \u003cem\u003eE. coli\u003c/em\u003e BL21 cells were transformed with recombinant plasmid pET\u0026ndash;\u003cem\u003eEt\u003c/em\u003eMIC2, constructed previously in our laboratory [27], to express the recombinant protein His\u0026ndash;\u003cem\u003eEt\u003c/em\u003eMIC2 as the negative control. Another, r\u003cem\u003eEt\u003c/em\u003eEsp was loaded in an empty glutathione-agarose column as the negative. \u0026nbsp;All the proteins were then resolved with 12% SDS-PAGE and detected with western blotting using the appropriate antibodies, as described above.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssay of \u003cem\u003eEt\u003c/em\u003eEsp secretion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFreshly excysted sporozoites (4 \u0026times;\u0026thinsp;10\u003csup\u003e6\u003c/sup\u003e) were incubated in 100 \u0026mu;L of complete medium (CM; Dulbecco\u0026rsquo;s modified Eagle\u0026rsquo;s medium [DMEM] supplemented with 10% fetal bovine serum [FBS], 100 U/mL penicillin/streptomycin, 2 mM l-glutamine) for 2 h at 41 \u0026deg;C under 5% CO\u003csub\u003e2 \u003c/sub\u003efor the secretion experiments. They were then incubated with 5, 10, or 20 \u0026mu;M staurosporine (Sigma; dissolved in dimethylsulfoxide [DMSO]) or an appropriate volume of carrier DMSO, as described previously [28]. The secretion of \u003cem\u003eEt\u003c/em\u003eMIC2 and \u003cem\u003eEt\u003c/em\u003eGRA(\u003cem\u003eTg\u003c/em\u003eGRA7 homologous protein) was used as the control. The sporozoites were then pelleted by centrifugation for 10 min at 6000 \u0026times; g. The supernatants and sporozoites were recovered and analyzed with western blotting using a rabbit anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody and rabbit anti-r\u003cem\u003eEt\u003c/em\u003eMIC2 antibody generated previously in our laboratory [27] and mouse anti-\u003cem\u003eTg\u003c/em\u003eGAR7 antibody which generated previous in other laboratory of our Institute .\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunofluorescence\u003c/strong\u003e \u003cstrong\u003estaining of parasites \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePurified differentially developed parasites (Spz, sporocysts [Sporo], and sMrz) were transferred to glass slides and air-dried, as previously described [10, 29]. Purified freshly sporozoites were infected DF-1 cells after incubation in CM for 2 h at 41\u0026deg;C. At different time points after infection, the DF-1 cells were collected, washed, transferred to glass slides, and air-dried. The slides were then fixed in 2% paraformaldehyde in phosphate-buffered saline (PBS) and placed in 1% Triton X-100 in PBS for 15 min to increase their permeability. Non permeabilized sporozoites and sporocysts were used as control. The slides were blocked with PBS containing 2% (w/v) bovine serum albumin for overnight at 4 \u0026deg;C. A rabbit anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody (1:100) was added and the cells were incubated for 1 h at 37 \u0026deg;C. A 1:500 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG antibody (Sigma-Aldrich) was then added and the cells incubated for 1 h at 37 \u0026deg;C. The cell nuclei were stained by incubation in 10 \u0026mu;g/mL 4\u0026cent;,6-diamidino-2-phenylindole (Beyotime) at room temperature for 10 min. After each step, the slides were washed three times for 10 min each with PBS containing 0.05% Tween 20. The slides were finally mounted with 50 \u0026mu;L of Fluoromount Aqueous Mounting Medium (Sigma-Aldrich) before observation with a fluorescence microscope (Olympus, Tokyo, Japan). At the same time, we performed the co-localization of \u003cem\u003eEt\u003c/em\u003eEsp and \u003cem\u003eEt\u003c/em\u003eAMA1 in sporozoites. Purified sporozoites were treated with mouse anti-r\u003cem\u003eEt\u003c/em\u003eAMA1 antibody (1:100) and rabbit anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody(1:100), then goat anti-rabbit IgG fluorescein isothiocyanate (FITC)-conjugated antibody(1:500) and goat anti-mouse IgG cyanine (Cy3)-conjugated antibody (Sigma-Aldrich)(1:500) were secondary antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInvasion inhibition assay\u003c/strong\u003e\u003cstrong\u003e \u003cem\u003ein vitro\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe invasion inhibition assay was based on previous reports the invasion of DF-1 cells by \u003cem\u003eE. tenella \u003c/em\u003esporozoites. Antibodies were purified with Protein A+G Agarose (Beyotime). DF-1 cells (2\u0026thinsp;\u0026times;\u0026thinsp;10\u003csup\u003e5 \u003c/sup\u003ecells per well) were cultured in 24-well plates (Corning) in CM for 24 h at 37 \u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The freshly purified sporozoites were counted and labeled with carboxyfluorescein diacetate succinimidyl ester (Beyotime). The labeled sporozoites were incubated at 37 \u0026deg;C with 50, 100, 200, 300 or 400 \u0026mu;g/mL purified IgG directed against r\u003cem\u003eEt\u003c/em\u003eEsp for 2 h. The same quantity of IgG from na\u0026iuml;ve rabbit serum (Sigma-Aldrich) was used as the negative control, and an equivalent volume of PBS as the normal control. After they were washed twice with sterile PBS, DF-1 cells (10\u003csup\u003e5\u003c/sup\u003e/well) were infected with the labeled sporozoites (10\u003csup\u003e5\u003c/sup\u003e/well) in 24-well plates and cultured for 16 h at 41 \u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e. The cells were then collected and analyzed with flow cytometry on a Cytomics\u0026trade; FC 500 (Beckman Coulter, Indianapolis, IN, USA). The controls were uninfected DF-1 cells. The infected cells, uninfected cells, and free sporozoites were gated with the CXP software to count the infected (labeled sporozoites) and uninfected (fluorescence-free) cells. All assays were performed in triplicate. The percentages of infected cells in the presence or absence of an anti-r\u003cem\u003eEt\u003c/em\u003eEsp polyclonal antibody were used to calculate the inhibition rates, as previously described [10].\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCloning and sequence analysis of full-length \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eEsp \u003c/strong\u003e\u003cstrong\u003ecDNA\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe 1108-bp full-length cDNA of \u003cem\u003eEt\u003c/em\u003eEsp was obtained with RACE. A sequence analysis showed that the full-length cDNA included a 5\u0026cent;-untranslated region (UTR) of 70 bp, a 3\u0026cent;-UTR of 542 bp with a poly(A) tail, and an ORF of 501 bp, which encoded 166 amino acids with a calculated molecular weight of 18.1 kDa and a theoretical isoelectric point of 4.2 (Fig. 1). Analysis of the amino acid sequence showed a signal peptide of 19 amino acids at the N-terminus and no transmembrane region. Searches in the Motif Database and the Conserved Domain Database revealed the presence of one N-glycosylation site, five casein kinase II phosphorylation sites, six N-myristoylation sites, one tyrosine kinase phosphorylation site, one intein DOD-type homing endonuclease domain, and no conserved domains (Fig. 1). A BLAST search of the \u003cem\u003eE. tenella\u003c/em\u003e genome database showed that the ORF sequence shared 100% sequence identity with ETH_00016590, which encodes an \u003cem\u003eEimeria\u003c/em\u003e-specific protein, on supercontig Eth_scaff124: 9216\u0026ndash;10141.\u003c/p\u003e\n\u003cp\u003eThe amino acid sequence shared 100% homology with the \u003cem\u003eE. tenella Eimeria\u003c/em\u003e-specific protein (XP_013228647.1) and 92% (152/170) identity with the \u003cem\u003eE. necatrix\u003c/em\u003e \u003cem\u003eEimeria\u003c/em\u003e-specific protein (XP_013435139.1) in NCBI. Therefore, this gene was designated \u003cem\u003eEt\u003c/em\u003eEsp and submitted to NCBI GenBank (GenBank accession no. MN161778). It also shared 68% (106/157) amino acid identity with \u003cem\u003eE. brunetti\u003c/em\u003e conserved hypothetical protein (CDJ53027.1), 63% (108/172) identity with \u003cem\u003eE. praecox\u003c/em\u003e conserved hypothetical protein (CDI81636.1), 74% (97/131) identity with \u003cem\u003eE. maxima\u003c/em\u003e conserved hypothetical protein (XP_013336310.1), and 73% (91/124) identity with \u003cem\u003eE. acervulina\u003c/em\u003e conserved hypothetical protein (XP_013248166.1). Also, this protein is not found in other apicomplexa parasites. These results show that the protein is conserved in \u003cem\u003eEimeria\u003c/em\u003e spp.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExpression and characterization of \u003c/strong\u003e\u003cstrong\u003erecombinant \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eEsp\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003er\u003cem\u003eEt\u003c/em\u003eEsp was expressed as a His6-tagged fusion protein. SDS-PAGE showed that r\u003cem\u003eEt\u003c/em\u003eEsp was mainly present in the soluble fraction of the bacterial lysate. After the purification of r\u003cem\u003eEt\u003c/em\u003eEsp with Ni-NTA chromatography, a protein of approximately 21 kDa was observed with SDS-PAGE. Because 3 kDa of the fusion protein was derived from the vector, the predicted molecular mass of \u003cem\u003eEt\u003c/em\u003eEsp was about 18.1 kDa. Western blotting showed that purified r\u003cem\u003eEt\u003c/em\u003eEsp was recognized by rabbit serum directed against sporozoites and by a monoclonal anti-His6 antibody. Na\u0026iuml;ve rabbit serum failed to recognize any protein corresponding to the expected size of r\u003cem\u003eEt\u003c/em\u003eEsp (Fig. 2). These results indicate that r\u003cem\u003eEt\u003c/em\u003eEsp was recognized specifically by rabbit serum directed against a soluble sporozoite protein and by a monoclonal anti-His antibody.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eEsp mRNA and protein expression at different developmental stages of \u003cem\u003eE. tenella\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eqPCR was used to analyze the UO, SO, Spz, and sMrz stages of \u003cem\u003eE. tenella\u003c/em\u003e for the presence of \u003cem\u003eEt\u003c/em\u003eEsp mRNA. The levels of \u003cem\u003eEt\u003c/em\u003eEsp mRNA were much higher in the sMrz stage than in the other three stages, and \u003cem\u003eEt\u003c/em\u003eEsp mRNA was almost undetectable in UO (Fig. 3a).\u003c/p\u003e\n\u003cp\u003eThe expression of \u003cem\u003eEt\u003c/em\u003eEsp in the four developmental stages was also determined with immunoblotting using rabbit antiserum against r\u003cem\u003eEt\u003c/em\u003eEsp. A monoclonal anti-\u0026alpha;-tubulin antibody was used as the control. Western blotting showed that the anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody reacted with a band of approximately 18 kDa in the parasite lysates prepared from the four different developmental stages of \u003cem\u003eE. tenella\u003c/em\u003e The expression levels of \u003cem\u003eEt\u003c/em\u003eEsp were higher in sporozoites than in other three stages(Fig. 3b,c).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfirmation of the interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo characterize the interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp \u003cem\u003ein vivo\u003c/em\u003e, a BiFC assay was performed. For the BiFC assay, fragments of the \u003cem\u003eEt\u003c/em\u003eEsp ORF and the \u003cem\u003eEt\u003c/em\u003eAMA1 ectodomain sequence were cloned into the plasmids pBiFC-VN155 and pBiFC-VC155, respectively, to generate the constructs pBiFC-VN155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp and pBiFC-VC155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1, respectively. The total proteins were extracted from DF-1 cells transfected separately with one or other construct. Western blotting showed that the two constructs were expressed individually in the DF-1 cells at 48 h after transfection (Fig. 4a). Strong green fluorescence was observed in DF-1 cells 48 h after they were cotransfected with both constructs. Green fluorescence was also observed in the positive control. However, there was no visible fluorescence in the DF-1 cells cotransfected with pBiFC-VC155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 and pBiFC-VN155 empty vector or pBiFC-VN155\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp and pBiFC-VC155 empty vector. These results indicate that \u003cem\u003eEt\u003c/em\u003eEsp interacts with \u003cem\u003eEt\u003c/em\u003eAMA1 in cells (Fig. 4b).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGST pull-down\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo confirm the interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp \u003cem\u003ein vitro\u003c/em\u003e, a GST pull-down assay was performed. GST\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 and His\u0026ndash;\u003cem\u003eEt\u003c/em\u003eEsp were expressed individually in \u003cem\u003eE. coli\u003c/em\u003e and purified. GST\u0026ndash;\u003cem\u003eEt\u003c/em\u003eAMA1 was bound to an equilibrated glutathione\u0026ndash;agarose column, and then His\u0026ndash;\u003cem\u003eEt\u003c/em\u003eESP was added to the column. The proteins bound to the glutathione\u0026ndash;agarose, and any nonspecifically bound proteins were removed by elution with buffer. The proteins retained on the column were then eluted and detected with immunoblotting using anti-r\u003cem\u003eEt\u003c/em\u003eAMA1 and anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibodies (Fig. 5). The results clearly indicated a direct interaction between the \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eEsp is not secreted from the microneme\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine the secretion of \u003cem\u003eEt\u003c/em\u003eEsp, sporozoites were incubated in CM at 41 \u0026deg;C. The supernatant containing the excretory\u0026ndash;secretory antigens (ESA) from the incubated sporozoites and sporozoites pellets were analyzed with western blotting. Immunoblots of the ESA samples and sporozoites were probed with an anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody and showed that \u003cem\u003eEt\u003c/em\u003eEsp was secreted when the sporozoites were incubated at 41 \u0026deg;C under 5% CO\u003csub\u003e2\u003c/sub\u003e in CM. Rabbit serum raised against the micronemal protein \u003cem\u003eEt\u003c/em\u003eMIC2 was used as the experimental control [27]. To demonstrate whether \u003cem\u003eEt\u003c/em\u003eEsp secretion is dependent on the micronemal pathway, we added staurosporine to the CM because staurosporine is a protein kinase inhibitor known to specifically inhibit microneme secretion [28]. In the parasites treated with 5, 10, or 20 \u0026mu;M staurosporine, the secretion of \u003cem\u003eEt\u003c/em\u003eEsp and \u003cem\u003eEt\u003c/em\u003eGRA into the supernatant was not affected, but the secretion of \u003cem\u003eEt\u003c/em\u003eMIC2 in supernatant was significant reduced compared with their secretion in the presence of the DMSO solvent only (Fig. 6a,b). These results show that \u003cem\u003eEt\u003c/em\u003eEsp is a secreted protein, but not a micronemal protein.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImmunolocalization of \u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eEt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eEsp\u003c/strong\u003e\u003cstrong\u003e at different developmental stages of\u003cem\u003e E. tenella\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the localization and distribution of the\u003cem\u003e Et\u003c/em\u003eEsp protein in different development stages of \u003cem\u003eE. tenella\u003c/em\u003e, including sporozoites, second-generation merozoites, immature schizonts, and mature schizonts, it was localized with immunofluorescence\u003cem\u003e in vitro\u003c/em\u003e using an antibody against r\u003cem\u003eEt\u003c/em\u003eEsp. The \u003cem\u003eEt\u003c/em\u003eEsp protein was mainly distributed on the surfaces of the permeabilized parasite sporozoites, sporocysts, and second-generation merozoites (Fig. 7a-a, b-a, k,). The protein were also mainly located on the surface of non permeabilized sporozoites and sporocysts (Fig. 7a-b, b-b). After incubation in CM for 2 h, the fluorescence increased and mainly localized to the anterior and surface of parasites (Fig. 7c). \u003cem\u003eEt\u003c/em\u003eEsp protein was also mainly located on the surfaces of parasites 2 h after their invasion of DF-1 cells (Fig. 7d). At 12 h after the sporozoites were added to DF-1 cells, \u003cem\u003eEt\u003c/em\u003eEsp also localized to the cytoplasm of the sporozoites, except for the refractile body in the posterior section of the parasites, and the intensity of \u003cem\u003eEt\u003c/em\u003eEsp staining had increased (Fig. 7e). At 24\u0026ndash;72 h postinfection, the \u003cem\u003eEt\u003c/em\u003eEsp protein was uniformly distributed in trophozoites, immature schizonts, and mature schizonts, and the protein\u0026rsquo;s expression had increased (Fig. 7e\u0026ndash;j).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCo-localization of \u003cem\u003eEt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eAMA1\u003c/strong\u003e\u003cstrong\u003e and \u003cem\u003eEt\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003eEsp\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIFAs were performed to determine the location of \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp. Purified sporozoites were treated with mouse anti-r\u003cem\u003eEt\u003c/em\u003eAMA1 antibody and rabbit anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody. The results showed \u003cem\u003eEt\u003c/em\u003eEsp was mainly located on the surface of sporozoites, \u003cem\u003eEt\u003c/em\u003eAMA1 was distributed throughout the cytoplasm and the membrane of sporozoites with the exception of refractile bodies (Fig 8).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAnti-r\u003cem\u003eEt\u003c/em\u003eEsp antibodies inhibit DF-1 cell invasion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the effect of the \u003cem\u003eEt\u003c/em\u003eEsp protein on the invasion of DF-1 cells by \u003cem\u003eE. tenella\u003c/em\u003e sporozoites, an invasion inhibition assay of sporozoites was performed \u003cem\u003ein vitro\u003c/em\u003e. When the sporozoites were incubated with purified anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody before infection, their capacity to invade the DF-1 cells was significantly reduced. After pretreatment with 50, 100, 200, 300, or 400 \u0026mu;g/mL anti-\u003cem\u003erEt\u003c/em\u003eEsp IgG antibody, their invasion of cells was highly significantly reduced compared with that of sporozoites treated with na\u0026iuml;ve rabbit IgG (negative control) (P \u0026lt; 0.01). Under these experimental conditions, an inhibition plateau of 62.9% was reached at an antibody concentration of 300 \u0026mu;g/mL. In a comparative analysis, the same dose of the na\u0026iuml;ve rabbit serum IgG antibody did not significantly affect invasion (Fig. 9).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we cloned and characterized the\u003cem\u003e E. tenella\u003c/em\u003e \u003cem\u003eEimeria\u003c/em\u003e-specific protein, a putative \u003cem\u003eEt\u003c/em\u003eAMA1-interacting protein, using a yeast two-hybrid system in our laboratory [22]. Although the yeast two-hybrid system is a widely used and powerful method for identifying the partners of proteins in regulatory complexes and in the analysis of protein\u0026ndash;protein interactions [30], the system has several limitations, including the possibility of isolating very large numbers of clones with no biological relevance [31]. Therefore, the interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp required validation with an alternative technique, such as a BiFC assay or GST pull-down assay. The BiFC assay is a versatile technique for investigating protein\u0026ndash;protein interactions in living systems, and is based on the reconstitution of a fluorescent protein \u003cem\u003ein vivo\u003c/em\u003e [32]. GST pull-down is amenable to more specific investigations of protein\u0026ndash;protein interactions \u003cem\u003ein vitro\u003c/em\u003e, but relies on purified proteins that may not fully mimic the protein\u0026rsquo;s native conformation or posttranslational modification, which mediate its interactions [33]. Although these assays have some advantages in identifying protein\u0026ndash;protein interactions, each also has its drawbacks. Therefore, in many research fields, these methods are often combined to identify the interactions between two proteins [33\u0026ndash;35]. In this study, the interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp was confirmed with a GST pull-down assay \u003cem\u003ein vitro\u003c/em\u003e and a BiFC assay \u003cem\u003ein vivo\u003c/em\u003e. These results indicated that \u003cem\u003eEt\u003c/em\u003eEsp interacts with \u003cem\u003eEt\u003c/em\u003eAMA1.\u003c/p\u003e\n\u003cp\u003eProteins perform a vast number of cellular functions through their interactions with one or multiple binding partners. Moreover, many protein\u0026ndash;protein interactions are regulated by posttranscriptional modification (e.g., phosphorylation) of the protein of interest, and these modifications are induced by exposure to certain circumstances [36]. In this study, an amino acid sequence analysis predicted that \u003cem\u003eEt\u003c/em\u003eEsp contains one N-glycosylation site, five casein kinase II phosphorylation sites, six N-myristoylation sites, one tyrosine kinase phosphorylation site and one intein DOD-type homing endonuclease domain. Inteins, also called protein introns, are parasitic genetic elements that excise themselves at the protein level by self-splicing, allowing the formation of functional, nondisrupted proteins[37]. These data suggest that its functions may be regulated by posttranslational modification. We supposed that the interaction of \u003cem\u003eEt\u003c/em\u003eAMA1 with \u003cem\u003eEt\u003c/em\u003eEsp may be regulated by posttranslational modification.\u003c/p\u003e\n\u003cp\u003eTo understand the expression of \u003cem\u003eEt\u003c/em\u003eEsp in the different developmental stages of parasite, we examined its expression patterns with qPCR and western blotting. Our results indicated that \u003cem\u003eEt\u003c/em\u003eEsp mRNA levels were higher in second-generation merozoites and sporozoites than in sporulated oocysts or unsporulated oocysts. But western blotting showed that the expression of \u003cem\u003eEt\u003c/em\u003eEsp was higher in sporozoites than other developmental stages of \u003cem\u003eE.tenella.\u003c/em\u003e Immunofluorescent localization showed that the expression of the protein increased with the development of the parasites in DF-1 cells. Previous proteomic and transcriptomic data confirm that \u003cem\u003eEt\u003c/em\u003eAMA paralogues are tightly stage-regulated [38, 39]. \u003cem\u003eEt\u003c/em\u003eAMA1 is a sporozoite-specific protein involved in the invasion process of sporozoites [10, 19, 38]. While another \u003cem\u003eEt\u003c/em\u003eAMA1 paralogues, \u003cem\u003eEt\u003c/em\u003eAMA2 is a merozoites-specific protein not involved in the parasite invasion. All these finding indicate that \u003cem\u003eE. tenella\u003c/em\u003e parasites harbour stage-specific AMA proteins that could be relevant during specific phases of the parasite cycle [19]. In this study, \u003cem\u003eEt\u003c/em\u003eEsp is differentially expressed during the distinct phases of the parasite life cycle, and may be very important in the invasion and development of the parasite life cycle.\u003c/p\u003e\n\u003cp\u003eImmunofluorescent localization showed that \u003cem\u003eEt\u003c/em\u003eEsp was located on the surface of the sporozoite and concentrated around the anterior of the parasite during its incubation in CM. However, the protein has no transmembrane region or glycophosphatidyl inositol (GPI)-anchor sequence, but has a signal peptide, and six N-myristoylation sites. The presence of a signal peptide is necessary for the translocation of proteins from their ribosomal sites of translation into the lumen of the endoplasmic reticulum, from where they are trafficked in the endomembrane system to their final locations within the cell or beyond [40]. We speculated that \u003cem\u003eEt\u003c/em\u003eEsp also undergoes posttranslational modification according to the amino acid sequence analysis, including phosphorylation, myristoylation, and glycosylation. Among these modifications, myristoylation is the key factor in the membrane localization of signal-transducing proteins [41]..\u003c/p\u003e\n\u003cp\u003eMost surface antigens are involved in the invasion, pathogenesis, and immune evasion of parasites. For example, in \u003cem\u003ePlasmodium\u003c/em\u003e, merozoite surface proteins are critical for parasite invasion, and represent attractive targets for antibody-based therapies against clinical malaria [42]. We also found that the expression of \u003cem\u003eEt\u003c/em\u003eEsp increased and that the protein mainly localized on the anterior and surface of the parasites after incubation in CM for 2 h. This suggests that the protein is involved in the sporozoite invasion of host cells. To investigate the function of \u003cem\u003eEt\u003c/em\u003eEsp in the invasion process, we performed an invasion test \u003cem\u003ein vitro\u003c/em\u003e and found that polyclonal rabbit anti-r\u003cem\u003eEt\u003c/em\u003eEsp serum efficiently reduced the sporozoite invasion of cultured DF-1 cells. \u003cem\u003eEt\u003c/em\u003eAMA1 also localized to the anterior of the sporozoites after their invasion of DF-1 cells [10]. Previous reports have shown that monospecific mouse anti-r\u003cem\u003eEt\u003c/em\u003eAMA1 serum or polyclonal rabbit antiserum against r\u003cem\u003eEt\u003c/em\u003eAMA1 also blocked the invasion of host cells \u003cem\u003ein vitro\u003c/em\u003e [10,19].\u003cem\u003e. \u003c/em\u003eIn this study, \u003cem\u003eEt\u003c/em\u003eEsp is involved in invasion as demonstrated by using antibodies raised against \u003cem\u003eEt\u003c/em\u003eEsp in vitro. We tested that \u003cem\u003eEt\u003c/em\u003eEsp is an interacting protein with \u003cem\u003eEt\u003c/em\u003eAMA1 by using BiFC, GST-pull down and yeast two-hybrid system.. Therefore, we speculated that\u003cem\u003e Et\u003c/em\u003eEsp mediates sporozoites invasion in host cells by interacting with \u003cem\u003eEt\u003c/em\u003eAMA1. The exact function of \u003cem\u003eEt\u003c/em\u003eEsp needs further study.\u003c/p\u003e\n\u003cp\u003ePrevious studies have shown that in \u003cem\u003eT. gondii \u003c/em\u003eand \u003cem\u003ePlasmodium\u003c/em\u003e, AMA1 interacts directly with rhoptry neck protein 2 (RON2), which is secreted from the parasite rhoptries and specifically localizes at the moving junction. The RON2\u0026ndash;AMA1 interaction is a critical step in the moving-junction-dependent invasion of host cells by apicomplexan parasites [43, 44]. Although the interaction between AMA1 and RON2 has not been reported in \u003cem\u003eEimeria\u003c/em\u003e spp., \u003cem\u003eE. tenella\u003c/em\u003e is an apicomplexan and AMA1 is conserved in this phylum. Therefore, we inferred that \u003cem\u003eEt\u003c/em\u003eAMA1 may also interact with \u003cem\u003eEt\u003c/em\u003eRON2 and specifically localize to the moving junction during the invasion of host cells by\u003cem\u003e E. tenella\u003c/em\u003e. In this study, we have shown that \u003cem\u003eEt\u003c/em\u003eEsp is an \u003cem\u003eEt\u003c/em\u003eAMA1-interacting protein, but whether it localizes to the moving junction during invasion requires further study.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, we have shown that interacts with \u003cem\u003eEt\u003c/em\u003eAMA1 using a BIFC assay \u003cem\u003ein vivo\u003c/em\u003e and a GST pull-down assay \u003cem\u003ein vitro\u003c/em\u003e. Using staurosporine, we showed that \u003cem\u003eEt\u003c/em\u003eEsp is a secreted protein of sporozoites but not from micronemes. An invasion inhibition assay revealed that an antibody against r\u003cem\u003eEt\u003c/em\u003eEsp also blocked the parasite invasion of its host cells by more than 62%. These data have implications for the use of \u003cem\u003eEt\u003c/em\u003eAMA1 or \u003cem\u003eEt\u003c/em\u003eAMA1-interacting proteins as targets in therapeutic intervention strategies against avian coccidiosis.\u003c/p\u003e"},{"header":"Supplementary Information ","content":"\u003cp\u003e\u003cstrong\u003eAdditional file 1\u003c/strong\u003e: Table S. Primers sequence used in this study.\u003c/p\u003e"},{"header":"Abbreviations ","content":"\u003cp\u003e\u003cem\u003eEt\u003c/em\u003eAMA1: \u003cem\u003eEimeria tenella\u003c/em\u003e apical membrane antigen 1; \u003cem\u003eEt\u003c/em\u003eEsp: \u003cem\u003eE. tenella Eimeria\u003c/em\u003e-specific protein; BiFC: bimolecular fluorescence complementation; GST pull-down: glutathione S-transferase pull-down; RACE: random amplification of PCR ends; UO: unsporulated oocysts; SO: sporulated oocysts; Spz: sporozoites; sMrz: second-generation merozoites; NCBI: the National Center for Biotechnology Information; ORF: open reading frame; CM: complete medium.\u003c/p\u003e"},{"header":"Declarations ","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank all organizations which funded this work and all the teachers who cooperated in technical assistance and Dr Wang quan who provided mouse anti-TgGRA7 antibody of \u003cem\u003eT.gondii\u003c/em\u003e .\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe protocol was approved and authorized by the Animal Care and Use Committee of the Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant Nos. 31572266 and 31672551) and National Key Research and Development Program of China (2018YFD0500302) and National Sharing Service Platform for Parasite Resources (No. TDRC-22) and Shanghai Minhang District talent development special funds.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHYH and HD conceived and designed the study. CL, QPZ, SHZ and QJW performed the experiments. CL, BH and HZZ analyzed the data.CL, QPZ, HXW, SLY, YY and SSL collected parasites. CL, QPZ and HYH wrote the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003csup\u003e1\u003c/sup\u003eShanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Key Laboratory of Animal Parasitology of Ministry of Agriculture, Minhang, Shanghai 200241, PR China. \u003csup\u003e2\u003c/sup\u003eCollege of Life and Environment Sciences, Shanghai Normal University, Shanghai 200234, China.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003e\n\u003cp\u003eQuiroz-Castaneda RE, Dantan-Gonzalez E. Control of avian coccidiosis: future and present natural alternatives. 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Molecular characterization and protective efficacy of the microneme 2 protein from \u003cem\u003eEimeria tenella\u003c/em\u003e. Parasite. 2018; 25:60.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eCarruthers VB1, Giddings OK, Sibley Secretion of micronemal proteins is associated with toxoplasma invasion of host cells.Cell Microbiol. 1999;1:225-35..\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003ePeroval M; Pery P; Labbe M. The heat shock protein 90 of \u003cem\u003eEimeria tenella\u003c/em\u003e is essential for invasion of host cell and schizont growth. Int J Parasitol. 2006; 36:1205-15.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eMiller J, Stagljar I. Using the yeast two-hybrid system to identify interacting proteins. Methods Mol Biol. 2004; 261:247-62.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eFerro E, Baldni E, Trabalzini L. Use of the yeast two-hybrid technology to isolate molecular interactions of Ras GTPases. Methods Mol Biol\u003cem\u003e.\u003c/em\u003e 2014;1120: 97-120.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003ePham CD. Detection of Protein-Protein Interaction Using Bimolecular Fluorescence Complementation Assay. Methods Mol Biol. 2015; 1278:483-95.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eBain AL, Harris JL, Khanna KK. Identification of ATM-Interacting Proteins by Co-immunoprecipitation and Glutathione-S-Transferase (GST) Pull-Down Assays. Methods Mol Biol. 2017; 1599:163-81.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eLv L, Huang B, Zhao Q, Zhao Z, Dong H, Zhu S, et al. Identification of an interaction between calcium-dependent protein kinase 4 (\u003cem\u003eEt\u003c/em\u003eCDPK4) and serine protease inhibitor (\u003cem\u003eEt\u003c/em\u003eSerpin) in \u003cem\u003eEimeria tenella\u003c/em\u003e. Parasit Vectors. 2018; 11:259.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eMa F, Ni L, Liu L, Li X, Zhang H, Zhang A, et al. ZmABA2, an interacting protein of ZmMPK5, is involved in abscisic acid biosynthesis and functions. Plant Biotechnol J. 2016; 14:771-82.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eTakahashi Y. Co-immunoprecipitation from Transfected Cells. Methods Mol Biol. 2015; 1278:381-9.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eNaor A, Altman-Price N, Soucy SM, Green AG, Mitiagin Y, Turgeman-Grott I, et al.. Impact of a homing intein on recombination frequency and organismal fitness. Proc Natl Acad Sci U S A. 2016; 113.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eLal K, Bromley E, Oakes R, Prieto JH, Sanderson SJ, Kurian D, et al. Proteomic comparison of four \u003cem\u003eEimeria tenella\u003c/em\u003e life-cycle stages: unsporulated oocyst, sporulated oocyst,sporozoite and second-generation merozoite. Proteomics. 2009; 9: 4566-76.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eReid AJ, Blake DP, Ansari HR, Billington K, Browne HP, Bryant J, et al. Genomic analysis of the causative agents of coccidiosis in domestic chickens. Genome Res. 2014, 24:1676-85.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eMarugan-Hernandez V, Long E, Blake D, Crouch C, Tomley F. \u003cem\u003eEimeria tenella\u003c/em\u003e protein trafficking: differential regulation of secretion versus surface tethering during the life cycle. Sci Rep. 2017; 7:4557.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eMcCabe JB, Berthiaume N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not to lipid rafts/caveolae. Mol Biol Cell. 2001; 12:3601-17.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eBabacar M, Fall MM, Varela ML, Loucoubar C, Joos C, Fall B, et al. Analysis of antibody responses to selected \u003cem\u003ePlasmodium falciparum\u003c/em\u003e merozoite surface antigens in mild and cerebral malaria and associations with clinical outcomes Clin Exp Immunol. 2018; 23.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eTonkin ML, Roques M, Lamarque MH, Pugni\u0026egrave;re M, Douguet D, Crawford J, et al. Host cell invasion by apicomplexan parasites: insights from the co-structure of AMA1 with a RON2 peptide. Science. 2011; 333:463-7.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eShen B, Sibley The moving junction, a key portal to host cell invasion by apicomplexan parasites. Curr Opin Microbiol. 2012; 15:449-55.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp style=\"margin-top: 12.0pt; text-align: left; line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003eTable 1. Primers sequence used in this study\u003c/span\u003e\u003c/p\u003e\n\u003ctable style=\"border-collapse: collapse; border: none;\" width=\"599\"\u003e\n\u003ctbody\u003e\n\u003ctr style=\"height: 20.4pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 0in 0in 2.9pt; height: 20.4pt;\" width=\"231\"\u003e\n\u003cp style=\"text-align: center; line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003ePrimer name\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border-top: solid windowtext 1.0pt; border-left: none; border-bottom: solid windowtext 1.0pt; border-right: none; padding: 0in 0in 0in 2.9pt; height: 20.4pt;\" width=\"369\"\u003e\n\u003cp style=\"text-align: center; line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003ePrimer sequence\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 19.85pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; padding: 0in 0in 0in 2.9pt; height: 19.85pt;\" width=\"231\"\u003e\n\u003cp style=\"text-align: left; line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003eGS5P (5' Primer)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; padding: 0in 0in 0in 2.9pt; height: 19.85pt;\" width=\"369\"\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-ATGGTCTCGGGCCAGTTCTCGTTCA -3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 20.6pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"231\"\u003e\n\u003cp style=\"text-align: left; line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003eGS5N (5' Nested Primer)\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"369\"\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-GGAGATGAGACCCAGGCGGATGAAA -3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 20.6pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"231\"\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cem\u003e\u003cspan style=\"line-height: 150%;\"\u003eEt\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"line-height: 150%;\"\u003eEsp-UP\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"369\"\u003e\n\u003cp style=\"margin-left: -.85pt; line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-GCGGATCCATGAAGGGCCTGTTCTTCACCGTCG-3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 20.6pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"231\"\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cem\u003e\u003cspan style=\"line-height: 150%;\"\u003eEt\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"line-height: 150%;\"\u003eEsp-LP\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"369\"\u003e\n\u003cp style=\"margin-left: -.85pt; line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-GCCTCGAGCGAATCTACTTCAAGAAAAGCCACG-3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 20.6pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"231\"\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cem\u003e\u003cspan style=\"line-height: 150%;\"\u003eEt\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"line-height: 150%;\"\u003eEsp\u003c/span\u003e\u003cspan style=\"line-height: 150%;\"\u003e-SP \u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"369\"\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-CCCCGACTACCTCAAGTTCCTCAGC -3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 20.6pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"231\"\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"color: #000000; font-family: verdana, geneva; font-size: 10pt;\"\u003e\u003cem\u003e\u003cspan style=\"line-height: 150%;\"\u003eEt\u003c/span\u003e\u003c/em\u003e\u003cspan style=\"line-height: 150%;\"\u003eEsp\u003c/span\u003e\u003cspan style=\"line-height: 150%;\"\u003e-AP \u003c/span\u003e\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"369\"\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-TGGGTCCGTCTCCCCCTCCTTGGTG -3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 20.6pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"231\"\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e18S-SP \u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"369\"\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-TGTAGTGGAGTCTTGGTGATTC-3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003ctr style=\"height: 20.6pt;\"\u003e\n\u003ctd style=\"width: 173.0pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"231\"\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e18S-AP \u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003eBf\u003cem\u003eEt\u003c/em\u003eESp-UP\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003eBf\u003cem\u003eEt\u003c/em\u003eESp-LP\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003eBf\u003cem\u003eEt\u003c/em\u003eAMA1-UP\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003eBf\u003cem\u003eEt\u003c/em\u003eAMA1-LP\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003ctd style=\"width: 276.45pt; border: none; border-bottom: solid windowtext 1.0pt; padding: 0in 0in 0in 2.9pt; height: 20.6pt;\" width=\"369\"\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-CCTGCTGCCTTCCTTAGATG-3'\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-GCGAATTCGGGCCACCATGAAGGGCCTGTTCTT-3'\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-GCAGATCTGCTGCTCGCGTTGCCAGCAGAT -3'\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-GCGAATTCGGGCCACCATGCAGCCGCCCTAT-3'\u003c/span\u003e\u003c/p\u003e\n\u003cp style=\"line-height: 150%; margin: 0in .25in .0001pt -.85pt;\"\u003e\u003cspan style=\"line-height: 150%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e5'-GCCTCGAGGGTATTCCTGGTCCAG-3'\u003c/span\u003e\u003c/p\u003e\n\u003c/td\u003e\n\u003c/tr\u003e\n\u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp style=\"text-align: left; line-height: 200%; text-autospace: none;\"\u003e\u003cspan style=\"line-height: 200%; font-family: verdana, geneva; color: #000000; font-size: 10pt;\"\u003e\u0026nbsp;\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasites-and-vectors","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"parv","sideBox":"Learn more about [Parasites \u0026 Vectors](http://parasitesandvectors.biomedcentral.com/)","snPcode":"13071","submissionUrl":"https://submission.nature.com/new-submission/13071/3","title":"Parasites \u0026 Vectors","twitterHandle":"@bugbittentweets","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Eimeria tenella, Apical membrane antigen 1, Eimeria -specific protein","lastPublishedDoi":"10.21203/rs.2.17982/v3","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.2.17982/v3","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eAvian coccidiosis is a widespread, economically significant disease of poultry, caused by several species of the protozoan parasite \u003cem\u003eEimeria\u003c/em\u003e. Among these species, \u003cem\u003eE. tenella\u003c/em\u003e causes hemorrhagic pathologies and high mortality. These parasites have complex and diverse lifestyles that require the invasion of their host cells. This is mediated by various proteins secreted from apical secretory organelles. Apical membrane antigen 1 (AMA1), which is released from micronemes and is conserved across all apicomplexans, plays a central role in the host cell invasion. In a previous study, some putative \u003cem\u003eEt\u003c/em\u003eAMA1-interacting proteins of \u003cem\u003eE. tenella\u003c/em\u003e were screened. In this study, we characterized one putative \u003cem\u003eEt\u003c/em\u003eAMA1-interacting protein, \u003cem\u003eE. tenella Eimeria\u003c/em\u003e -specific protein (\u003cem\u003eEt\u003c/em\u003eEsp).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp \u003cem\u003ein vivo\u003c/em\u003e and\u003cem\u003e in vitro.\u003c/em\u003e The expression of \u003cem\u003eEt\u003c/em\u003eEsp was analyzed in different developmental stages of \u003cem\u003eE. tenella\u003c/em\u003e with quantitative PCR and western blotting. The secretion of \u003cem\u003eEt\u003c/em\u003eEsp protein\u003cem\u003e \u003c/em\u003ewas tested with staurosporine when sporozoites were incubated in complete medium at 41 °C.The localization of \u003cem\u003eEt\u003c/em\u003eEsp was analyzed with an immunofluorescence assay. An \u003cem\u003ein vitro\u003c/em\u003e invasion inhibition assay was conducted to assess the ability of antibodies against \u003cem\u003eEt\u003c/em\u003eEsp to inhibit cell invasion by \u003cem\u003eE. tenella\u003c/em\u003e sporozoites.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The interaction between \u003cem\u003eEt\u003c/em\u003eAMA1 and \u003cem\u003eEt\u003c/em\u003eEsp was confirmed with BiFC \u003cem\u003ein vivo\u003c/em\u003e and by GST pull-down \u003cem\u003ein vitro\u003c/em\u003e. Our results show that \u003cem\u003eEt\u003c/em\u003eEsp is differentially expressed during distinct phases of the parasite life cycle. An immunofluorescence analysis showed that the \u003cem\u003eEt\u003c/em\u003eEsp protein is mainly distributed on the parasite surface, and that the expression of this protein increases during the development of the parasite in the host cells. Using staurosporine,\u003cem\u003e \u003c/em\u003ewe showed that \u003cem\u003eEt\u003c/em\u003eEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-r\u003cem\u003eEt\u003c/em\u003eEsp antibody attenuated the capacity of \u003cem\u003eE. tenella\u003c/em\u003e to invade host cells \u003cem\u003ein vitro\u003c/em\u003e. \u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eIn this study, we show that \u003cem\u003eEt\u003c/em\u003eEsp interacts with \u003cem\u003eEt\u003c/em\u003eAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in the sporozoite invasion of host cells and maybe involved in \u0026nbsp;the growth of the parasite in the host. These data have implications for the use of \u003cem\u003eEt\u003c/em\u003eAMA1 or \u003cem\u003eEt\u003c/em\u003eAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.\u003c/p\u003e","manuscriptTitle":"Eimeria tenella Eimeria-specific protein that interacts with apical membrane antigen 1 (EtAMA1) is involved in host cell invasion","msid":"","msnumber":"","nonDraftVersions":[{"code":3,"date":"2020-06-13 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