Molecular characterization and functional analysis of subunit 7 of eukaryotic initiation factor 3 from Eimeria tenella

Kong, Chunlin; Dong, Hui; Zhu, Shanfeng; Zhao, Qiping; Zhai, Qixiao; Liang, Siting; Li, Sha; Yang, Shi-Han; Huang, Bing

doi:10.1016/j.exppara.2015.04.002

Cited by 18 publications

(9 citation statements)

References 42 publications

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“…The authors also found that the secretion of Et Mic2 and Et Hsp90 was stimulated by FCS [ 15 ]. In our lab previous reports, we found that the different treatments with PBS or CM could affect the distribution of Et Serpin, Et PDIL, and Et eIF3s7 [ 12 , 31 , 32 ]. Moreover, the staining of Et CHP559 in the second-generation merozoites was increased after incubated in CM compared to them in PBS.…”

Section: Discussionmentioning

confidence: 97%

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella

et al. 2016

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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.

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Section: Discussionmentioning

confidence: 97%

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella

et al. 2016

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“…The purified r Et Esp and protein lysates were separated with SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes. Rabbit antiserum (1:100) against sporozoite proteins, previously produced in our laboratory [ 26 ], a rabbit anti-r Et Esp antibody (1:100), a mouse monoclonal anti-α-tubulin antibody (1:1000) (Sigma-Aldrich), and a monoclonal anti-His antibody (1:1000) were used as the primary antibodies to detect r Et Esp or native Et Esp. Naïve rabbit serum (1:100) was used as the negative control.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2020

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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.

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“…After blocking with 5% (w/v) skimmed milk powder in phosphate‐buffered saline (PBS) for 2 h at 37 °C, the membranes were incubated in SZ‐immunized rabbit sera (1:100 dilution) which observed in our laboratory (Han et al. ) or r Et STP‐immunized rabbit sera (1:100 dilution) for 2 h at 37 °C. At the same time, anti‐GST monoclonal antibody (1:1000 dilution), naive rabbit sera (1:100 dilution) were used as controls or mouse monoclonal anti‐α‐tubulin (1:2000 dilution) (Beyotime) was used as internal reference for each group.…”

Section: Methodsmentioning

confidence: 99%

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella

Zhao

Zhu

et al. 2020

J Eukaryotic Microbiology

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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.

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Molecular characterization and functional analysis of subunit 7 of eukaryotic initiation factor 3 from Eimeria tenella

Cited by 18 publications

References 42 publications

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella

Molecular Characterization and Immune Protection of a New Conserved Hypothetical Protein of Eimeria tenella

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

Molecular characterization of serine/threonine protein phosphatase of Eimeria tenella

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