Identification and characterization of Eimeria tenella apical membrane antigen-1 (AMA1).
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SUBMITTER: Jiang L
PROVIDER: S-EPMC3400601 | biostudies-literature | 2012
REPOSITORIES: biostudies-literature
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Identification and characterization of Eimeria tenella apical membrane antigen-1 (AMA1).
Jiang Lianlian L Lin Jiaojiao J Han Hongyu H Dong Hui H Zhao Qiping Q Zhu Shunhai S Huang Bing B
PloS one 20120719 7
Apical membrane antigen-1 (AMA1) is a micronemal protein of apicomplexan parasites that appears to be essential during the invasion of host cells. In this study, a full-length cDNA of AMA1 was identified from Eimeria tenella (Et) using expressed sequence tag and the rapid amplification of cDNA ends technique. EtAMA1 had an open reading frame of 1608 bp encoding a protein of 535 amino acids. Quantitative real-time PCR analysis revealed that EtAMA1 was expressed at higher levels in sporozoites tha ...[more]
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Project description: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.
| S-EPMC7731912 | biostudies-literature
Project description:BackgroundAvian 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).MethodsBimolecular 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.ResultsThe 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.ConclusionIn 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.
| S-EPMC7382093 | biostudies-literature
Project description:Apical membrane antigen 1 (AMA1) is an essential component of the moving junction complex used by Apicomplexan parasites to invade host cells. We report the 2.0 A resolution x-ray crystal structure of the full ectodomain (domains I, II, and III) of AMA1 from the pervasive protozoan parasite Toxoplasma gondii. The structure of T. gondii AMA1 (TgAMA1) is the most complete of any AMA1 structure to date, with more than 97.5% of the ectodomain unambiguously modeled. Comparative sequence analysis reveals discrete segments of divergence in TgAMA1 that map to areas of established functional importance in AMA1 from Plasmodium vivax (PvAMA1) and Plasmodium falciparum (PfAMA1). Inspection of the TgAMA1 structure reveals a network of apical surface loops, reorganized in both size and chemistry relative to PvAMA1/PfAMA1, that appear to serve as structural filters restricting access to a central hydrophobic groove. The terminal portion of this groove is formed by an extended loop from DII that is 14 residues shorter in TgAMA1. A pair of tryptophan residues (Trp(353) and Trp(354)) anchor the DII loop in the hydrophobic groove and frame a conserved tyrosine (Tyr(230)), forming a contiguous surface that may be critical for moving junction assembly. The minimalist DIII structure folds into a cystine knot that probably stabilizes and orients the bulk of the ectodmain without providing excess surface area to which invasion-inhibitory antibodies can be generated. The detailed structural characterization of TgAMA1 provides valuable insight into the mechanism of host cell invasion by T. gondii.
| S-EPMC2865318 | biostudies-literature
Project description:Apicomplexa parasite Eimeria is the causative agent of coccidiosis, which leads to enteritis in animals and causes huge economic burden to the farm industry. The Apicomplexan Apetala2/ERF (ApiAP2) transcription factors have proved to play key roles in various processes in other Apicomplexa parasites. However, little is known about the function of ApiAP2s in Eimeria species. In this study, we functionally characterized an ApiAP2 by direct knockout mediated by CRISPR-Cas9. Our results showed that the invasion efficiency, total oocyst output and virulence to the host were significantly impaired after EtAP2-S1 depletion. RNA-Seq and CUT&Tag analyze showed that EtAP2-S1 targets on the promoters of numerous genes and its knockout results in upregulation of 59 sag genes. Additionally, the knockout strain exhibits significantly reduced virulence but provides excellent immune protection, which makes it a good vaccine candidate. This study demonstrates that EtAP2-S1 is a fitness coffering gene that suppress the expression of sag genes in E. tenella, and this is the first practice in developing gene knockout vaccine for the control of coccidiosis.
2024-12-01 | GSE282226 | GEO
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: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: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:BackgroundCytauxzoon felis is a tick-transmitted apicomplexan that causes cytauxzoonosis in domestic cats (Felis catus). Even with intensive care, the mortality rate of acute cytauxzoonosis approaches 40% in domestic cats, while bobcats (Lynx rufus), the natural intermediate host of C. felis, remain clinically asymptomatic. However, multiple reports of domestic cats surviving acute disease without any treatment exist. One hypothesis for survival of these cats is infection with unique C. felis genotypes of lower pathogenicity. Prior studies have identified genetically distinct C. felis isolates containing polymorphisms within internal transcribed spacer regions (ITS) of the rRNA operon. However, these polymorphisms do not correlate with the clinical outcome of cytauxzoonosis, and so additional genetic markers are needed to test this hypothesis. We selected C. felis apical membrane antigen-1 (ama1) as a potential genetic marker of differential pathogenicity. AMA1 is a vaccine candidate for relatives of C. felis within Plasmodium spp.; however its historically high level of genetic polymorphism has resulted in escape from vaccine-induced immunity. While such diversity has hindered vaccine development, the expected polymorphism within the ama1 gene may be useful to evaluate population genetics.ResultsA 677 bp sequence of the C. felis ama1 gene was PCR-amplified from 84 domestic cats and 9 bobcats and demonstrated 99.9% sequence identity across all samples. A single nucleotide polymorphism (SNP) was identified in domestic cats and bobcats with evidence for co-infection with both genotypes identified in two domestic cats. The prevalence of the two genotypes varied with geographical distribution in domestic cats. Nucleotide diversity (π) and haplotype diversity (H) were calculated for C. felis ama1 and ama1 of related apicomplexans to assess genetic diversity. Based on these values (π = 0.00067 and H = 0.457), the diversity of the C. felis ama1 gene region analyzed is considerably lower than what is documented in related apicomplexans.ConclusionsIn surprising contrast to related apicomplexans, our results support that the sequence of the C. felis ama1 gene is highly conserved. While lack of genetic diversity limits utility of C. felis AMA1 as a genetic marker for clinical outcome, it supports further investigation as a vaccine candidate for cytauxzoonosis.
| S-EPMC6423858 | biostudies-literature
Project description:Apicomplexan parasites are the causative agents of globally prevalent diseases including malaria and toxoplasmosis. These obligate intracellular pathogens have evolved a sophisticated host cell invasion strategy that relies on a parasite-host cell junction anchored by interactions between apical membrane antigens (AMAs) on the parasite surface and rhoptry neck 2 (RON2) proteins discharged from the parasite and embedded in the host cell membrane. Key to formation of the AMA1-RON2 complex is displacement of an extended surface loop on AMA1 called the DII loop. While conformational flexibility of the DII loop is required to expose the mature RON2 binding groove, a definitive role of this substructure has not been elucidated. To establish a role of the DII loop in Toxoplasma gondii AMA1, we engineered a form of the protein where the mobile portion of the loop was replaced with a short Gly-Ser linker (TgAMA1ΔDIIloop). Isothermal titration calorimetry measurements with a panel of RON2 peptides revealed an influential role for the DII loop in governing selectivity. Most notably, an Eimeria tenella RON2 (EtRON2) peptide that showed only weak binding to TgAMA1 bound with high affinity to TgAMA1ΔDIIloop. To define the molecular basis for the differential binding, we determined the crystal structure of TgAMA1ΔDIIloop in complex with the EtRON2 peptide. When analyzed in the context of existing AMA1-RON2 structures, spatially distinct anchor points in the AMA1 groove were identified that, when engaged, appear to provide the necessary traction to outcompete the DII loop. Collectively, these data support a model where the AMA1 DII loop serves as a structural gatekeeper to selectively filter out ligands otherwise capable of binding with high affinity in the AMA1 apical groove. These data also highlight the importance of considering the functional implications of the DII loop in the ongoing development of therapeutic intervention strategies targeting the AMA1-RON2 invasion complex.
| S-EPMC4425356 | biostudies-literature
Project description:BackgroundApical membrane antigen 1 (AMA1) is one of the best-studied blood-stage malaria vaccine candidates. When an AMA1 vaccine was tested in a malaria naïve population, it induced functionally active antibodies judged by Growth Inhibition Assay (GIA). However, the same vaccine failed to induce higher growth-inhibitory activity in adults living in a malaria endemic area. Vaccination did induce functionally active antibodies in malaria-exposed children with less than 20% inhibition in GIA at baseline, but not in children with more than that level of baseline inhibition.MethodsTotal IgGs were purified from plasmas collected from the pediatric trial before and after immunization and pools of total IgGs were made. Another set of total IgGs was purified from U.S. adults immunized with AMA1 (US-total IgG). From these total IgGs, AMA1-specific and non-AMA1 IgGs were affinity purified and the functional activity of these IgGs was evaluated by GIA. Competition ELISA was performed with the U.S.-total IgG and non-AMA1 IgGs from malaria-exposed children.ResultsAMA1-specific IgGs from malaria-exposed children and U.S. vaccinees showed similar growth-inhibitory activity at the same concentrations. When mixed with U.S.-total IgG, non-AMA1 IgGs from children showed an interference effect in GIA. Interestingly, the interference effect was higher with non-AMA1 IgGs from higher titer pools. The non-AMA1 IgGs did not compete with anti-AMA1 antibody in U.S.-total IgG in the competition ELISA.ConclusionChildren living in a malaria endemic area have a fraction of IgGs that interferes with the biological activity of anti-AMA1 antibody as judged by GIA. While the mechanism of interference is not resolved in this study, these results suggest it is not caused by direct competition between non-AMA1 IgG and AMA1 protein. This study indicates that anti-malaria IgGs induced by natural exposure may interfere with the biological effect of antibody induced by an AMA1-based vaccine in the target population.
| S-EPMC3113848 | biostudies-literature