Molecular characterization and protective efficacy of a new conserved hypothetical protein of Eimeria tenella.
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ABSTRACT:
SUBMITTER: Zhao H
PROVIDER: S-EPMC8095096 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The genome sequences of Eimeria tenella have been sequenced, but >70% of these genes are currently categorized as having an unknown function or annotated as conserved hypothetical proteins, and few of them have been studied. In the present study, a conserved hypothetical protein gene of E. tenella, designated EtCHP559, was cloned using rapid amplification of cDNA 5'-ends (5'RACE) based on the expressed sequence tag (EST). The 1746-bp full-length cDNA of EtCHP559 contained a 1224-bp open reading frame (ORF) that encoded a 407-amino acid polypeptide with the predicted molecular weight of 46.04 kDa. Real-time quantitative PCR analysis revealed that EtCHP559 was expressed at higher levels in sporozoites than in the other developmental stages (unsporulated oocysts, sporulated oocysts and second generation merozoites). The ORF was inserted into pCold-TF to produce recombinant EtCHP559. Using western blotting, the recombinant protein was successfully recognized by rabbit serum against E. tenella sporozoites. Immunolocalization by using EtCHP559 antibody showed that EtCHP559 was mainly distributed on the parasite surface in free sporozoites and became concentrated in the anterior region after sporozoites were incubated in complete medium. The EtCHP559 became uniformly dispersed in immature and mature schizonts. Inhibition of EtCHP559 function using anti-rEtCHP559 polyclonal antibody reduced the ability of E. tenella sporozoites to invade host cells by >70%. Animal challenge experiments demonstrated that the recombinant EtCHP559 significantly increased the average body weight gain, reduced the oocyst outputs, alleviated cecal lesions of the infected chickens, and resulted in anticoccidial index >160 against E. tenella. These results suggest that EtCHP559 plays an important role in sporozoite invasion and could be an effective candidate for the development of a new vaccine against E. tenella.
| S-EPMC4910997 | biostudies-literature
Project description:Microneme proteins play an important role in the adherence of apicomplexan parasites to host cells during the invasion process. In this study, the microneme 2 protein from the protozoan parasite Eimeria tenella (EtMIC2) was cloned, characterized, and its protective efficacy as a DNA vaccine investigated. The EtMIC2 gene, which codes for a 35.07 kDa protein in E. tenella sporulated oocysts, was cloned and recombinant EtMIC2 protein (rEtMIC2) was produced in an Escherichia coli expression system. Immunostaining with an anti-rEtMIC2 antibody showed that the EtMIC2 protein mainly localized in the anterior region and membrane of sporozoites, in the cytoplasm of first- and second-generation merozoites, and was strongly expressed during first-stage schizogony. In addition, incubation with specific antibodies against EtMIC2 was found to efficiently reduce the ability of E. tenella sporozoites to invade host cells. Furthermore, animal-challenge experiments demonstrated that immunization with pcDNA3.1(+)-EtMIC2 significantly increased average body weight gain, while decreasing the mean lesion score and oocyst output in chickens. Taken together, these results suggest that EtMIC2 plays an important role in parasite cell invasion and may be a viable candidate for the development of new vaccines against E. tenella infection in chickens.
| S-EPMC6254105 | biostudies-literature
Project description:BackgroundSilent information regulator 2 (SIR2) proteins are a family of NAD + -dependent protein deacetylases that are considered potential targets for anti-parasitic agents. In this study, we cloned and characterized SIR2A of the protozoan parasite Eimeria tenella (EtSIR2A) and investigated its protective efficacy as a DNA vaccine.MethodsThe EtSIR2A gene encoding 33.37 kDa protein from E. tenella second-generation merozoites was cloned, and recombinant EtSIR2A protein (rEtSIR2A) was produced in an Escherichia coli expression system. The rEtSIR2A was used to immunize rabbits. Anti-rEtSIR2A antibodies were used to determine the immunolocolization of EtSIR2A in the parasite by immunofluorescence assay (IFA). Transcript and protein expression of EtSIR2A in different development stages of E. tenella were observed by quantitative real-time PCR (qPCR) and western blot (WB) analysis, respectively. The recombinant plasmid pCAGGS-EtSIR2A was constructed and its efficacy against E. tenella infection in chickens was evaluated.ResultsqPCR and WB analysis revealed EtSIR2A expression was developmentally regulated at both the mRNA and protein levels. EtSIR2A mRNA levels were higher in unsporulated oocysts than at other developmental stages, including sporulated oocysts, sporozoites and second-generation merozoites. In contrast, EtSIR2A protein expression levels were highest in second-generation merozoites, moderate in unsporulated oocysts and sporulated oocysts and lowest in sporozoites. Immunostaining with anti-rEtSIR2A antibody indicated that EtSIR2A was mainly located in the cytoplasm of sporozoites and second-generation merozoites, and was strongly expressed during first stage schizogony. Animal-challenge experiments demonstrated that immunization with pCAGGS-EtSIR2A significantly increased average body-weight gain, and decreased mean lesion score and oocyst output in chickens.ConclusionsThese results suggest that EtSIR2A may play an important role in parasite cell survival and may be an effective candidate for the development of new vaccines against E. tenella infection in chickens.
| S-EPMC5123391 | biostudies-literature
Project description:Protein disulfide isomerase (PDI) and PDI-like proteins are members of the thioredoxin superfamily. They contain thioredoxin-like domains and catalyze the physiological oxidation, reduction and isomerization of protein disulfide bonds, which are involved in cell function and development in prokaryotes and eukaryotes. In this study, EtPDIL, a novel PDI-like gene of Eimeria tenella, was cloned using rapid amplification of cDNA ends (RACE) according to the expressed sequence tag (EST). The EtPDIL cDNA contained 1129 nucleotides encoding 216 amino acids. The deduced EtPDIL protein belonged to thioredoxin-like superfamily and had a single predicted thioredoxin domain with a non-classical thioredoxin-like motif (SXXC). BLAST analysis showed that the EtPDIL protein was 55-59% identical to PDI-like proteins of other apicomplexan parasites. The transcript and protein levels of EtPDIL at different development stages were investigated by real-time quantitative PCR and western blot. The messenger RNA and protein levels of EtPDIL were higher in sporulated oocysts than in unsporulated oocysts, sporozoites or merozoites. Protein expression was barely detectable in unsporulated oocysts. Western blots showed that rabbit antiserum against recombinant EtPDIL recognized only a native 24 kDa protein from parasites. Immunolocalization with EtPDIL antibody showed that EtPDIL had a disperse distribution in the cytoplasm of whole sporozoites and merozoites. After sporozoites were incubated in complete medium, EtPDIL protein concentrated at the anterior of the sporozoites and appeared on the surface of parasites. Specific staining was more intense and mainly located on the parasite surface after merozoites released from mature schizonts invaded DF-1 cells. After development of parasites in DF-1 cells, staining intensified in trophozoites, immature schizonts and mature schizonts. Antibody inhibition of EtPDIL function reduced the ability of E. tenella to invade DF-1 cells. These results suggested that EtPDIL might be involved in sporulation in external environments and in host cell adhesion, invasion and development of E. tenella.
| S-EPMC4059736 | biostudies-literature
Project description: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.
| S-EPMC9929201 | biostudies-literature
Project description:Microneme proteins (MICs) of Eimeria tenella play key roles in motility, migration, attachment, and invasion processes. More than 20 apicomplexan parasite's MICs have been identified, with nine Eimeria MICs being reported. In this study, a novel E. tenella MIC was identified, and its gene structural features, developmental expression levels, localization, role in adhesion and invasion, and immunogenicity were studied. The results showed that the open reading frame was 1,650 bp, encoding 550 amino acids. It contains a signal sequence, a transmembrane region, four low-complexity boxes, and five epidermal growth factor-like domains (EGF). Subcellular localization revealed its distribution on the membrane surface of the parasite. These characteristics are consistent with the common features of MICs and are named EtMIC8. Anti-EtMIC8 antibodies recognized a specific binding of about 100 kDa in E. tenella, which was twice as large as the prokaryotic expression (about 50 kDa), suggesting that MIC8 may exist naturally as a dimer. EtMIC8 was expressed at higher levels in sporozoites (3.08-fold) and merozoites (2.1-fold) than in sporulated oocysts. The attachment assays using a yeast surface display of MIC8 and its different domains showed that the adherence rates of EtMIC8 to host cells were significantly higher than those of the control (3.17-fold), which was the full contribution of EGF, but neither was alone. Anti-EtMIC8 antibodies significantly reduced the invasion rate of sporozoites into host cells compared to those of the control (P < 0.01). Recombinant EtMIC8-EGF peptides could provide moderate protective efficacy (anticoccidial index [ACI]: 169.7), induce humoral responses, and upregulate CD3+CD8+ lymphocyte cells.
| S-EPMC8562341 | biostudies-literature
Project description:Eimeria tenella is an obligate intracellular parasite that actively invades cecal epithelial cells of chickens. The basis of cell invasion is not completely understood, but some key molecules of host cell invasion have been discovered. This paper investigated the characteristics of calcium-dependent protein kinase 4 (EtCDPK4), a critical molecule in E. tenella invasion of host cells. A full-length EtCDPK4 cDNA was identified from E. tenella using rapid amplification of cDNA ends. EtCDPK4 had an open reading frame of 1803 bp encoding a protein of 600 amino acids. Quantitative real-time PCR and western blotting were used to explore differences in EtCDPK4 transcription and translation in four developmental stages of E. tenella. EtCDPK4 was expressed at higher levels in sporozoites, but translation was higher in second-generation merozoites. In vitro invasion inhibition assays explored whether EtCDPK4 was involved in invasion of DF-1 cells by E. tenella sporozoites. Polyclonal antibodies against recombinant EtCDPK4 (rEtCDPK4) inhibited parasite invasion, decreasing it by approximately 52%. Indirect immunofluorescence assays explored EtCDPK4 distribution during parasite development after E. tenella sporozoite invasion of DF-1 cells in vitro. The results showed that EtCDPK4 might be important in sporozoite invasion and development. To analyze EtCDPK4 functional domains according to the structural characteristics of EtCDPK4 and study the kinase activity of rEtCDPK4, an in vitro phosphorylation system was established. We verified that rEtCDPK4 was a protein kinase that was completely dependent on Ca2+ for enzyme activity. Specific inhibitors of rEtCDPK4 activity were screened by kinase activity in vitro. Some specific inhibitors were applied to assays of DF-1 cell invasion by E. tenella sporozoites to confirm that the inhibitors functioned in vitro. W-7, H-7, H-89, and myristoylated peptide inhibited DF-1 invasion by E. tenella sporozoites. The experimental results showed that EtCDPK4 may be involved in E. tenella invasion of chicken cecal epithelial cells.
| S-EPMC5158193 | biostudies-literature
Project description:BACKGROUND:Avian coccidiosis is often caused by co-infection with several species of Eimeria worldwide. Developing a multivalent vaccine with an antigen common to multiple Eimeria species is a promising strategy for controlling clinical common co-infection of Eimeria. In the previous study, 14-3-3 was identified as one of the immunogenic common antigen in E. tenella, E. acervulina and E. maxima. The aim of the present study was to evaluate the immunogenicity and protective efficacy of Ea14-3-3 in the form of DNA vaccine against infection with three species of Eimeria both individually and simultaneously. RESULTS:After vaccination with pVAX-Ea14-3-3, the Ea14-3-3 gene was transcribed and expressed in the injected muscles. Vaccination with pVAX-Ea14-3-3 significantly increased the proportion of CD4+ and CD8+ T lymphocytes and produced a strong IgY response in immunized chickens. Similarly, pVAX-Ea14-3-3 stimulated the chicken's splenocytes to produce high levels of Th1-type (IFN-?, IL-2) and Th2-type (IL-4) cytokines. The vaccine-induced immune response was responsible to increase weight gain, decreased the oocyst output, and alleviated enteric lesions significantly in immunized chickens as compared to control group, in addition to induce moderate anti-coccidial index (ACI). CONCLUSION:These results indicate that Ea14-3-3 is highly immunogenic and capable to induce significant immune responses. Furthermore, Ea14-3-3 antigen can provide effective protection against infection with Eimeria tenella, Eimeria acervulina, Eimeria maxima both individually and in combination with three Eimeria species. Significant outcomes of our study provide an effective candidate antigen for developing a multivalent Eimeria vaccine against mixed infection with various Eimeria species under natural conditions.
| S-EPMC6233286 | biostudies-literature
Project description:Rhoptry proteins (ROPs) of Apicomplexa are crucial secreted virulence factors and sources of vaccine candidates. To date, Eimeria tenella ROPs are not well studied. This study identified and characterized a novel E. tenella ROP (EtROP35), which showed the highest levels among 28 putative ROPs in previous sporozoite and merozoite transcriptomes. Sequence analysis showed that EtROP35 contains an N-terminal secretory signal and a protein kinase domain including eight conserved ROP35-subfamily motifs. Subsequent experiments confirmed that it is a secretory protein. Subcellular localization revealed it localized at the apical end of the sporozoites and merozoites, which was consistent with the ROPs of other Apicomplexan parasites. To further understand the biological meaning of EtROP35, expression levels in different developmental stages and sporozoite invasion-blocking assay were investigated. EtROP35 showed significantly higher levels in sporozoites (6.23-fold) and merozoites (7.00-fold) than sporulated oocysts. Sporozoite invasion-blocking assay revealed that anti-EtROP35 polyclonal antibody significantly reduced the sporozoite invasion rate, suggesting it might participate in host cell invasion and be a viable choice as a vaccine candidate. The immunological protective assays showed that EtROP35 could induce a high level of serum IgY and higher mean body weight gain, and lower cecum lesion score and oocysts excretion than the challenged control group. These data indicated that EtROP35 had good immunogenicity and may be a promising vaccine candidate against E. tenella.
| S-EPMC9505231 | biostudies-literature
Project description:Avian coccidiosis causes significant economic losses on the global poultry breeding industry. Exploration of new-concept vaccines against coccidiosis has gradually become a research hotspot. In this study, an Enterococcus faecalis strain (MDXEF-1) showing excellent performance isolated from chicken intestinal tract was used as a vector to deliver Eimeria target protein. The plasmid pTX8048-SP-DCpep-NAΔ3-1E-CWA harboring dendritic cell-targeting peptide (DCpep) fusion with Eimeria tenella NAΔ3-1E gene (3-1E protein-coding gene without start codon ATG and terminator codon TAA) was electrotransformed into MDXEF-1 to generate the recombinant bacteria MDXEF-1/pTX8048-SP-DCpep-NAΔ3-1E-CWA in which NAΔ3-1E protein was covalently anchored to the surface of bacteria cells by cell wall anchor (CWA) sequence. The expression of target fusion protein DCpep-NAΔ3-1E-CWA was detected by Western blot. Each chicken was immunized 3 times at 2-wk intervals with live E. faecalis expressing DCpep-NAΔ3-1E fusion protein (DCpep-NAΔ3-1E group), live E. faecalis expressing NAΔ3-1E protein (NAΔ3-1E group), and live E. faecalis containing empty vector only. The 3 immunized groups were then challenged with homologous E. tenella sporulated oocyst after immunizations, and the immune response and protective efficacy in each group were evaluated. The results showed that serum IgG levels, secretory IgA levels in cecal lavage, proportion of CD4+ and CD8α+ cells in peripheral blood, and mRNA expression levels of IL-2 and IFN-γ in the spleen were significantly higher in chickens in the DCpep-NAΔ3-1E group than in chickens of the NAΔ3-1E group (P < 0.05). Oral immunization to chickens with live E. faecalis expressing DCpep-NAΔ3-1E offered more protective efficacy against homologous challenge including significant improved body weight gain, increased oocyst decrease ratio, and reduced average lesion scores in cecum compared with chickens with live E. faecalis expressing NAΔ3-1E protein. These results suggest that recombinant E. faecalis expressing dendritic cell-targeting peptide fusion with E. tenella 3-1E protein could be a potential approach for prevention of Eimeria infection.
| S-EPMC7597732 | biostudies-literature