Host protein EPCAM interacting with EtMIC8-EGF is essential for attachment and invasion of Eimeria tenella in chickens.

The five epidermal growth factor-like domains (EGF) of Eimeria tenella microneme protein 8 (EtMIC8) (EtMIC8-EGF) plays a vital role in host cell attachment and invasion. These processes require interactions between parasite proteins and receptors on the surface of host cells. In this study, five chicken membrane proteins potentially interacting with EtMIC8-EGF were identified using the GST pull-down assay and mass spectrometry analysis, and only chicken (Gallus gallus) epithelial cell adhesion molecule (EPCAM) could bind to EtMIC8-EGF. EPCAM-specific antibody and recombinant EPCAM protein (rEPCAM) inhibited the EtMIC8-EGF binding to host cells in a concentration-dependent manner. Furthermore, the rEPCAM protein showed a binding activity to sporozoites in vitro, and a significant reduction of E. tenella invasion in DF-1 cells was further observed after pre-incubation of sporozoites with rEPCAM. The specific anti-EPCAM antibody further significantly decreased weight loss, lesion score and oocyst output during E. tenella infection, displaying partial inhibition of E. tenella infection. These results indicate that chicken EPCAM is an important EtMIC8-interacting host protein involved in E. tenella-host cell adhesion and invasion. The findings will contribute to a better understanding of the role of adhesion-associated microneme proteins in E. tenella.

A candidate virulence factor of Eimeria tenella (EtROP30) predicted by virulence enhancement of transgenic Toxoplasma gondii.

Difficulties of in vitro culture and genetic manipulation of Eimeria tenella have hindered the screening of virulence factors in this parasite. In this study, the E. tenella rhoptry protein 30 (EtROP30) was expressed in Toxoplasma gondii (RH∆Ku80-EtROP30), and its effect on the proliferation and virulence of parasites was investigated. The results revealed that the expression of EtROP30 had no impact on the invasion and egress processes. However, the RH∆Ku80-EtROP30 strain formed larger plaques compared to the RH∆Ku80, indicating that the EtROP30 expression promotes T. gondii proliferation. Furthermore, the RH∆Ku80-EtROP30 strain exhibited greater pathogenicity, resulting in earlier mortality and shorter overall survival time compared to RH∆Ku80. These results imply that EtROP30 expression facilitates parasite intracellular proliferation and virulence in mice, suggesting that EtROP30 might be a candidate virulence factor of E. tenella.

Label-free quantitative detection and comparative analysis of lysine acetylation during the different life stages of Eimeria tenella.

Proteome-wide lysine acetylation has been documented in apicomplexan parasite Toxoplasma gondii and Plasmodium falciparum. Here, we conducted the first lysine acetylome in unsporulated oocysts (USO), sporulated 7 h oocysts (SO 7h), sporulated oocysts (SO), sporozoites (S), and the second generation merozoites (SMG) of Eimeria tenella through a 4D label-free quantitative technique. Altogether, 8532 lysine acetylation sites on 2325 proteins were identified in E. tenella, among which 5445 sites on 1493 proteins were quantified. In addition, 557, 339, 478, 248, 241, and 424 differentially expressed proteins were identified in the comparisons SO7h vs USO, SO vs SO7h, SO vs USO, S vs SO, SMG vs S, and USO vs SMG, respectively. The bioinformatics analysis of the acetylome showed that the lysine acetylation is widespread on proteins of diverse functions. Moreover, the dynamic changes of lysine acetylome among E. tenella different life stages revealed significant regulation during the whole process of E. tenella growth and stage conversion. This study provides a beginning for the investigation of the regulate role of lysine acetylation in E. tenella and may provide new strategies for anticoccidiosis drug and vaccine development. Raw data are publicly available at iProX with the data set identifier PXD040368.

Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony.

BACKGROUND: The apicomplexan parasites Eimeria spp. are the causative agents of coccidiosis, a disease with a significant global impact on the poultry industry. The complex life cycle of Eimeria spp. involves exogenous (sporogony) and endogenous (schizogony and gametogony) stages. Unfortunately, the genetic regulation of these highly dynamic processes, particularly for genes involved in specific developmental phases, is not well understood. METHODS: In this study, we used RNA sequencing (RNA-Seq) analysis to identify expressed genes and differentially expressed genes (DEGs) at seven time points representing different developmental stages of Eimeria tenella. We then performed K-means clustering along with co-expression analysis to identify functionally enriched gene clusters. Additionally, we predicted apicomplexan AP2 transcription factors in E. tenella using bioinformatics methods. Finally, we generated overexpression and knockout strains of ETH2_0411800 to observe its impact on E. tenella development. RESULTS: In total, we identified 7329 genes that are expressed during various developmental stages, with 3342 genes exhibiting differential expression during development. Using K-means clustering along with co-expression analysis, we identified clusters functionally enriched for oocyte meiosis, cell cycle, and signaling pathway. Among the 53 predicted ApiAP2 transcription factors, ETH2_0411800 was found to be exclusively expressed during sporogony. The ETH2_0411800 overexpression and knockout strains did not exhibit significant differences in oocyst size or output compared to the parental strain, while the resulting ETH2_0411800 knockout parasite showed a relatively small oocyst output. CONCLUSIONS: The findings of our research suggest that ETH2_0411800 is not essential for the growth and development of E. tenella. Our study provides insights into the gene expression dynamics and is a valuable resource for exploring the roles of transcription factor genes in regulating the development of Eimeria parasites.

Refractile bodies of Eimeria tenella are proteinaceous membrane-less organelles that undergo dynamic changes during infection.

Introduction: Refractile bodies (RB) are large membrane-less organelles (MLO) of unknown function found as a prominent mismatched pair within the sporozoite stages of all species of Eimeria, parasitic coccidian protozoa. Methods: High resolution imaging methods including time-lapse live confocal microscopy and serial block face-scanning electron microscopy (SBF-SEM) were used to investigate the morphology of RB and other intracellular organelles before and after sporozoite invasion of host cells. Results: Live cell imaging of MDBK cells infected with E. tenella sporozoites confirmed previous reports that RB reduce from two to one post-infection and showed that reduction in RB number occurs via merger of the anterior RB with the posterior RB, a process that lasts 20-40 seconds and takes place between 2- and 5-hours post-infection. Ultrastructural studies using SBF-SEM on whole individual sporozoites, both pre- and post-host cell invasion, confirmed the live cell imaging observations and showed also that changes to the overall sporozoite cell shape accompanied RB merger. Furthermore, the single RB post-merger was found to be larger in volume than the two RB pre-merger. Actin inhibitors were used to investigate a potential role for actin in RB merger, Cytochalasin D significantly inhibited both RB merger and the accompanying changes in sporozoite cell shape. Discussion: MLOs in eukaryotic organisms are characterised by their lack of a membrane and ability to undergo liquid-liquid phase separation (LLPS) and fusion, usually in an actin-mediated fashion. Based on the changes in sporozoite cell shape observed at the time of RB merger together with a potential role for actin in this process, we propose that RB are classed as an MLO and recognised as one of the largest MLOs so far characterised.

Combined oral immunization with probiotics Entercoccus faecalis delivering surface-anchored Eimeria tenella proteins provide protective efficacies against homologous infection in chickens.

Background and Objectives: Avian coccidiosis is an intestinal parasitic disease exerting a highly negative impact on the global poultry industry. The aim of the present study is to evaluate the immune protective efficacies against Eimeria tenella infection in chickens orally immunized with combined recombinant probiotics Entercoccus faecalis (E. faecalis) delivering surface-anchored E. tenella proteins. Methods: Four kinds of novel probiotics vaccines that surface-expressing four Eimeria tenella (E. tenella) proteins EtAMA1, EtIMP1, EtMIC2 and Et3-1E were produced, respectively. The expression of four target proteins on the surface of recombinant bacteria was detected by Western blot and indirect immunofluorescence assay (IFA). Then the four kinds of recombinant E. faecalis were combined to immunize chickens via oral route in different combinations. The immunizations were performed three times at two-week intervals, and each for three consecutive days. After immunizations, chickens in each immunized group were orally challenged with E. tenella sporulated oocysts. The immune responses and protective efficacies against homologous infection were evaluated. Results: The results showed that three or four live recombinant E. faecalis induced effective antigen-specific humoral, intestinal mucosal immune responses, stimulated peripheral T lymphocytes proliferation, and displayed partial protections against homologous challenge as measured by cecal lesions, oocyst shedding, and body weight gain (BWG). Notably, higher levels of protective efficacies were observed when the four recombinant E. faecalis delivering target proteins were combined. Conclusion: Chickens orally administrated with three or four, especially the four combined recombinant E. faecalis stimulated specific immune responses, which provided anti-coccidial effects. This study offers an idea for future development of novel vaccines based on multi-antigens delivered by probiotic bacteria.

Long noncoding RNA profiling reveals that LncRNA BTN3A2 inhibits the host inflammatory response to Eimeria tenella infection in chickens.

Coccidiosis is a widespread parasitic disease that causes serious economic losses to the poultry industry every year. Long noncoding RNAs (lncRNAs) play important roles in transcriptional regulation and are involved in a variety of diseases and immune responses. However, the lncRNAs associated with Eimeria tenella (E. tenella) resistance have not been identified in chickens. In addition, the expression profiles and functions of lncRNAs during E. tenella infection remain unclear. In the present study, high-throughput sequencing was applied to identify lncRNAs in chicken cecal tissues from control (JC), resistant (JR), and susceptible (JS) groups on day 4.5 post-infection (pi), and functional tests were performed. A total of 564 lncRNAs were differentially expressed, including 263 lncRNAs between the JS and JC groups, 192 between the JR and JS groups, and 109 between the JR and JC groups. Functional analyses indicated that these differentially expressed lncRNAs were involved in pathways related to E. tenella infection, including the NF-kappa B signaling, B cell receptor signaling and natural killer cell-mediated cytotoxicity pathways. Moreover, through cis regulation network analysis of the differentially expressed lncRNAs, we found that a novel lncRNA termed lncRNA BTN3A2 was significantly increased in both cecum tissue and DF-1 cells after coccidia infection or sporozoite stimulation. Functional test data showed that the overexpression of lncRNA BTN3A2 reduced the production of inflammatory cytokines, including IL-6, IL-1ß, TNF-α and IL-8, while lncRNA BTN3A2 knockdown promoted the production of these inflammatory cytokines. Taken together, this study identify the differentially expressed lncRNAs during E. tenella infection in chickens for the first time and provide the direct evidence that lncRNA BTN3A2 regulates the host immune response to coccidia infection.

Cellular electron tomography of the apical complex in the apicomplexan parasite Eimeria tenella shows a highly organised gateway for regulated secretion.

The apical complex of apicomplexan parasites is essential for host cell invasion and intracellular survival and as the site of regulated exocytosis from specialised secretory organelles called rhoptries and micronemes. Despite its importance, there are few data on the three-dimensional organisation and quantification of these organelles within the apical complex or how they are trafficked to this specialised region of plasma membrane for exocytosis. In coccidian apicomplexans there is an additional tubulin-containing hollow barrel structure, the conoid, which provides a structural gateway for this specialised apical secretion. Using a combination of cellular electron tomography and serial block face-scanning electron microscopy (SBF-SEM) we have reconstructed the entire apical end of Eimeria tenella sporozoites; we report a detailed dissection of the three- dimensional organisation of the conoid and show there is high curvature of the tubulin-containing fibres that might be linked to the unusual comma-shaped arrangement of protofilaments. We quantified the number and location of rhoptries and micronemes within cells and show a highly organised gateway for trafficking and docking of rhoptries, micronemes and microtubule-associated vesicles within the conoid around a set of intra-conoidal microtubules. Finally, we provide ultrastructural evidence for fusion of rhoptries directly through the parasite plasma membrane early in infection and the presence of a pore in the parasitophorous vacuole membrane, providing a structural explanation for how rhoptry proteins may be trafficked between the parasite and the host cytoplasm.

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.

The interaction between free Ca2+ in host cells and invasion of E. tenella.

Eimeria tenella is the most pathogenic and common coccidia that causes chicken coccidiosis. The intracellular free Ca2+ of the host cell is closely related to the invasion, development, and proliferation of intracellular parasites. To determine the dynamic changes of intracellular free Ca2+ and its function in the process of E. tenella invading host cells, we established a chick embryo cecal epithelial cells model of E. tenella infection. Chick embryo cecal epithelial cells were treated with different Ca2+ signal inhibitor, respectively, and then infected with E. tenella. The results showed that extracellular Ca2+, Ca2+ channels on the cell membrane, IP3R ion channels on the endoplasmic reticulum membrane, and RyR ion channels regulated the free Ca2+ in cecal epithelial cells. Through fluorescence labeling and invasion rate detection, we found that the intracellular Ca2+ did not change significantly during the invasion of E. tenella, but its stability was critical to the invasion of parasites.

Eimeria proteins: order amidst disorder.

Apicomplexans are important pathogens that cause severe infections in humans and animals. The biology and pathogeneses of these parasites have shown that proteins are intrinsically modulated during developmental transitions, physiological processes and disease progression. Also, proteins are integral components of parasite structural elements and organelles. Among apicomplexan parasites, Eimeria species are an important disease aetiology for economically important animals wherein identification and characterisation of proteins have been long-winded. Nonetheless, this review seeks to give a comprehensive overview of constitutively expressed Eimeria proteins. These molecules are discussed across developmental stages, organelles and sub-cellular components vis-à-vis their biological functions. In addition, hindsight and suggestions are offered with intention to summarise the existing trend of eimerian protein characterisation and to provide a baseline for future studies.

The role of Eimeria tenella EtCab protein in the attachment and invasion of host cells.

Calcium-binding proteins (CaBPs) containing the specific calcium-binding motif (EF-hand) play a crucial role in important physiological events such as secretion, storage and signal transduction of cells. Recently, CaBPs have been found to be associated with host cell invasions in some parasites. In this study, an Eimeria tenella membrane-associated calcium-binding protein (EtCab) was cloned and its expression at different developmental stages, adhesive functions and host cell invasion in vitro were investigated. The results of the sequence analysis showed that EtCab contains six EF-hand motifs and the HDEL ER-retention signal belonging to the CREC (45 kDa calcium-binding protein, reticulocalbin, ER calcium-binding protein of 55 kDa, and calumenin) family. An indirect immunofluorescence assay (IFA) using specific polyclonal antibodies under permeabilized and nonpermeabilized conditions labeled EtCab on the surface of sporozoites. Quantitative real-time PCR and western blotting indicated that EtCab was highly transcribed and expressed in sporozoites. The attachment assay using a yeast surface display model showed that the adherence rates of EtCab expressed on the surfaces of yeasts to host cells were 2.5-fold greater than the control. Invasion inhibition assays revealed that specific polyclonal antibodies against EtCab significantly reduced the invasion rate of sporozoites on host cells compared to the control group (P < 0.01). These results suggest that EtCab plays an important role in the attachment and invasion of E. tenella to host cells.

The structure of a major surface antigen SAG19 from Eimeria tenella unifies the Eimeria SAG family.

In infections by apicomplexan parasites including Plasmodium, Toxoplasma gondii, and Eimeria, host interactions are mediated by proteins including families of membrane-anchored cysteine-rich surface antigens (SAGs) and SAG-related sequences (SRS). Eimeria tenella causes caecal coccidiosis in chickens and has a SAG family with over 80 members making up 1% of the proteome. We have solved the structure of a representative E. tenella SAG, EtSAG19, revealing that, despite a low level of sequence similarity, the entire Eimeria SAG family is unified by its three-layer αßα fold which is related to that of the CAP superfamily. Furthermore, sequence comparisons show that the Eimeria SAG fold is conserved in surface antigens of the human coccidial parasite Cyclospora cayetanensis but this fold is unrelated to that of the SAGs/SRS proteins expressed in other apicomplexans including Plasmodium species and the cyst-forming coccidia Toxoplasma gondii, Neospora caninum and Besnoitia besnoiti. However, despite having very different structures, Consurf analysis showed that Eimeria SAG and Toxoplasma SRS families each exhibit marked hotspots of sequence hypervariability that map to their surfaces distal to the membrane anchor. This suggests that the primary and convergent purpose of the different structures is to provide a platform onto which sequence variability can be imposed.

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.

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.

Proteomic analysis of the second-generation merozoites of Eimeria tenella under nitromezuril and ethanamizuril stress.

BACKGROUND: Eimeria tenella is a highly pathogenic coccidian that causes avian coccidiosis. Both nitromezuril (NZL) and ethanamizuril (EZL) are novel triazine compounds with high anticoccidial activity, but the mechanisms of their action are still unclear. This study explored the response of E. tenella to NZL and EZL by the study of changes in protein composition of the second-generation merozoites. METHODS: Label-free quantification (LFQ) proteomics of the second-generation merozoites of E. tenella following NZL and EZL treatment were studied by LC-MS/MS to explore the mechanisms of action. The identified proteins were annotated and analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction (PPI) networks analysis. RESULTS: A total of 1430 proteins were identified by LC-MS/MS, of which 375 were considered as differential proteins in response to drug treatment (DPs). There were 26 only found in the NZL treatment group (N-group), 63 exclusive to the EZL treatment group (E-group), and 80 proteins were present in both drug groups. In addition, among the DPs, the abundant proteins with significantly altered expression in response to drug treatment (SDPs) were found compared with the C-group, of which 49 were upregulated and 51 were downregulated in the N-group, and 66 upregulated and 79 downregulated in the E-group. Many upregulated proteins after drug treatment were involved in transcription and protein metabolism, and surface antigen proteins (SAGs) were among the largest proportion of the downregulated SDPs. Results showed the top two enriched GO terms and the top one enriched pathway treated with EZL and NZL were related, which indicated that these two compounds had similar modes of action. CONCLUSIONS: LFQ proteomic analysis is a feasible method for screening drug-related proteins. Drug treatment affected transcription and protein metabolism, and SAGs were also affected significantly. This study provided new insights into the effects of triazine anticoccidials against E. tenella.

Identification and Application of Epitopes in EtMIC1 of Eimeria tenella Recognized by the Monoclonal Antibodies 1-A1 and 1-H2.

Eimeria tenella microneme-1 protein (EtMIC1) has been proposed to be a transmembrane protein, but this characteristic has not yet been confirmed experimentally. Furthermore, despite EtMIC1 being an important candidate antigen, its key epitope has not been reported. Here, two linear B-cell epitopes of EtMIC1, 91LITFATRSK99 and 698ESLISAGE705, were identified by Western blotting using specific monoclonal antibodies (MAbs) and were named epitope I (located in the I-domain) and epitope CTR (located in the CTR domain), respectively. Sequence comparative analyses of these epitopes among Eimeria species that infect chickens showed that epitope I differs greatly across species, whereas epitope CTR is relatively conserved. Point mutation assay results indicate that all the amino acid residues of the epitopes recognized by MAb 1-A1 or 1-H2 are key amino acids involved in recognition. Comparative analyses of indirect immunofluorescence assay (IFA) results for MAbs 1-A1 and 1-H2 under both nonpermeabilization and permeabilization conditions indicate that epitope I is located on the outer side of the sporozoite surface membrane whereas epitope CTR is located on the inner side, together providing experimental evidence that EtMIC1 is a transmembrane protein. IFA also labeled the EtMIC1 protein on the parasitophorous vacuole membrane and on the surface of schizonts, which suggests that the EtMIC1 protein may play an important role in parasitophorous vacuole formation and E. tenella development. Immunoprotective efficacy experiments revealed that epitope I has good immunogenicity, as evidenced by its induction of high serum antibody levels, blood lymphocyte proliferation, and CD4+ blood lymphocyte percentage.

Effects of host fatty acid-binding protein 4 on Eimeria tenella sporozoites invasion of cells.

In our previous study, proteomics analyses of host cells infected with Eimeria tenella sporozoites coupled with isobaric tags for relative and absolute quantitation, identified several host proteins related to Eimeria invasion. In this study, A 458-bp Gallus gallus fatty acid-binding protein 4 (FABP4) gene was cloned and subcloned to pET-28c(+) vector to construct the prokaryotic recombinant expression plasmid pET-28c(+)-FABP4. The 18.5 kDa recombinant FABP4 protein (rFABP4) was expressed and identified by western blotting. Expression of FABP4 in E. tenella sporozoite-infected DF-1 cells was downregulated significantly than in non-infected cells detected by western blotting and immunohistochemistry. The antibody inhibition assay showed that antibodies against FABP4 at 50, 100, 200, 300, and 400 µg/mL had no significant effect on sporozoite invasion. BMS-309403 and transforming growth factor-ß3 (TGF-ß3) was used to inhibit and improve the expression of FABP4 in DF-1 cells, respectively, and their effect on the sporozoite invasion of cells was detected by flow cytometry. Sporozoite invasion rate in the BMS-309403-treated group was not significantly affected; however, the invasion rate in the TGF-ß3-treated group declined significantly. These results show that host FABP4 plays a negative role in Eimeria invasion. However, further studies are needed to elucidate the exact mechanism of how FABP4 negatively regulates Eimeria invasion.

The effect of autophagy on the survival and invasive activity of Eimeria tenella sporozoites.

Autophagy is a cellular process that is vital for the maintenance of homeostasis in eukaryotic cells. Currently, autophagy-related genes (atgs) in the Eimeria tenella genome database have been reported, but very little is known about the effects of autophagy on the survival and invasive activity of this protozoan. In this study, we investigated the autophagy in E. tenella sporozoites under starvation and autophagy-modulators treatments and evaluated the autophagy influence on cellular adenosine triphosphate (ATP) levels, the survival rate and the invasive activity of the sporozoites. The results showed that the autophagy could be induced in the sporozoites by starvation or inducer rapamycin (RP), but it could be inhibited by 3-methyladenine (3-MA) treatment. The sporozoites after starvation and RP-treatment displayed punctate signals of EtATG8 and formed autophagosomes. The survival rate of the sporozoites under starvation was significantly lower than that in the control group, whereas the ATP levels in sporozoite were far greater than those in the control. The quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) showed that the invasive activity of the sporozoites was up- and down-regulated by RP and 3-MA induction, respectively. Our results indicate that autophagy has effects on the survival and invasive activity of E. tenella sporozoites, which may provide new insights into anti-coccidial drugs.

Recombinant lactococcus lactis expressing Eimeria tenella AMA1 protein and its immunological effects against homologous challenge.

Avian coccidiosis leads to severe economic losses for the global poultry industry. Apical membrane antigen 1 (AMA1) of E. tenella (EtAMA1) plays a vital role during invasion of parasites into host cells. In the present study, recombinant live Lactococcus lactis expressing cytoplasmic, secreted and cell wall-anchored EtAMA1 protein were respectively constructed. The three live bacteria were respectively administered orally to SPF chickens (100 µl bacteria containing 5 × 109 CFU per chicken) for three times at 10-day intervals. After immunization, the lymphocyte proliferative function, the percentage of CD4+ and CD8α+ T cells in peripheral blood, and the IgG titers in serum of chickens in each group were respectively measured. The protective effects of live bacteria expressing EtAMA1 protein against E. tenella challenge were evaluated based on body weight gain (BWG), lesion score in cecum, oocyst descrease ratio. The results showed that chickens immunized with three live bacteria, especially the bacteria expressing cell wall-anchored EtAMA1 protein, displayed higher IgG titers and CD4+ T cells proportions, thus provided more immune protective effects against homologous challenge compared with the PBS control group and vector control group (lactococci harboring pTX8048). The oocyst decrease ratio of 33.33% from chickens immunized with lactococci expressing cell wall-anchored EctoAMA1 was observed, which was higher than that of 27.67% and 25.37% from the other two bacteria-immunized groups, respectively. The above results suggested that cell wall-anchored EtAMA1 protein delivered by Lactococcus lactis could stimulate an effective immune responses against Eimeria infection.