Diclazuril-induced expression of CDK-related kinase 2 in the second-generation merozoites of Eimeria tenella

Background: Diclazuril is a classic anticoccidial drug. The key molecules of diclazuril in anticoccidial action allows target screening for the development of anticoccidial drugs. In the present study, a diclazuril anticoccidiosis animal model was established, and the transcription and translation levels of the CDK-related kinase 2 of Eimeria tenella (EtCRK2) were detected through quantitative real-time PCR and Western blot analysis, respectively. The localisation of EtCRK2 in merozoites was examined with immunouorescence techniques. Results: The mRNA and protein expression levels of EtCRK2 decreased in the infected/diclazuril group compared with those in the infected/control group. In addition, immunouorescence analysis showed that EtCRK2 was localised in the cytoplasm of the merozoites. The uorescence intensity of EtCRK2 in the infected/diclazuril group was signicantly weaker than that in the infected/control group. Conclusions: The anticoccidial drug diclazuril against E.tenella affects the expression pattern of EtCRK2 molecule, and EtCRK2 is a potential target for new drug development.


Background
Eimeria tenella is an obligate intracellular parasite that invades chicken caecal epithelial cells to survive and complete its lifecycle. This parasite causes a serious form of coccidiosis and huge economic losses to the poultry community [1]. The prevention and control of coccidiosis mainly rely on chemical drugs.
However, coccidial resistance is induced by the long-term use of anticoccidiosis drugs [2]. Therefore, novel drug targets and control strategies must be sought.
E. tenella has a complex lifecycle that involves an asexual stage (sporogony and schizogamy) and a sexual stage (gametogony) [3]. During schizogamy, under the precise regulation of cell cycle, E. tenella proliferates rapidly within host cells [4]; this process is similar to cell cycles in other eukaryotes regulated by cyclin-dependent kinases (CDKs) [5]. Cyclin-dependent kinases (CDKs) are serine/threonine kinases that regulate the activity of substrate proteins through phosphorylation [6,7], which regulates cell cycle progression, proliferation and differentiation and modulates transcription [8][9][10][11][12][13]. Several CDK-like kinases have been identi ed and characterised in parasitic protozoans. Toxoplasma gondii CRK1 (TgCRK1), TgCRK2, TgCRK4 and TgCRK6 are essential for tachyzoite replication and growth [14]. Plasmodium falciparum CDK-related kinase 3 (PfCRK-3) plays a key role in the intraerythrocytic development of P. falciparum [15]. Plasmodium berghei CDK-related kinase 5 (CRK5) is necessary for the S-and M-phases during gametogony and is required for mosquito transmission [16]. Theileria annulata CRK3 may be a regulator in gene transcription in all bovine intracellular life cycle stages [17]. CDK2 is involved in the differentiation of Giardia lamblia into cysts [8]. CDK-related kinase 2 (EtCRK2) is the only CDK that has been con rmed in E. tenella. It is expressed during the asexual and sexual states of E. tenella development [5,18]. E. tenella schizont development is completely inhibited under treatment with the special CDK inhibitor avopiridole at concentrations of 150 and 300 nM. Thus, E. tenella CDKs are considered chemically validated drug targets [4,5]. In our previous study, EtCDK was found to have differential expression according to the diclazuril anticoccidial cDNA library constructed through the suppression of subtractive hybridisation (data not shown). We investigated the expression pattern of EtCRK2 in response to diclazuril treatment against E. tenella infection.
Diclazuril, a classic anticoccidial drug, signi cantly induces merozoite apoptosis, effectively decreases the number of merozoites, and alleviates cecum damage induced by E. tenella [19][20][21][22][23][24]. Here, we reported changes in EtCRK2 in response to diclazuril treatment against E. tenella infection and observed the expression levels of EtCRK2 mRNA and protein and the spatial location of EtCRK2.

Results
Cloning of the EtCRK2 gene The 1015 bp EtCRK2 gene was ampli ed from second-generation merozoites and cloned into a pMD-19T vector. A DNA fragment of EtCRK2 ORF ampli ed from pMD-19-EtCRK2 was subcloned into pET-28a (+). The recombinant plasmid pET-28a-EtCRK2 was analysed through PCR with pET-28a (+) vector universal primers, and the product length was in accordance with ORF 891 bp plus vector sequence 360 bp ( Fig. 1).
A 891 bp insertion ORF was also identi ed by EcoR I and Hind III enzyme digestion (Fig. 2) and sequenced for 100% correctness.

Expression of recombinant EtCRK2 protein and polyclonal preparation
A high level of rEtCRK2 protein expression was achieved by inducing the rEtCRK2 protein at 37 °C with 0.5 mM IPTG for 4 h. The protein was then separated through SDS-PAGE (Fig. 3). The theoretical molecular weight of the rEtCRK2 protein was approximately 37.67 kDa. The puri ed rEtCRK2 protein (Fig. 4) was used for antibody preparation. The antiserum had a speci c binding with the recombinant protein (Fig. 5), and the titer of the antibody against rEtCRK2 exceeded 1:512K (Fig. 6).
Expression of EtCRK2 mRNA Fig. 7 shows that the mRNA expression level of EtCRK2 in the infected/diclazuril group was downregulated by 42.3% relative to that in the infected/control group (P < 0.01).

Western blot analysis
As shown in Fig. 8, the EtCRK2 protein expression level in the infected/diclazuril group was downregulated by 59.32% (P < 0.01) relative to that in the infected/control group.

Immuno uorescence analysis
The subcellular location of EtCRK2 in the second-generation merozoites was visualised through immuno uorescence microscopy. As shown in Fig. 9, EtCRK2 was widely distributed in the cytoplasm of the merozoites. The green uorescence in the merozoites of the infected/diclazuril group was darker than that in the merozoites of the infected/control group.

Discussion
Coccidiosis caused by the apicomplexan parasites of the genus Eimeria induces high economic problems by reducing poultry productivity and performance [25]. E. tenella is one of the most virulent species of Eimeria, mainly infects the chicken cecum. During secondgeneration schizogony, large amounts of released second-generation merozoites reinvade other uninfected caecal epithelial cells. Therefore, inhibiting the reproductive process of second-generation merozoites may be an effective strategy for controlling coccidiosis.
E. tenella utilises complex and distinctive mechanisms to regulate its replicative cycles [14]. CDKs participates in mediating cell proliferation and division in the parasite [7,[26][27][28]. In Plasmodium berghei, CRK2 mRNA is highly expressed in gametocytes and mosquito endophase; this characteristic suggests that CRK2 protein has a key role throughout the life cycle [29]. At the trophozoite/schizont stage, the inhibition of P. falciparum Cdc2-related kinase-1 (PfCRK-1) protein interferes with the schizogony of P. falciparum [30]. The depletion of Plasmodium falciparum CRK4 results in the complete blockage of nuclear division and intensively inhibits DNA replication during schizogony in the intraerythrocytic blood stage [31]. The downregulation of P. falciparum MO15-related protein kinase, Cdc2-related kinase PfPK5, P. falciparum CRK3 and other cyclins during dormancy arrests parasite progression at the G 1 phase and halts DNA synthesis [32]. The loss of cytoplasmic TgCRK2 results in T.
gondii cell cycle G1 phase arrest [14]. These results suggest that CDKs are important molecular switches that contribute to cell cycle progression in parasites [30]. CDK2 plays a pivotal role in regulating G1/S and S/G2 transitions during the cell cycle [12,13]. In A375 human melanoma cells, CRISP/Cas9 technology knockout CDK2 induces G0/G1 phase arrest and early apoptosis [31]. Gastric cancer cells culture in a low Cl − medium inhibits cell growth and causes the G0/G1 phase arrest by diminishing the expression of CDK2 and phosphorylated tumor suppressor gene Rb [32]. In this study, the levels of EtCRK2 mRNA and protein in the infected/diclazuril group decreased relative to those in the infected/control group. This effect indicates that diclazuril likely blocks cell cycle in merozoites in the G1 phase by interfering with the EtCRK2 pathway and induces the occurrence of apoptotic events [20,35], including decrease in mitochondrial membrane and chromatin agglutination [20], downregulation of serine/threonine protein phosphatase type 5 mRNA expression [3] and increase in glyceraldehyde-3-phosphate dehydrogenase transcription and protein expression [36]. Thus, the normal reproductive cycle of E. tenella is inhibited, and the number of second-generation merozoites subsequently decreases [19].
The spatial distribution of proteins affects protein function. The localisation of T. annulata CRK2 protein in parasite nuclei is upregulated transiently in midmerogony. This phenomenon suggests that TaCRK2 coordinated the parasite's nuclear division [17]. The protozoan G. lamblia CDK2 localised in the cytoplasm contributes to cyst formation and shows increased expression during encystation [8]. The members of P. falciparum CDK-like kinase (CLK) family, PfCLk-1 and PfCLK-2, are primarily localised in the nucleus, and the further dispersion of PfCLK-2 into the cytoplasm indicates that PfCLKs participate in gene regulation and the post-transcriptional modi cation of mRNA in the malarial blood stage [37]. In the present study, EtCRK2 was localised in the cytoplasm of second-generation merozoites, and the uorescence intensity of EtCDK2 decreased under diclazuril treatment. This phenomenon is consistent with the determined expression levels. These results implies that EtCRK2 plays an essential role in the asexual life cycle of E. tenella and using agents targeting CRK2 is potential therapeutic strategy against coccidiosis. However, the exact molecular mechanism needs to be further veri ed in a coccidial cell model in vitro using CDKs-speci c inhibitors.

Conclusion
Our results indicate that diclazuril downregulates the mRNA and protein expression levels of EtCRK2 and disrupts the cell cycle of second-generation merozoites. This action may be an aspect of the molecular mechanism of the anticoccidial action of diclazuril.

Inoculum and drug
Oocysts of E. tenella (Luoyang strain) were passaged before inoculation, and the obtained oocysts was sporulated in 2.5% K 2 Cr 2 O 7 solution at 29 °C for 48 h. Diclazuril ( > 99%; Shanghai Veterinary Research Institute, CAAS, China) was supplied at a dose of 1 mg/kg in broiler feed.

Experimental chickens and treatment
One-day-old male Chinese Yellow broiler chickens were obtained from Gonghua Commercial Hatchery, Luoyang, China. The chickens were kept in wire-oored batteries for 14 days under coccidiosis-free conditions. On the 14th day, healthy 90 chickens were randomly divided into two groups of 45 with three biological replicates of 15 in each group: (1) Chickens that were challenged with E. tenella sporulated oocysts and received commercial diet without drugs were included to the infected/control group. (2) Chickens that were challenged with E. tenella sporulated oocysts and treated with 1 mg/kg diclazuril in feed from 96 h to 120 h were included to the infected/diclazuril group. Inoculation was performed with a

Preparation of the second-generation merozoites
At 120 h postinoculation, chickens were killed through CO 2 asphyxiation, and the pooled caecal tissues from 10 randomly selected chickens in each replicate were used for the preparation of the second-generation merozoites in accordance with a previously described method [19][20][21]. Brie y, caecal tissues were cut into pieces, then incubated with enzyme digestion solution (0.12 mol/L NaCl, 3.0 mmol/L K 2 HPO 4 ·3H 2 O, 9 mmol/L CaCl 2 , 1 mg/mL hyaluronidase, 1 mg/mL BSA and 0.02 mol/L Tris; pH = 7.4) at 37 °C for 60 min. After the ltration and centrifugation of the digestion mixture, the collected sediment was incubated with red blood cell lysis buffer (Beyotime, Shanghai) at 4 °C for 10 min. Lysates were removed through centrifugation, and the merozoites were collected through density gradient centrifugation. The collected merozoite samples were subjected to total RNA preparation, Western blot and immuno uorescence assay successively. Three samples in each group were used for each experiment.
Total RNA preparation and cDNA synthesis The total RNA of merozoites was extracted with TRIzol® Reagent (Invitrogen, USA) according to the manufacturer's procedure, and cDNA was synthesised with EasyScript One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech, Beijing).

Ampli cation of the EtCRK2 gene
According to the sequence (GenBank: AY508221.1), speci c primers P1: 5′-AAGGGACTTACGGAGTGGTTTA-3′ and P2: 5′-TGAATTTACGTGAATATGTTGG-3′ were designed to amplify the EtCRK2 gene and an ORF from a cDNA template. Ampli ed fragments were separated through 1% agarose gel electrophoresis and isolated using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 according to the manufacturer's instructions. The puri ed ampli ed fragments were ligated into the pMD-19T vector and then transformed into Escherichia coli strain DH5α. The recombinant clone of pMD-19-EtCRK2 was identi ed through PCR and sequenced by Shanghai Sangon Biotech Co., Ltd. The positive plasmid was named pMD-19-EtCRK2.

Polyclonal antibody preparation
For the production of polyclonal antibodies, puri ed rEtCRK2 protein was used as an antigen in subsequent immune procedures. rEtCRK2 protein emulsi ed with the same volume of Freund's complete adjuvant (Sigma-Aldrich) was injected into New Zealand rabbits at a dose of 500 µg/rabbit. After 2 weeks, puri ed rEtCRK2 protein emulsi ed with Freund's incomplete adjuvant (Sigma-Aldrich) was injected to the rabbits at a dose of 500 µg/rabbit for secondary immunisation. After a 2 week hiatus, the third and fourth rounds of immunisation were performed separately. Ten days after the last immunisation, serum samples were collected for the determination of speci city antibody titer. The speci city of the antibody was determined with Western blot. Brie y, 25 ng of recombinant protein was used for SDS-PAGE, and preimmunisation rabbit serum and antiserum were used as the primary antibodies. HRP conjugated goat antirabbit IgG (Biolab, Beijing) was subsequently used. HRP activity was revealed by enhanced chemiluminescence system using BeyoECL Plus substrate (P0018S, Beyotime Biotechnology, China). The antibody titer was analysed with indirect enzyme linked immunosorbent assay (ELISA) [38]. Brie y, 500 ng/well rEtCRK2 protein was used to coat 96-well plates at 4 °C overnight, and the antiserum diluted with PBS at 1:2K, 1:4K, 1:8K, 1:16K, 1:32K, 1:64K, 1:128K, 1:256K, 1:512K, 1:1024K and 1:2048K were incubated with the antigen at 37 °C for 2 h. After washing with TBST, horse radish peroxidase (HRP)-conjugated goat antirabbit IgG were added to all microwells at 37 °C for 2 h. After washing, the substrate solution tetramethylbenzidine was added, and the microplate was read at 450 nm in a microplate reader (Multiskan FC, Thermo Scienti c, USA). Preimmunisation rabbit serum and PBS were used as the control.

EtCRK2 mRNA expression analysis
The mRNA expression level of EtCRK2 was quanti ed through real-time PCR with a CFX96 touch real-time PCR system (Bio-Rad, America) and TB Green ® Premix Ex Taq™ GC (Perfect Real Time; Takara, Beijing). Each reaction was performed in triplicate, and the entire experiment was carried out in triplicate. E. tenella 18S rRNA was used as the control. The primer sequences are shown in Table 1. Relative mRNA expression was determined using the ΔΔ Ct method.

Western blot analysis
Puri ed merozoites were treated with RIPA lysis buffer (Beyotime, Shanghai) for Western blot analysis and determined using a BCA protein assay kit (Cwbio, Beijing) for concentration assessment. Pyrolysis products were dissolved in SDS-PAGE sample buffer (Beyotime, Shanghai), heated at 96 °C for 5 min, separated on 12% SDS-PAGE and electrotransferred to a polyvinylidene di uoride membrane (Membrane Solutions, USA). The membrane was detected with rabbit antiserum against EtCRK2 as the primary antibody or anti-β-tubulin monoclonal antibody (1:1000 dilution, K200059 M, Solarbio, China), followed by HRP conjugated goat antirabbit IgG ((Biolab, Beijing). HRP activity was revealed by enhanced chemiluminescence system using BeyoECL Plus substrate (P0018S, Beyotime Biotechnology, China) and Image J Software. The independent experiments were performed in triplicate.

Immuno uorescence test
In accordance with our previously described method with minor modi cations [22], the merozoites were prepared into smears and xed with 4% paraformaldehyde. The merozoites were washed three times with PBS, then permeabilised with 1% Triton X-100 (Sangon-Biotech, Shanghai) and blocked with 2% BSA-PBS at 4 °C overnight. Rabbit antiserum against EtCRK2 (1:2000 dilution) was used as the primary antibody at 37 °C for 1 h, and FITC-conjugated goat antirabbit IgG (Servicebio, Wuhan) with 1:100 dilution was used as the second antibody at 37 °C in the dark for 1 h. Finally, the merozoites were stained with 4′,6′diamidino-2-phenylindole (Boster, China) at room temperature for 30 min, and 50 μL of anti-fade mounting medium (Sangon Biothch, Shanghai) was used to close the coverslip. Images were visualised with a confocal laser scanning microscope (LSM 800, ZEISS) at a l ex/em of 492 nm/520 nm and 358 nm/461 nm.

Statistical analysis
Data were expressed as mean ± standard deviation. Student's t test was used in statistical analyses. Values of P < 0.05 and P < 0.01 were considered signi cant.

Consent for publication
Not applicable.

Availability of data and materials
The data generated or analyzed during this study are included in this article.

Competing interests
No potential con ict of interest was reported by the authors.  Tables   Table 1 Primer