Identification and Characterization of the ATG8, a Marker of Eimeria tenella Autophagy

Autophagy plays an important role in maintaining cell homeostasis through degradation of denatured proteins and other biological macromolecules. In recent years, many researchers focus on mechanism of autophagy in apicomplexan parasites, but little was known about this process in avian coccidia. In our present study. The cloning, sequencing and characterization of autophagy-related gene ( Etatg8 ) were investigated by quantitative real-time PCR (RT-qPCR), western blotting (WB), indirect immunofluorescence assays (IFAs) and transmission electron microscopy (TEM), respectively. The results have shown 375-bp ORF of Etatg8 , encoding a protein of 124 amino acids in E. tenella , the protein structure and properties are similar to other apicomplexan parasites. RT-qPCR revealed Etatg8 gene expression during four developmental stages in E. tenella , but their transcriptional levels were significantly higher at the unsporulated oocysts stage. WB and IFA showed that EtATG8 was lipidated to bind the autophagosome membrane under starvation or rapamycin conditions, and aggregated in the cytoplasm of sporozoites and merozoites, however, the process of autophagosome membrane production can be inhibited by 3-methyladenine. In conclusion, we found that E. tenella has a conserved autophagy mechanism like other apicomplexan parasites, and EtATG8 can be used as a marker for future research on autophagy targeting avian coccidia. important role in the study of parasite autophagy. In the present study, the Etatg8 gene was cloned, sequenced and analyzed by bioinformatics tools, an anti-EtATG8 specific antibody was produced against recombinant EtATG8 recognized an 18 kDa native protein by WB. The autophagosomes of E. tenella sporozoites were detected by TEM and the expression levels of EtATG8 proteins were analyzed during four developmental stages of E.tenella . The expression profiles of EtATG8 under autophagy induction conditions and its subcellular localization in sporozoites and merozoites were also studied. EtATG8 evidence for formation of stress-induced autophagosomes in Eimeria conserved, typical ubiquitin-like homology three-dimensional binding E. tenella autophagosome.


Introduction
Avian coccidiosis, a parasitic disease, can be caused by intestinal infection with seven Eimeria species, namely; E. tenella, E. necatrix, E. acervulina, E. maxima, E. brunetti, E. mitis and E. praecox (Liu D et al., 2014). The infection is seriously harmful to the intestinal health of chickens, and inflicts huge economic losses to the poultry industry worldwide (Quiroz-Castañeda & Dantán-González, 2015;Tan et al., 2017). Chemoprophylaxis (ionophores and other chemicals) and vaccination (live attenuated or unattenuated vaccines) are the two major preventive strategies against coccidiosis, however, owing to emergence of resistant Eimeria strains and drug residue problems, there arise a need to explore future alternatives (Clark et al., 2017;Peek & Landman, 2011;Tan et al., 2017). Moreover, increasing public health concerns have put a question mark on the prophylactic use of drugs in food-producing animals and consequently restrictions are imposed for their usage in the developed world (Blake & Tomley, 2014;Quiroz-Castañeda & Dantán-González, 2015). Although, live vaccines are a major alternative for the effective control of coccidiosis, but their complex production processes, short-term storage issues, and the risk of the vaccine strains regaining their original pathogenicity, have limited their development and application (Tang et al., 2019;Qi et al., 2013;Tewari & Maharana, 2011). Thus, the discovery of new anticoccidial drugs and vaccines has become an urgent need in addition to the other alternatives for prevention and control of avian coccidiosis.
Autophagy, a "self-eating" process in eukaryotic cells, is involved in targeted degradation of damaged or senescent cells, organelles, denatured proteins, nucleic acids and other biological macromolecules for maintaining cell homeostasis (Brennand et al., 2011;Mizushima et al., 2010;Yu et al., 2018), including organelle clearance during cell differentiation and progression (Besteiro, 2012). This mechanism is evolutionarily conserved across all eukaryotes, including apicomplexan parasites (Latré de Laté et al., 2017;Sinai & Roepe, 2012) and several studies have proven the mechanism of autophagy in apicomplexan parasites is activated in response to starvation or anticoccidial drugs, which leads to programmed cell death (Besteiro, 2017). Thus, induction of autophagy in parasites could be a novel approach for the future anti-parasitic therapeutics.
Currently, more than 40 autophagy-related proteins (ATG) have been identified in eukaryotic cells; most of them are evolutionarily conserved from protozoans to mammals, having vital functions in the accomplishment of programmed cell death (Besteiro et al., 2011). For example, ATG5-ATG12 complex and ATG7-ATG3 complex are involved in autophagosome formation, the ATG17-ATG29-ATG31 complex is a pre-autophagosomal structure (PAS) formation, Beclin1-Vps34 complex is the main regulator of autophagy, ATG15, ATG22 and PEP4 in the breakdown of autophagy, and ATG22 and ATG33 are closely related to the formation of mitophagy (Meijer et al., 2007). ATG8, a key ubiquitin-like protein, can bind the autophagosome membrane after being activated by ATG3 and ATG7 (Yamaguchi et al., 2010), and has been considered as a marker for studies on autophagy.
Many autophagy studies on apicomplexans have shown that the biochemical function of ATG8 is conserved across all eukaryotes (Tomlins et al., 2013), but there are two views on the subcellular localization of ATG8; some researchers agree that ATG8 is localized in the apicoplasts of some parasites, and participates in the formation of apicoplast membranes (Walczak et al., 2018), while others think that it should be distributed in the cytoplasm, and ATG8-related punctate structures may exist in the cytoplasm under starvation or autophagy-induced treatment, which can be used as a marker for detecting autophagosomes (Sinai & Roepe, 2012). However, very little is known about autophagy in E. tenella. In a previous study, we identified about 11 ATG gene orthologs in E. tenella genome database (ETH_00016760, ETH_00018400, ETH_00011205, ETH_00021095, ETH_00032225, ETH_00019020, ETH_00038935, ETH_00030035, ETH_00026430, ETH_00026880, ETH_00041360), including Etatg8 (ETH_00016760) conserved in E. tenella and other apicomplexans. In the present study, we report the cloning, sequencing expression and characterization of Etatg8 of E. tenella, which will provide new insight for autophagy studies in Eimeria relevant to the prevention and control of avian coccidiosis.

Ethics approval
The study was conducted with the approval of China Guangdong Province Science and Technology Department (Permit Number: SYXK (Yue) 2016-0165).

Parasites and animals
The E. tenella GD strain used in this study was isolated from a ceca sample obtained at a chicken farm in Guangdong, China. Oocysts were passaged in 2-week-old Lingnan Yellow chickens for 7 days by infecting them Cloning, sequencing and bioinformatics analysis PCR products purified using Gel Extraction Kit (OMEGA) were cloned using the pMD18-T Vector Cloning kit (Takara) following the manufacturer's instructions, then transformed into DH5α chemically competent cells (Transgen Biotech, China). The positive clones identified by PCR (same protocol as PCR-amplification) were sent to Invitrogen (China) biotechnology company for sequencing, the sequences were analyzed using the SignalP 4.1 (http://www. cbs.dtu.dk/services/SignalP/), the TMHMM Server v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/) for protein signal peptide, protein transmembrane domain and protein structure identification, respectively. In addition, the TargetP 1.1 server (http://www.cbs.dtu.dk/services/TargetP/) was used for predicting the subcellular location of the protein, and the basic local alignment search (BLAST, http://blast.ncbi.nlm.nih.gov/Blast.cgi) and MEGA 5.05 (Tamura et al., 2011) were used for molecular phylogenetic analysis.

Expression and purification of recombinant EtATG8
Protein expression and purification was conducted as described previously (Sun et al., 2016). Briefly, the Etatg8 open reading frame was amplified by PCR with primers which incorporated EcoRI and XhoI restriction sites ( Table 1). The PCR product was cloned into the pET28a expression vector (Novagen, Darmstadt, Germany), and recombinant EtATG8 (rEtATG8) with an N-terminal His tag was expressed in the Escherichia coli BL21 strain, and then purified under denaturing conditions by Ni-NTA column chromatography (Novagen) according to the manufacturer's instructions. The target protein (100 μg) was separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 12% gels and visualized after staining with coomassie brilliant blue, and detected by Western Blotting (WB) using a mouse anti-His-Tag moloclonal antibody (diluted 1:1000, Cwbiotech, China) and goat anti-mouse IgG-HRP (diluted 1:2000, Santa Cruz Biotechnology, USA). with 5×10 4 sporulated oocysts. The chicks (day-old hatchlings) were purchased from Guangdong WIZ Agricultural Science & Technology Co., Ltd. (Guangzhou, China). The oocysts were harvested and purified as described previously, and the saturated salt solution used as floatation solution (Qi et al., 2019;Sun et al., 2016). The free sporozoites prepared from sporulated oocysts by in vitro excystation were purified by passage through a G3 filter funnel (Jingke, Guangzhou, China). Merozoites were collected from ceca of chickens that had been infected with 5×10 4 sporulated oocysts per chicken at 120 h post-infection. The purification procedure was the same as previously described (Sun et al., 2016).

Transmission electron microscopy (TEM)
Following incubation in Dulbecco' s modified Eagle' s medium (DMEM) or Hank' s balanced salt solution (HBSS) at 41 °C for 8 h, the E. tenella sporozoites were fixed in 2.5% glutaraldehyde with 4% parafomaldehyde in 0.1 M cacodylate buffer for 3 h followed by post fixation in 1% osmium tetroxide for 3 h. Parasites were dehydrated in a series of ethanol formulations followed by 100% epoxy ethane, infiltrated with Spurr's resin and polymerized for 12 h at 70 °C. Ultra-thin sections (70 nm) were placed and stained with 1% uranyl acetate or 0.25% lead citrate, then observed with HT7700 transmission electron microscope (TEM, HITACHI, Japan).

RNA extraction and RT -PCR amplification
The homologous gene to Etatg8 (ETH_00016760) was used to design specific primers ( Table 1) for amplifying the open reading frame of Etatg8 gene. Total RNA was extracted from the four life stages of E. tenella (unsporulated oocysts, sporulated oocysts, sporozoites and merozoites) using the HiPure Total RNA Micro kit (MAGEN, China). The RNA was treated with DNase I (Takara), and cDNA was generated from it using PrimeScript II 1st Strand cDNA Synthesis kit (Takara) with random primers. The Etatg8 gene was PCR-amplified by using LATaq®DNA polymerase (Takara) using following procedure: an initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final elongation at 72°C for 7 min. The PCR products were analyzed by 3% agarose gel electrophoresis.
Transcriptional profile of Etatg8 in four life stages of E. tenella The transcriptional profile of Etatg8 in the four life stages of E. tenella was detected using quantitative real-time PCR (RT-qPCR) with SYBR green I (Liu R et al., 2014;Xu et al., 2008). Briefly, (Xu et al., 2008) the primers were designed using Primer Premier 5.0 ( Table 1), and RT-qPCR was performed in a 20 μL reaction volume containing 10 μL 2×Taq PCR MasterMix (Takara), 1.0 μL cDNA, each primer (1 μL, 10 μM), and 7 μL nuclease free H 2 O, which was performed using the CFX Connect system (Bio-Rad). Nuclease free H 2 O and the Etactin gene were used as the negative and internal controls, all samples were run in triplicate. The amplification was performed using initial denaturation at 95 °C for 10 sec, followed by 40 cycles of 95 °C for 5 sec, and 60 °C for 30 sec. Finally, the transcriptional level of the Etatg8 gene was calculated according to the 2 -△△CT method, the data were statistically analyzed with one-way ANOVA (p<0.05, n = 3).

Protein expressional profile of EtATG8 in four life stages of E. tenella
Total proteins from the four life stages of E. tenella were prepared using the Cell Lysis Buffer system (Beyotime, China), and the protein concentrations were determined using the BCA protein assay kit (Beyotime, China), then total protein extracts of each stage (100 μg) were subsequently separated on 12% SDS-PAGE. The EtATG8 expression profile in the four life stages was determined by WB with polyclonal rabbit anti-rEtATG8 antibody (diluted 1:500). A rabbit monoclonal anti-Actin antibody (diluted 1:500, Sigma, USA) and goat anti-rabbit IgG-HRP (diluted 1:2000, SantaCruz Biotechnology, USA) was used as control and secondary antibody, respectively.

Expression of EtATG8 protein instarvation-or inducer-induced sporozoites
Following the induction of starvation and/or inducer treatment, the total proteins from sporozoites incubated in DMEM, HBSS, HBSS with 3-methyladenine (3-MA, 25 mM, Sigma, USA), DMEM with rapmycin (RP, 10μM, Sigma, USA), and DMEM with RP and 3-MA were prepared using the Cell Lysis Buffer system (Beyotime, China), and the protein concentrations were determined using the BCA protein assay kit (Beyotime, China), then total protein extracts of each sample (100 μg) were separated on 12% SDS-PAGE with 6 M urea to visualise the lipid-conjugated form of EtATG8, or on 12% SDS-PAGE without urea to visualise the total EtATG8. The EtATG8 was determined by Western blot with polyclonal rabbit anti-rEtATG8 antibody (diluted 1:500, the polyclonal antibodies derived from Et ATG8-vaccinated rabbits). A rabbit monoclonal anti-Actin antibody (diluted 1:500, Sigma, USA) was used as the control, the goat anti-rabbit IgG-HRP (diluted 1:2000, SantaCruz Biotechnology, USA) was used as the secondary antibody.

Indirect immunofluorescence assays
Indirect immunofluorescence assays (IFAs) were performed either on sporozoites or on merozoites of E. tenella under different conditions as described previously (Xu et al., 2008). Briefly, the extracellular sporozoites and merozoites were harvested after being incubated in DMEM, HBSS, DMEM with rapamycin (RP, 10 μM), or DMEM with monensin (Mon, 100 ng/mL, Sigma, USA) at 41 °C for 8 h. The parasites were fixed for 20 min with 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS), permeabilized with 1% Triton X-100 for 10 min after being adhered onto poly-L-lysine slides for 20 min, and then blocked with 0.1% (w/v) BSA in PBS for 30 min at 37°C. The anti-rEtATG8 antibody (diluted 1:500) and donkey anti-rabbit IgG-R (diluted 1:400, SantaCruz Biotechnology, USA) were incubated successively with the parasites for 1 h at 37°C. Parasitic DNA was labeled for 5 min with DAPI solution (10 μg/mL, Beyotime, China). All images were acquired with a Zeiss LSM710 confocal system (CarlZeiss).

Morphological evidence of autophagosomes in E. tenella
To confirm the autophagy of E. tenella under starvation, we examined the subcellular ultrastructure of E. tenella sporozoites by TEM. The results showed that there were some food vacuoles (FV) in the sporozoites cytoplasm without autophagosome-like structure after been treated with DMEM, (Figure 1A). After starvation in HBSS, more FV ( Figure 1B) were formed in sporozoite compared to DMEM control, and a double membrane autophagosome in the sporozoite is marked as a red solid arrow, which contains partially digested material ( Figure 1B), including some degraded materials (damaged organelles).

Cloning and characterization of Etatg8
The ortholog of the atg8 gene (ETH_00016760) in E. tenella genome database was identified as 375-bp open reading frame (Figure 2A), which encodes a protein of 124 amino acid residues. Bioinformatics software analyses showed that the Etatg8 gene encodes non-secreted protein, without signal peptide ( Figure 2B) and transmembrane domain ( Figure 2D), and it is most likely located in the parasite's cytoplasm ( Figure 2C). In the phylogenetic tree established by NJ method, all Eimeria species clustered on one branch, and had the closest genetic relationship with other apicomplexan parasites (Figure 3). The SWISS-MODEL analysis indicated the highest quality of the model, where 90.8% of the residues were located in the most favored regions, 8.3% in the additionally-allowed regions, and 0.9% in the disallowed regions ( Figure 4A). The EtATG8 structure ( Figure 4B) was very similar to PfATG8 (Figure 4C), which contains four alpha helices and six beta sheets ( Figure 4D), including two N-terminal alpha helices (α1 and α2), a C-terminal ubiquitin-like domain, and two short beta strands (β3 and β4) between α3 and β5, where the domain (shown in blue frame) between α1 and α3 is the binding region for ATG3 ( Figure 4E).   The rEtATG8 expressed under induction with 1 mM isopropyl β-D-1-thiogalactopyranoside, was identified as a band of about 18 kDa on 12% SDS-PAGE gel ( Figure 5A). The purified proteins were obtained from the bacterial lysates using a Ni-NTA chromatography column, and a specific band around the 18kDa size was detected by Western blot using the anti-His-Tag monoclonal antibody against His-tagged rATG8( Figure 5B).

Transcriptional profile of Etatg8 in four stages of E. tenella
The RT-qPCR results showed that the transcription level of Etatg8 was highest at the unsporulated oocyst stage (Figure 6), which was about 200 times higher than that in the merozoite stage, but less than 10 times higher than those in the sporulated oocyst and sporozoite stages (p<0.05).

EtATG8 of E. tenella sporozoites was lipidated under different conditions
The results showed that EtATG8 could be separated into a single band of about 14 kDa by SDS-PAGE without urea under starvation (HBSS) condition ( Figure 8A, upper), whereas it could be separated into non-lipidated (form I) and lipidated (form II) proteins by SDS-PAGE with 6 M urea ( Figure 8A, middle). However, form II-EtATG8 increased significantly in the presence of inducer RP or under starvation condition than other groups ( Figure 8A, middle). After starvation for 8 h, the ratio of form II/form I was about 0.44, which was significantly higher than that in HBSS + 3-MA group (0.07) and DMEM control group ( Figure 8B). The ratio of form II/form I in DMEM+RP group was about 0.63 ( Figure 8B), which was higher than that in DMEM+RP+3-MA group (0.28) and DMEM control group.

Subcellular location of EtATG8 in the sporozoites and merozoites
Subcellular location of EtATG8 in the sporozoites or merozoites was shown in Figure 9A and 9B. The results showed that EtATG8 signals were aggregated in the sporozoite cytoplasm after incubation in HBSS (Fig. 9A2), DMEM+RP (Fig. 9A3) or DMEM+Mon (Fig. 9A4), while they were evenly distributed throughout sporozoites after incubation in DMEM control (Fig. 9A1), and the EtATG8 signals were aggregated in the merozoites cytoplasm after incubation in HBSS (Figure 9 B2), DMEM+RP (Figure 9 B3) or DMEM+Mon (Figure 9 B4), while they were evenly distributed throughout merozoites after incubation in DMEM control (Figure 9 B1).

Discussion
In recent years, researches on autophagy have shown the importance of this process in the survival of apicomplexan parasites (Nguyen et al., 2017). The ATG8, as a marker for detection of autophagosomes, plays an important role in the study of parasite autophagy. In the present study, the Etatg8 gene was cloned, sequenced and analyzed by bioinformatics tools, an anti-EtATG8 specific antibody was produced against recombinant EtATG8 recognized an 18 kDa native protein by WB. The autophagosomes of E. tenella sporozoites were detected by TEM and the expression levels of EtATG8 proteins were analyzed during four developmental stages of E.tenella. The expression profiles of EtATG8 under autophagy induction conditions and its subcellular localization in sporozoites and merozoites were also studied.
During the complex mechanism of autophagy, autophagosome formation is a key step, which results in enclosure of materials to be degraded for delivery to the lysosome (Ichimura et al., 2000;Tooze & Yoshimori, 2010). In the present study, the autophagosome-like structure with bilayer membranes is prominent under TEM in the E. tenella sporozoites, the expression of EtATG8 protein in starvation-or inducer-induced sporozoites was detected by WB, and the results have shown that EtATG8 was lipidated under these conditions, which provide evidence for formation of stress-induced autophagosomes in Eimeria. Sequence analysis of the Etatg8 gene has revealed that it is highly conserved, and encodes a typical non-secreted, ubiquitin-like protein without transmembrane domain. The homology modeling showed that EtATG8 has a conserved three-dimensional structure and a similar binding region of ATG3 to ATG8 of P. falciparum, which provides indirect evidence that EtATG8 plays a similar role in the formation of E. tenella autophagosome.
Autophagy plays an important role during cell differentiation and development (Qi et al., 2019). In this study, we found that Etatg8 mRNA levels in the unsporulated oocysts stage were significantly higher than those in the other three developmental stages. These results are consistent with the analysis of the coccidial transcriptome in EuPathDB (ETH_00016760). We predict that autophagy has an important role in the sporulation of oocysts, which requires  the storage of a large number of atg8 mRNAs to express enough ATG8 proteins for autophagosome formation. However, the expression levels of EtATG8 protein during four developmental stages were inconsistent with the results of mRNA levels. This may be due to two reasons: (1) post-transcriptional processing and modification of the Etatg8 gene, including transcriptional degradation, results in inconsistent levels of transcription and expression; (2) the expression process of EtATG8 protein would maintain at a stable level during all the developmental stages, and would show variation only when undergoing cell differentiation. These hypotheses need further investigations.
The subcellular presence of ATG8 in yeast and mammalian cells have shown it's localization in the cytoplasm (Lévêque et al., 2015), whereas in case of T. gondii and P. falciparum, ATG8 is localized in the apicoplast membrane (Kitamura et al., 2012). Here, we investigated the subcellular localization of EtATG8 in sporozoites and merozoites, and found that it is generally distributed in the cytoplasm of the E. tenella parasites in DMEM cultures. After treatment with HBSS, monensin or rapamycin, EtATG8 gathered in the cytoplasm of these parasites.

Conclusions
To summarize, we successfully cloned and expressed the autophagy marker protein gene (Etatg8) from unsporulated and sporulated oocyst, sporozoite and merozoite developmental stages of E. tenella (EtATG8). It was confirmed that E.tenella sporozoites can form autophagosomes after induction either by starvation or autophagy inducer, EtATG8 protein is lipidated and accumulates in sporozoite and merozoite cytoplasm, while autophagy inhibitor (3-MA) can inhibit the production of sporozoite autophagosomes. These findings confirmed a conserved autophagy mechanism in E. tenella, and also have proven that ATG8 can be used as a marker protein for autophagy research in avian coccidia, which will serve as a basis for future investigations in this field.