Eimeria tenella pyrroline -5-carboxylate reductase is a secreted protein and involved in host cell invasion.

Chicken coccidiosis, which caused by Eimeria spp, is a parasitic protozoal disease. At present, control measures of this disease depend mainly on anticoccidial drugs and live vaccines. But these control strategies have drawbacks such as drug resistance and limitations in live vaccines production. Therefore, novel control approaches are urgently need to study to control this disease effectively. In this study, the function and characteristics of the pyrroline-5-carboxylate reductase of Eimeria tenella (EtPYCR) protein were preliminary analyzed. The transcription and translation level were analyzed by using qPCR and Western blot. The results showed that the mRNA transcription and translation levels of EtPYCR were higher in unsporulated oocysts (UO) and second generation merozoites (Mrz) than that in sporulated oocysts (SO) and sporozoites. Enzyme activity showed that the enzyme activity of EtPYCR was also higher in the UO and Mrz than that in the SO and sporozoites. Immunofluorescence localization showed EtPYCR was mainly located on the top of sporozoites and the whole cytoplasm and surface of Mrz. The secretion assay indicated that EtPYCR was secretion protein, but not from micronemes. Invasion inhibition assay showed that rabbit anti-rEtPYCR polyclonal antibodies can effectively inhibit sporozoite invasion of DF-1 cells. These results showed that EtPYCR possess several important roles that separate and distinct from its conversion 1-pyrroline-5-carboxylate (P5C) into proline and maybe involved in the host cell invasion and development of parasites in host cells.

Integrated application of transcriptomics and metabolomics provides insight into the mechanism of Eimeria tenella resistance to maduramycin.

Avian coccidiosis, caused by Eimeria parasites, continues to devastate the poultry industry and results in significant economic losses. Ionophore coccidiostats, such as maduramycin and monensin, are widely used for prophylaxis of coccidiosis in poultry. Nevertheless, their efficacy has been challenged by widespread drug resistance. However, the underlying mechanisms have not been revealed. Understanding the targets and resistance mechanisms to anticoccidials is critical to combat this major parasitic disease. In the present study, maduramycin-resistant (MRR) and drug-sensitive (DS) sporozoites of Eimeria tenella were purified for transcriptomic and metabolomic analysis. The transcriptome analysis revealed 5016 differentially expressed genes (DEGs) in MRR compared to DS, and KEGG pathway enrichment analysis indicated that DEGs were involved in spliceosome, carbon metabolism, glycolysis, and biosynthesis of amino acids. In the untargeted metabolomics assay, 297 differentially expressed metabolites (DEMs) were identified in MRR compared to DS, and KEGG pathway enrichment analysis indicated that these DEMs were involved in 10 pathways, including fructose and mannose metabolism, cysteine and methionine metabolism, arginine and proline metabolism, and glutathione metabolism. Targeted metabolomic analysis revealed 14 DEMs in MRR compared to DS, and KEGG pathway analysis indicated that these DEMs were involved in 20 pathways, including fructose and mannose metabolism, glycolysis/gluconeogenesis, and carbon metabolism. Compared to DS, energy homeostasis and amino acid metabolism were differentially regulated in MRR. Our results provide gene and metabolite expression landscapes of E. tenella following maduramycin induction. This study is the first work involving integrated transcriptomic and metabolomic analyses to identify the key pathways to understand the molecular and metabolic mechanisms underlying drug resistance to polyether ionophores in coccidia.

Proteomic Analysis of Fractionated Eimeria tenella Sporulated Oocysts Reveals Involvement in Oocyst Wall Formation.

Eimeria tenella is the most pathogenic intracellular protozoan parasite of the Eimeria species. Eimeria oocyst wall biogenesis appears to play a central role in oocyst transmission. Proteome profiling offers insights into the mechanisms governing the molecular basis of oocyst wall formation and identifies targets for blocking parasite transmission. Tandem mass tags (TMT)-labeled quantitative proteomics was used to analyze the oocyst wall and sporocysts of E. tenella. A combined total of 2865 E. tenella proteins were identified in the oocyst wall and sporocyst fractions; among these, 401 DEPs were identified, of which 211 were upregulated and 190 were downregulated. The 211 up-regulated DEPs were involved in various biological processes, including DNA replication, fatty acid metabolism and biosynthesis, glutathione metabolism, and propanoate metabolism. Among these proteins, several are of interest for their likely role in oocyst wall formation, including two tyrosine-rich gametocyte proteins (EtGAM56, EtSWP1) and two cysteine-rich proteins (EtOWP2, EtOWP6). Concurrently, 96 uncharacterized proteins may also participate in oocyst wall formation. The present study significantly expands our knowledge of the proteome of the oocyst wall of E. tenella, thereby providing a theoretical basis for further understanding of the biosynthesis and resilience of the E. tenella oocyst wall.

Molecular characterization of methionine aminopeptidase1 from Eimeria tenella.

Coccidiosis, a serious intestinal parasitic disease caused by Eimeria spp., can result in huge annual economic losses to the poultry industry worldwide. At present, coccidiosis is mainly controlled by anticoccidial drugs. However, drug resistance has developed in Eimeria because of the long-term and unreasonable use of the drugs currently available. In our previous study, RNA-seq showed that the expression of methionine aminopeptidase1 (EtMetAP1) was up-regulated in diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains compared to drug-sensitive (DS) strain of Eimeria tenella. In this study, EtMetAP1 was cloned and expressed, and the function and characteristics of the EtMetAP1 protein were analyzed. The transcription and translation levels of EtMetAP1 in DS strain of E. tenella at different developmental stages were analyzed by qPCR and western blotting. We found that the transcription and translation levels of EtMetAP1 in second-generation merozoites (SM) were higher than those of the other three stages (unsporulated oocyst, sporulated oocyst, and sporozoites). Simultaneously, qPCR was used to analyze the mRNA transcription levels of EtMetAP1 in DS, DZR, MRR, and salinomycin-resistant (SMR) strain. The results showed that compared to the sensitive strain, the transcription levels of EtMetAP1 in DZR and MRR were up-regulated. There was no significant difference in transcription level in SMR. Indirect immunofluorescence localization showed that the protein was mainly localised in the cell membrane and cytoplasm of sporozoites and SM. An invasion inhibition test showed that anti-rEtMetAP1 polyclonal antibody could effectively inhibit the sporozoite invasion of host cells. These results suggest that the protein may be involved in the growth and development of parasites in host cells, the generation of drug resistance, and host cell invasion.

Molecular characterization and analysis of drug resistance-associated protein enolase 2 of Eimeria tenella.

Eimeria tenella, an intestinal parasite, has brought huge economic losses to the poultry industry. The prevalence and severity of the development of drug resistance has increased the challenge of coccidiosis control. We previously identified the enolase 2 of E. tenella (EtENO2) was differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In this study, the expression of EtENO2 in diclazuril-resistant (DZR), maduramicin-resistant (MRR), and salinomycin-resistant (SMR) strains was analyzed by quantitative real-time PCR (qRT-PCR) and western blots. EtENO2 was highly expressed in several drug-resistant strains compared with the DS strain. The qRT-PCR showed that the transcription level of EtENO2 in the field-isolated resistant strains was upregulated compared with the DS strain. The enzyme activity results indicated that the catalytic activity of EtENO2 in the drug-resistant strains was higher than in the DS strain. In addition, qRT-PCR and western blots showed that the expression level of EtENO2 was higher in second generation merozoites (SM) and unsporulated oocysts (UO) than that in sporozoites (SZ) and sporulated oocysts (SO). Immunofluorescence localization revealed that EtENO2 was distributed throughout SZ and SM and on the surface of the parasites. After the SZ invasion DF-1 cells, it was also observed on the parasitophorous vacuole membrane. Our secretion experiments found that EtENO2 could be secreted outside the SZ. This study indicated that EtENO2 might be related to the interaction between E. tenella and host cells and be involved in the development of E. tenella resistance to some anticoccidial drugs.

Characteristics analyses of Eimeria tenella 14-3-3 protein and verification of its interaction with calcium-dependent protein kinase 4.

Avian coccidiosis is a common disease caused by Eimeria spp. In the genus Eimeria, the species Eimeria tenella is an obligate intracellular parasite that invades mostly chicken cecal epithelial cells. The 14-3-3 protein is one of the most common adaptor proteins. It is involved in regulating protein phosphorylation and is associated with phosphorylated proteins to regulate signal transduction. Previous reports have shown that 14-3-3 protein has a direct regulatory effect on calcium-dependent protein kinases (CDPKs) activity by interacting with CDPKs. In this study, the characteristics of the E. tenella 14-3-3 protein including transcription and translation analyses, localization in different developmental stages etc were analyzed. The interaction between E. tenella 14-3-3 (Et14-3-3) and E. tenella calcium-dependent protein kinase 4 (EtCDPK4) which is a critical molecule in E. tenella invasion of host cells was verified by Bimolecular Fluorescent Complimentary (BiFC), Co-Immunoprecipitation (co-IP), and Glutathione S-transferase (GST) pull-down. The transcription and translation levels were analyzed using real-time quantitative PCR and western blot. The results showed that the mRNA transcription level of Et14-3-3 was highest in the sporozoite, and the translation level was higher in the unsporulated oocyst than in the other stages. Indirect immunolocalization found that Et14-3-3 was located mainly at the anterior of sporozoites and on the surface of second-generation merozoites. As the sporozoites developed in cells, the fluorescence intensity of Et14-3-3 gradually darkened. BiFC results showed green fluorescence under microscopy in 293T cells co-transfected with pBiFC-VN155-Et14-3-3 and pBiFC-VC155-EtCDPK4. Co-IP and GST pull-down showed that Et14-3-3 interacted with EtCDPK4, which is consistent with the BiFC results. These results indicated that Et14-3-3 had significant interactions with EtCDPK4. Co-localization of Et14-3-3 with EtCDPK4 in sporozoites revealed that they were located in the same position. The secretion assay results indicated that Et14-3-3 was a secreted protein but was not secreted from micronemes. These results lay the foundation for further research on the mechanism of action of EtCDPK4 with Et14-3-3 and the functions of Et14-3-3 in the lifecycle of E. tenella.

Molecular characterization of glyceraldehyde-3-phosphate dehydrogenase from Eimeria tenella.

Chicken coccidiosis is an extremely common and lethally epidemic disease caused by Eimeria spp. The control measures of coccidiosis depend mainly on drugs. However, the ensuing drug resistance problem has brought considerable economic loss to the poultry industry. In our previous study, comparative transcriptome analyses of a drug-sensitive (DS) strain and two drug-resistant strains (diclazuril-resistant (DZR) and maduramicin-resistant (MRR) strains) of Eimeria tenella were carried out by transcriptome sequencing. The expression of glyceraldehyde-3-phosphate dehydrogenase of E. tenella (EtGAPDH) was upregulated in the two resistant strains. In this study, we cloned and characterized EtGAPDH. Indirect immunofluorescence localization was used to observe the distribution of EtGAPDH in E. tenella. The results showed that the protein was distributed mainly on the surface of sporozoites and merozoites, and in the cytoplasm of merozoites. qPCR was performed to detect the transcription level of EtGAPDH in the different developmental stages of the E. tenella DS strain. The transcription level of EtGAPDH was significantly higher in second-generation merozoites than in the other three stages. The transcription level of EtGAPDH in the different drug-resistant strains and DS strain of E. tenella was also analyzed by qPCR. The results showed that the transcription level was significantly higher in the two drug-resistant strains (MRR and DZR) than in the DS strain. As the concentration of diclazuril and maduramicin increased, the transcription levels also increased. Western blot results showed that EtGAPDH protein was upregulated in the DZR and MRR strains. Enzyme activity showed that the enzyme activity of EtGAPDH was higher in the two resistant strains than in the DS strain. These results showed that EtGAPDH possess several roles that separate and distinct from its glycolytic function and maybe involved in the development of E. tenella resistance to anticoccidial drugs.

Effects of host vimentin on Eimeria tenella sporozoite invasion.

BACKGROUND: Chicken coccidiosis is a parasitic disease caused by Eimeria of Apicomplexa, which has caused great economic loss to the poultry breeding industry. Host vimentin is a key protein in the process of infection of many pathogens. In an earlier phosphorylation proteomics study, we found that the phosphorylation level of host vimentin was significantly regulated after Eimeria tenella sporozoite infection. Therefore, we explored the role of host vimentin in the invasion of host cells by sporozoites. METHODS: Chicken vimentin protein was cloned and expressed. We used qPCR, western blotting, and indirect immunofluorescence to detect levels of mRNA transcription, translation, and phosphorylation, and changes in the distribution of vimentin after E. tenella sporozoite infection. The sporozoite invasion rate in DF-1 cells treated with vimentin polyclonal antibody or with small interfering RNA (siRNA), which downregulated vimentin expression, was assessed by an in vitro invasion test. RESULTS: The results showed that vimentin transcription and translation levels increased continually at 6-72 h after E. tenella sporozoite infection, and the total phosphorylation levels of vimentin also changed. About 24 h after sporozoite infection, vimentin accumulated around sporozoites in DF-1 cells. Treating DF-1 cells with vimentin polyclonal antibody or downregulating vimentin expression by siRNA significantly improved the invasion efficiency of sporozoites. CONCLUSION: In this study, we showed that vimentin played an inhibitory role during the invasion of sporozoites. These data provided a foundation for clarifying the relationship between Eimeria and the host.

Molecular characterization and immune protection by cystathionine ß-synthase from Eimeria tenella.

Eimeria tenella is an obligate intracellular apicomplexan parasite that causes avian coccidiosis and leads to severe economic losses in the global poultry industry. Cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CGL) act together to generate H2S in the reverse transsulfuration pathway. In this study, E. tenella Cystathionine ß-synthase (EtCBS) was cloned using rapid amplification of cDNA 5'-ends (5'RACE) and characterized, and its immunoprotective effects were evaluated. The recombinant EtCBS protein (rEtCBS) was expressed and successfully recognized by anti-sporozoites (Spz) protein rabbit serum. EtCBS mRNA levels were highest in Spz by qPCR, and the protein expression levels were higher in unsporulated oocysts (UO) than in other stages by Western blot. Indirect immunofluorescence showed that EtCBS protein was found on the surface of Spz and second-generation merozoites (Mrz). The invasion inhibition assays showed that rabbit anti-rEtCBS polyclonal antibodies effectively inhibited parasite invasion host cells. Chickens immunized with rEtCBS protein showed prominently increased weight gains and decreased oocyst output compared to nonimmunized and infected control group. The results suggest that EtCBS could be a potential vaccine candidate against E. tenella.

Molecular characterization and protective efficacy of a new conserved hypothetical protein of Eimeria tenella.

Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. This parasite encodes a genome of more than 8000 genes. However, more than 70% of the gene models for this species are currently annotated as hypothetical proteins. In this study, a conserved hypothetical protein gene of E. tenella, designated EtCHP18905, was cloned and identified, and its immune protective effects were evaluated. The open reading frame of EtCHP18905 was 1053bp and encoded a protein of 350 amino acids with a molecular weight of 38.7kDa. The recombinant EtCHP18905 protein (rEtCHP18905) was expressed in E. coli. Using western blot, the recombinant protein was successfully recognized by anti GST-Tag monoclonal antibody and anti-sporozoites protein rabbit serum. Real-time quantitative PCR analysis revealed that the EtCHP18905 mRNA levels were higher in sporozoites than in unsporulated oocysts, sporulated oocysts and second-generation merozoites. Western blot analysis showed that EtCHP18905 protein expression levels were lower in sporozoites than in other stages. Immunofluorescence analysis indicated that the EtCHP18905 protein was located on the surface of sporozoites and second-generation merozoites. Inhibition experiments showed that the ability of sporozoites to invade host cells was significantly decreased after treatment with the anti-rEtCHP18905 polyclonal antibody. Vaccination with rEtCHP18905 protein was able to significantly decrease mean lesion scores and oocyst outputs as compared to non-vaccinated controls. The results suggest that the rEtCHP18905 protein can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.

TITLE: Caractérisation moléculaire et efficacité protectrice d'une nouvelle protéine hypothétique conservée d'Eimeria tenella. ABSTRACT: Eimeria tenella est un parasite intracellulaire obligatoire qui envahit activement les cellules épithéliales du caecum des poulets. Ce parasite code un génome de plus de 8000gènes. Cependant, plus de 70% des modèles de gènes de cette espèce sont actuellement annotés en tant que protéines hypothétiques. Dans cette étude, un gène de protéine hypothétique conservé d'E. tenella, désigné par EtCHP18905, a été cloné et identifié, et ses effets immuno-protecteurs ont été évalués. Le cadre de lecture ouvert d'EtCHP18905 était de 1053 pb et codait pour une protéine de 350 acides aminés avec un poids moléculaire de 38,7kDa. La protéine recombinante EtCHP18905 (rEtCHP18905) a été exprimée dans E. coli. En utilisant le Western blot, la protéine recombinante a été reconnue avec succès par un anticorps monoclonal anti-GST-Tag et un sérum de lapin anti-protéines de sporozoïtes. Une analyse PCR quantitative en temps réel a révélé que les niveaux d'ARNm d'EtCHP18905 étaient plus élevés dans les sporozoïtes que dans les oocystes non sporulés, les oocystes sporulés et les mérozoïtes de deuxième génération. L'analyse par Western blot a montré que les niveaux d'expression de la protéine EtCHP18905 étaient plus faibles dans les sporozoïtes que dans les autres stades. L'analyse par immunofluorescence a indiqué que la protéine EtCHP18905 était localisée à la surface des sporozoïtes et des mérozoïtes de deuxième génération. Des expériences d'inhibition ont montré que la capacité des sporozoïtes à envahir les cellules hôtes était significativement diminuée après le traitement par l'anticorps polyclonal anti-rEtCHP18905. La vaccination avec la protéine rEtCHP18905 a permis de réduire significativement les scores moyens des lésions et les sorties d'oocystes par rapport aux témoins non vaccinés. Les résultats suggèrent que la protéine rEtCHP18905 peut induire une protection immunitaire partielle contre l'infection par E. tenella et pourrait être un candidat efficace pour le développement de nouveaux vaccins.

Molecular characterization and analysis of the ATPase ASNA1 homolog gene of Eimeria tenella in a drug sensitive strain and drug resistant strains.

The widespread use of drugs has exacerbated the resistance of Eimeria tenalla to anti-coccidial drugs. Using RNA-seq, we previously found the ATPase ASNA1 homolog of E. tenella (EtASNA1) was differentially expressed in resistant strains and drug sensitive (DS) strain. In our study, we used western blotting and quantitative real-time PCR (qRT-PCR) to analyze the translational and transcriptional levels of EtASNA1 in a diclazuril-resistant (DZR) strain, maduramicin-resistant (MRR) strain, salinomycin-resistant (SMR) strain, and DS strain and found EtASNA1 was highly expressed in three drug-resistant strains. The qRT-PCR and western blotting results also showed that the expression levels of EtASNA1 increased with increasing drug concentration, and the transcription levels of the DZR strains isolated from the field were higher than those of the DS strain. In addition, we used in vivo and in vitro tests to analyze the changes of EtASNA1 expression after DZR, MRR, and DS strain infections in chickens, and in vitro inoculation of DF-1 cells in the presence of drugs. The addition of drugs caused expression to be upregulated. The results of qRT-PCR and western blotting also showed that the expression levels of EtASNA1 in second-generation merozoites (SM) and unsporulated oocysts (UO) were significantly higher than those in the other two developmental stages. The immunofluorescence localization of EtASNA1 indicated that the protein was distributed throughout the sporozoites (SZ) and SM, except for the refractile bodies of SZ. In vitro inhibition experiments showed that anti-EtASNA1 antibody incubation significantly inhibited SZ invasion of DF-1 cells. The above results showed that EtASNA1 may be related to host cell invasion of E. tenella and may be involved in the development of E. tenella resistance to some drugs.

Molecular characterization and functional analysis of Eimeria tenella citrate synthase.

Chicken coccidiosis, caused by an obligate intracellular protozoan parasite of the genus Eimeria, is a major parasitic disease in the intensively reared poultry industry. Due to the widespread use of anticoccidial drugs, resistance has become an inevitable problem. In our previous study, Eimeria tenella citrate synthase (EtCS) was found to be up-expressed in two drug-resistant strains (diclazuril-resistant and maduramycin-resistant strains) compared to drug-sensitive strain by RNA sequence. In this study, we cloned and expressed EtCS and obtain its polyclonal antibodies. Quantitative real-time polymerase chain (qPCR) reactions and Western blots were used to analyze the transcription and translation levels of EtCS in sensitive and three drug-resistant strains. Compared with the sensitive strain, the transcription of EtCS was both significantly upregulated in diclazuril-resistant and maduramycin-resistant strains, but was not significantly different in salinomycin-resistant strain. No significant difference was seen in translation level in the three drug-resistant strains. Indirect immunofluorescence indicated that EtCS was mainly located in the cytoplasm of sporozoites except for posterior refractile bodies and in the cytoplasm and surface of merozoites. Anti-rEtCS antibody has inhibitory effects on E. tenella sporozoite invasion of DF-1 cells and the inhibition rate is more than 83%. Binding of the protein to chicken macrophage (HD11) cells was confirmed by immunofluorescence assays. When macrophages were treated with rEtCS, secretion of nitric oxide and cell proliferation of the macrophages were substantially reduced. These results showed that EtCS may be related to host cell invasion of E. tenella and involve in the development of E.tenella resistance to some drugs.

Identification and Characterization of a Novel Apical Membrane Antigen 3 in Eimeria tenella.

Eimeria tenella is an obligate intracellular parasite in the phylum Apicomplexa. As described for other members of Apicomplexa, apical membrane antigen 1 (AMA1) has been shown to be critical for sporozoite invasion of host cells by E. tenella. Recently, an E. tenella paralogue of AMA1 (EtAMA1), dubbed sporoAMA1 (EtAMA3), was identified in proteomic and transcriptomic analyses of E. tenella, but not further characterized. Here, we show that EtAMA3 is a type I integral membrane protein that has 24% -38% identity with other EtAMAs. EtAMA3 has the same pattern of Cys residues in domains I and II of AMA1 orthologs from apicomplexan parasites, but high variance in domain III, with all six invariant Cys residues absent. EtAMA3 expression was developmentally regulated at the mRNA and protein levels. EtAMA3 protein was detected in sporulated oocysts and sporozoites, but not in the unsporulated oocysts or second-generation merozoites. EtAMA3 is secreted by micronemes and is primarily localized to the apical end of sporozoites during host-cell invasion. Additionally, pretreatment of sporozoites with rEtAMA3-specific antibodies substantially impeded their invasion into host cells. These results suggest EtAMA3 is a sporozoite-specific protein that is involved in host-cell sporozoite invasion.

Further investigation of the characteristics and biological function of Eimeria tenella apical membrane antigen 1.

Apical membrane antigen 1 (AMA1) is a type I integral membrane protein that is highly conserved in apicomplexan parasites. Previous studies have shown that Eimeria tenella AMA1 (EtAMA1) is critical for sporozoite invasion of host cells. Here, we show that EtAMA1 is a microneme protein secreted by sporozoites, confirming previous results. Individual and combined treatment with antibodies of EtAMA1 and its interacting proteins, E. tenella rhoptry neck protein 2 (EtRON2) and Eimeria-specific protein (EtESP), elicited significant anti-invasion effects on the parasite in a concentration-dependent manner. The overexpression of EtAMA1 in DF-1 cells showed a significant increase of sporozoite invasion. Isobaric tags for relative and absolute quantitation (iTRAQ) coupled with LC-MS/MS were used to screen differentially expressed proteins (DEPs) in DF-1 cells transiently transfected with EtAMA1. In total, 3953 distinct nonredundant proteins were identified and 163 of these were found to be differentially expressed, including 91 upregulated proteins and 72 downregulated proteins. The DEPs were mainly localized within the cytoplasm and were involved in protein binding and poly(A)-RNA binding. KEEG analyses suggested that the key pathways that the DEPs belonged to included melanogenesis, spliceosomes, tight junctions, and the FoxO and MAPK signaling pathways. The data in this study not only provide a comprehensive dataset for the overall protein changes caused by EtAMA1 expression, but also shed light on EtAMA1's potential molecular mechanisms during Eimeria infections.

TITLE: Étude approfondie des caractéristiques et de la fonction biologique de l'antigène 1 de la membrane apicale d'Eimeria tenella. ABSTRACT: L'antigène 1 de la membrane apicale (AMA1) est une protéine membranaire intégrale de type I hautement conservée chez les parasites Apicomplexa. Des études antérieures ont montré que l'AMA1 d'Eimeria tenella (EtAMA1) était importante pour l'invasion des cellules hôtes par les sporozoïtes. Nous montrons ici qu'EtAMA1 est une protéine des micronèmes sécrétée par les sporozoïtes, confirmant les résultats précédents. Un traitement individuel et combiné avec des anticorps d'EtAMA1 et de ses protéines en interaction, la protéine 2 du cou des rhoptries d'E. tenella (EtRON2) et la protéine spécifique d'Eimeria (EtESP), a provoqué des effets anti-invasion significatifs et dépendants de la concentration sur le parasite. La surexpression d'EtAMA1 dans les cellules DF-1 a montré une augmentation significative de l'invasion par les sporozoïtes. Des marqueurs isobares pour la quantification relative et absolue (iTRAQ) couplés à LC-MS/MS ont été utilisés pour cribler des protéines exprimées différentiellement (PED) dans des cellules DF-1 transfectées de manière transitoire avec EtAMA1. Au total, 3953 protéines non redondantes distinctes ont été identifiées et 163 d'entre elles se sont révélées exprimées de manière différentielle, dont 91 régulées à la hausse et 72 régulées à la baisse. Les PED étaient principalement localisées dans le cytoplasme et étaient impliquées dans la liaison aux protéines et la liaison au poly (A)-ARN. Les analyses de KEEG ont suggéré que les voies clés auxquelles appartenaient les PED comprenaient la mélanogenèse, les épissosomes, les jonctions étroites et les voies de signalisation FoxO et MAPK. Les données de cette étude fournissent non seulement un ensemble de données complet pour les modifications globales des protéines causées par l'expression d'EtAMA1, mais mettent également en lumière les mécanismes moléculaires potentiels d'EtAMA1 pendant les infections par Eimeria.

Molecular characterization of 60S ribosomal protein L12 of E. tenella.

This study analyzed the large-subunit (60S) ribosomal protein L12 of Eimeria tenella (Et60s-RPL12). A full-length cDNA was cloned, and the recombinant protein was expressed in E. coli BL21 and inoculated in rabbits to produce the polyclonal antibody. Quantitative real-time polymerase chain reaction and western blotting were used to analyze the transcription levels of Et60s-RPL12 and translation levels in different developmental stages of E. tenella. The results showed that the mRNA transcription level of Et60s-RPL12 was highest in second-generation merozoites, whereas the translation level was highest in unsporulated oocysts. Indirect immunofluorescence showed that Et60s-RPL12 was localized to the anterior region and surface of sporozoites, except for the two refractile bodies. As the invasion of DF-1 cells progressed, fluorescence intensity was increased, and Et60s-RPL12 was localized to the parasitophorous vacuole membrane (PVM). The secretion assay results using staurosporine indicated that this protein was secreted, but not from micronemes. The role of Et60s-RPL12 in invasion was evaluated in vitro. The results of the invasion assay showed that polyclonal antibody inhibited host cell invasion by the parasite, which reached about 12%. However, the rate of invasion was not correlated with the concentration of IgG.

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

BACKGROUND: Avian coccidiosis is a widespread, economically significant disease of poultry, caused by several Eimeria species. These parasites have complex and diverse life-cycles that require 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 EtAMA1-interacting proteins of E. tenella were screened. In this study, we characterized one putative EtAMA1-interacting protein, E. tenella Eimeria -specific protein (EtEsp). METHODS: Bimolecular fluorescence complementation (BiFC) and glutathione S-transferase (GST) fusion protein pull-down (GST pull-down) were used to confirm the interaction between EtAMA1 and EtEsp in vivo and in vitro. The expression of EtEsp was analyzed in different developmental stages of E. tenella with quantitative PCR and western blotting. The secretion of EtEsp protein was tested with staurosporine when sporozoites were incubated in complete medium at 41 °C. The localization of EtEsp was analyzed with an immunofluorescence assay (IFA). An in vitro invasion inhibition assay was conducted to assess the ability of antibodies against EtEsp to inhibit cell invasion by E. tenella sporozoites. RESULTS: The interaction between EtAMA1 and EtEsp was confirmed with BiFC and by GST pull-down. Our results show that EtEsp is differentially expressed during distinct phases of the parasite life-cycle. IFA showed that the EtEsp 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 EtEsp is a secreted protein, but not from micronemes. In inhibition tests, a polyclonal anti-rEtEsp antibody attenuated the capacity of E. tenella to invade host cells. CONCLUSION: In this study, we show that EtEsp interacts with EtAMA1 and that the protein is secreted protein, but not from micronemes. The protein participates in sporozoite invasion of host cells and is maybe involved in the growth of the parasite. These data have implications for the use of EtAMA1 or EtAMA1-interacting proteins as targets in intervention strategies against avian coccidiosis.

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella.

Avian coccidiosis is a widespread and economically significant poultry disease caused by several Eimeria species, including Eimeria tenella. Previously, E. tenella serine/threonine protein phosphatase (EtSTP) was found to be differentially expressed in drug-sensitive (DS) and drug-resistant strains using RNA-seq. In the present study, we found that transcription and translation levels of EtSTP were higher in diclazuril-resistant (DZR) strains and maduramicin-resistant (MRR) strains than in DS strains using quantitative real-time PCR (qPCR) and Western blotting. Enzyme activity results indicated that the catalytic activity of EtSTP was higher in the two drug-resistant strains than in DS strains. Western blot and qPCR analysis also showed that expression levels of EtSTP were higher in unsporulated oocysts (UO) and second-generation merozoites (SM). Indirect immunofluorescence localization showed that EtSTP was located in most areas of the parasite with the exception of refractile bodies, and fluorescence intensity was enhanced during development. In vitro inhibition experiments showed that the ability of sporozoites (SZ) to invade cells was significantly decreased after treatment with anti-rEtSTP antibody. These results indicated that EtSTP acted mainly during the developmental and reproductive stages of the parasite and may be related to the resistance of coccidia to external drug pressure.

Comparative Transcriptome Analyses of Drug-sensitive and Drug-resistant Strains of Eimeria tenella by RNA-sequencing.

Avian coccidiosis is a widespread and economically significant disease in poultry. At present, treatment of coccidiosis mainly relies on drugs. Anticoccidial drugs can be divided into two categories: ionophorous compounds and synthetic drugs. However, the emergence of drug-resistant strains has become a challenge for coccidiosis control with anticoccidial drugs. To gain insights into the molecular mechanism governing the drug resistance of Eimeria tenella, two drug-resistant strains of E. tenella, one maduramicin-resistant (MRR) strain and one diclazuril-resistant (DZR) strain, were generated. We carried out comparative transcriptome analyses of a drug-sensitive strain (DS) and two drug-resistant MRR and DZR strains of E. tenella using RNA-sequencing. A total of 1,070 differentially expressed genes (DEGs), 672 upregulated and 398 downregulated, were identified in MRR vs. DS, and 379 DEGs, 330 upregulated and 49 downregulated, were detected in DZR vs. DS. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed to better understand the functions of these DEGs. In the comparison of DZR vs. DS, some DEGs were involved in peroxisome, biosynthesis of unsaturated fatty acids, and fatty acid metabolism. In the comparison of MRR vs. DS, some DEGs were involved in glycolysis/gluconeogenesis, regulation of actin cytoskeleton, and DNA replication. In addition, some DEGs coded for surface antigens that were downregulated in two drug-resistant strains involved invasion, pathogenesis, and host-parasite interactions. These results provided suggestions for further research toward unraveling the molecular mechanisms of drug resistance in Eimeria species and contribute to developing rapid molecular methods to detect resistance to these drugs in Eimeria species in poultry.

Eimeria tenella Translation Initiation Factor eIF-5A That Interacts With Calcium-Dependent Protein Kinase 4 Is Involved in Host Cell Invasion.

Eimeria tenella is an apicomplexan, parasitic protozoan known to infect poultry worldwide. An important calcium-dependent protein kinase (CDPK) has been identified in plants, green algae, ciliates and apicomplexan, such as E. tenella. CDPKs are effector molecules involved in calcium signaling pathways, which control important physiological processes such as gliding motility, reproduction, and host cell invasion. Given that CDPKs are not found in the host, studying the functions of CDPKs in E. tenella may serve as a basis for developing new therapeutic drugs and vaccines. To assess the function of CDPK4 in E. tenella (EtCDPK4), a putative interactor, translation initiation factor eIF-5A (EteIF-5A), was screened by both co-immunoprecipitation (co-IP) and His pull-down assays followed by mass spectrometry. The interaction between EteIF-5A and EtCDPK4 was determined by bimolecular fluorescence complementation (BiFC), GST pull-down, and co-IP. The molecular characteristics of EteIF-5A were then analyzed. Quantitative real-time polymerase chain reaction and western blotting were used to determine the transcription and protein levels of EteIF-5A in the different developmental stages of E. tenella. The results showed that the transcription level of EteIF-5A mRNA was highest in second-generation merozoites, and the protein expression level was highest in unsporulated oocysts. Indirect immunofluorescence showed that the EteIF-5A protein was found throughout the cytoplasm of sporozoites, but not in the refractile body. As the invasion of DF-1 cells progressed, EteIF-5A fluorescence intensity increased in trophozoites, decreased in immature schizonts, and increased in mature schizonts. The secretion assay results, analyzed by western blotting, indicated that EteIF-5A was a secreted protein but not from micronemes. The results of invasion inhibition assays showed that rabbit anti-rEteIF-5A polyclonal antibodies effectively inhibited cell invasion by sporozoites, with an inhibition rate of 48%.

Molecular characterization and immune protection of an AN1-like zinc finger protein of Eimeria tenella.

Coccidiosis is caused by multiple species of the apicomplexan protozoa Eimeria. Among them, Eimeria tenella is frequently considered to be the most pathogenic. Zinc finger proteins (ZnFPs) are a type of protein containing zinc finger domains. In the present study, a putative Eimeria tenella AN1-like ZnFP (E. tenella AN1-like zinc finger domain-containing protein, putative partial mRNA, EtAN1-ZnFP) was cloned and characterized, and its immune protective effects were evaluated. The 798-bp ORF sequence of EtAN1-ZnFP that encoded a protein of approximately 27.0 kDa was obtained. The recombinant EtAN1-ZnFP protein (rEtAN1-ZnFP) was expressed in Escherichia coli. Western blot analysis showed that the recombinant protein was recognized by the anti-GST monoclonal antibody and anti-sporozoite protein rabbit serum. qPCR analysis revealed that EtAN1-ZnFP was highly expressed in unsporulated oocysts and sporozoites. Immunostaining with an anti-rEtAN1-ZnFP antibody indicated that EtAN1-ZnFP was uniformly distributed in the cytoplasm of sporozoites, except for the refractive body; furthermore, this protein was evenly distributed in the cytoplasm of immature schizonts but seldom distributed in mature schizonts. The results of the in vitro invasion inhibition assay indicated that the antibodies against rEtAN1-ZnFP efficiently reduced the ability of E. tenella sporozoites to invade host cells. Animal challenge experiments demonstrated that the chickens immunized with rEtAN1-ZnFP protein significantly decreased mean lesion scores and fecal oocyst output compared with challenged control group. The results suggest that EtAN1-ZnFP can induce partial immune protection against infection with E. tenella and could be an effective candidate for the development of new vaccines.