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SDS-PAGE analysis of the proteins of chicken jejunal epithelial cells co-cultured with the soluble proteins of E. maxima sporozoites. Twenty micrograms of protein samples were subjected to a 12% gel. Lane M: standard protein molecular marker; Lane 1: the protein samples from jejunal epithelial cells co-cultured with the soluble protein of E. maxima sporozoites
Source publication
Background:
Eimeria maxima initiates infection by invading the jejunal epithelial cells of chicken. However, the proteins involved in invasion remain unknown. The research of the molecules that participate in the interactions between E. maxima sporozoites and host target cells will fill a gap in our understanding of the invasion system of this par...
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Citations
... A large number of avian Eimeria MIC have been identified and characterized, and some MIC are unique for coccidial parasites. To date, 10 MIC of E. tenella have been reported (Zhao et al., 2021), and 6 have been identified in E. maxima (Pasamontes et al., 1993;Witcombe et al., 2003;Li et al., 2013;Huang et al., 2015a;Huang et al., 2015b;Huang et al., 2018a). Within the MIC family of coccidia parasites, MIC3 has garnered the most extensive research attention and is seemingly paramount in mediating parasite invasion and determining tissue tropism (Lai et al., 2011;Zhang et al., 2016;Huang et al., 2018b). ...
Eimeria maxima microneme protein 3 (EmMIC3) is pivotal in the initial recognition and attachment of E. maxima sporozoites to host cells. EmMIC3 comprises 5 tandem Type I microneme adhesive repeat (MAR) domains, among which MAR2 of EmMIC3 (EmMAR2) has been identified as the primary determinant of EmMIC3-mediated tissue tropism. Nonetheless, the mechanisms through which EmMAR2 guides the parasite to its invasion site through interactions with host receptors remained largely uncharted. In this study, we employed yeast two-hybrid (YTH) screening assays and shotgun LC-MS/MS analysis to identify EmMAR2 receptors in chicken intestine epithelial cells. ATPase H+ transporting V1 subunit G1 (ATP6V1G1), receptor accessory protein 5 (REEP5), transmembrane p24 trafficking protein (TMED2), and delta 4-desaturase sphingolipid 1 (DEGS1) were characterized as the 4 receptors of EmMAR2 by both assays. By blocking the interaction of EmMAR2 with each receptor using specific antibodies, we observed varying levels of inhibition on the invasion of E. maxima sporozoites, and the combined usage of all 4 antibodies resulted in the most pronounced inhibitory effect. Additionally, the spatio-temporal expression profiles of ATP6V1G1, REEP5, TMED2, and DEGS1 were assessed. The tissue-specific expression patterns of EmMAR2 receptors throughout E. maxima infection suggested that ATP6V1G1 and DEGS1 might play a role in early-stage invasion, whereas TMED2 could be involved in middle and late-stage invasion and REEP5 and DEGS1 may participate primarily in late-stage invasion. Consequently, E. maxima may employ a multitude of ligand-receptor interactions to drive invasion during different stages of infection. This study marks the first report of EmMAR2 receptors at the interface between E. maxima and the host, providing insights into the invasion mechanisms of E. maxima and the pathogenesis of coccidiosis.
... Two gametocyte antigens (GAM56 and GAM82) from the sexual stages of Eimeria parasites are important components of the oocyst wall and potential vaccine candidates as transmission-blocking vaccines against coccidiosis [120,121]. Chickens which were vaccinated with these two antigens showed significantly decreased oocyst shedding and enhanced serum antibody and lymphocyte proliferation response in E. maxima-infected chickens [120,122,123]. In addition, transgenic plants (tobacco leaves) expressing Gam 56 and Gam 82 antigens elicited a protective immune response in immunized chickens (but not in non-vaccinated chickens) as measured by increased body weight gain and reduced fecal oocyst output [124]. ...
... Two gametocyte antigens (GAM56 and GAM82) from the sexual stages of Eimeria parasites are important components of the oocyst wall and potential vaccine candidates as transmission-blocking vaccines against coccidiosis [120,121]. Chickens which were vaccinated with these two antigens showed significantly decreased oocyst shedding and enhanced serum antibody and lymphocyte proliferation response in E. maxima-infected chickens [120,122,123]. In addition, transgenic plants (tobacco leaves) expressing Gam 56 and Gam 82 antigens elicited a protective immune response in immunized chickens (but not in non-vaccinated chickens) as measured by increased body weight gain and reduced fecal oocyst output [124]. ...
Coccidiosis is an avian intestinal disease caused by several distinct species of Eimeria parasites that damage the host’s intestinal system, resulting in poor nutrition absorption, reduced growth, and often death. Increasing evidence from recent studies indicates that immune-based strategies such as the use of recombinant vaccines and various dietary immunomodulating feed additives can improve host defense against intracellular parasitism and reduce intestinal damage due to inflammatory responses induced by parasites. Therefore, a comprehensive understanding of the complex interactions between the host immune system, gut microbiota, enteroendocrine system, and parasites that contribute to the outcome of coccidiosis is necessary to develop logical strategies to control coccidiosis in the post-antibiotic era. Most important for vaccine development is the need to understand the protective role of the local intestinal immune response and the identification of various effector molecules which mediate anti-coccidial activity against intracellular parasites. This review summarizes the current understanding of the host immune response to coccidiosis in poultry and discusses various non-antibiotic strategies which are being developed for coccidiosis control. A better understanding of the basic immunobiology of pertinent host–parasite interactions in avian coccidiosis will facilitate the development of effective anti-Eimeria strategies to mitigate the negative effects of coccidiosis.
... Some of the identified proteins were suggested as candidate biomarkers for early diagnosis of Eimeria infection. A recent study investigating the interaction between Eimeria maxima and chicken jejunal epithelial cells utilized shotgun LC-MS/MS and Western blot analysis to reveal 35 annotated (Gene Ontology) peptides, 22 of which were associated with binding activity and 15 with catalytic activity (Huang et al., 2018). This revealed novel findings of host-parasite interaction on a cellular level. ...
Technological advances in the field of proteomics have enabled researchers to visualize and characterize diverse physiological states in organisms. In the field of avian research, the proteome responsible for egg composition, embryonic development, behavior, and plumage has been further expounded. In addition, production traits of the modern broiler chickens including reproduction as well as meat production continue to be investigated using high-throughput proteomic technology. As performance of modern meat birds reaches historic heights, disease, myopathy, and infection threaten the challenged broiler physique. Proteomic research allows for culpable biological pathways to be identified and investigated at the tissue of origin. Avian research also extends to a growing area of concern, animal welfare. With the use of proteomics, avian welfare can not only be measured but also defined. With the identification of biomarkers and molecular signatures in disease and welfare states, proteomics provides valuable approaches to prevention, treatment, and interpretation of animal husbandry in the poultry industry. Proteomics continues to offer novel insights into the challenges facing the poultry industry and the inquiries of avian biology through elucidating the interplay between genome, transcriptome, and proteome of birds. The versatility and range of proteomics tells the story of proteins in a way that has transformed our interpretation of the building blocks of life and their impact.
... After trypsin hydrolyses the protein, MS is used to assay each peptide with great efficiency (Hayes and Yates, 2002). The shotgun proteomic approach in the parasite field has been very successful in constructing differential expression profiles and screening for specific antigens such as schistosomiasis, Eimeria maxima, and Fasciola hepatica (Campos et al., 2017;Liu et al., 2017;Huang et al., 2018). Most of the recent studies have been single screenings of specific antigens; however, the interactions between the host and the parasite involve a large number of proteins, and these proteins are involved in an interconnected network. ...
Taenia solium cysts were collected from pig skeletal muscle and analyzed via a shotgun proteomic approach to identify known proteins in the cyst fluid and to explore host-parasite interactions. Cyst fluid was aseptically collected and analyzed with shotgun liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene alignment and annotation were performed using Blast2GO software followed by gene ontology analysis of the annotated proteins. The pathways were further analyzed with the Kyoto Encyclopedia of Genes and Genomes (KEGG), and a protein-protein interaction (PPI) network map was generated using STRING software. A total of 158 known proteins were identified, most of which were low-molecular-mass proteins. These proteins were mainly involved in cellular and metabolic processes, and their molecular functions were predominantly related to catalytic activity and binding functions. The pathway enrichment analysis revealed that the known proteins were mainly enriched in the PI3K-Akt and glycolysis/gluconeogenesis signaling pathways. The nodes in the PPI network mainly consisted of enzymes involved in sugar metabolism. The cyst fluid proteins screened in this study may play important roles in the interaction between the cysticerci and the host. The shotgun LC-MS/MS, gene ontology, KEGG, and PPI network map data will be used to identify and analyze the cyst fluid proteome of cysticerci, which will provide a basis for further exploration of the invasion and activities of T. solium.
... High LDH expression has been reported in mature oocysts in another coccidian, C. parvum [75,100]. An E. maxima study showed that LDH and fructose bisphosphate aldolase in sporozoites bound chicken jejunal epithelial cells [101] and it is known that immunization with E. acervulina LDH can induce protective immunity [102]. Fructose bisphosphate aldolase was found to be important for energy production and invasion in T. gondii [103] and P. falciparum [104]. ...
... Microneme proteins in Eimeria spp. sporozoites interact with chicken jejunal cells, indicating an important binding role to cells [101,120]. Therefore, the upregulation of micronemes in mature oocysts may indicate that fully-formed packaged sporozoites express these proteins, necessary for invasion. ...
Eimeria parasites cause enteric disease in livestock and the closely related Cyclospora cayetanensis causes human disease. Oocysts of these coccidian parasites undergo maturation (sporulation) before becoming infectious. Here, we assessed transcription in maturing oocysts of Eimeria acervulina , a widespread chicken parasite, predicted gene functions, and determined which of these genes also occur in C . cayetanensis . RNA-Sequencing yielded ~2 billion paired-end reads, 92% of which mapped to the E . acervulina genome. The ~6,900 annotated genes underwent temporally-coordinated patterns of gene expression. Fifty-three genes each contributed >1,000 transcripts per million (TPM) throughout the study interval, including cation-transporting ATPases, an oocyst wall protein, a palmitoyltransferase, membrane proteins, and hypothetical proteins. These genes were enriched for 285 gene ontology (GO) terms and 13 genes were ascribed to 17 KEGG pathways, defining housekeeping processes and functions important throughout sporulation. Expression differed in mature and immature oocysts for 40% (2,928) of all genes; of these, nearly two-thirds (1,843) increased their expression over time. Eight genes expressed most in immature oocysts, encoding proteins promoting oocyst maturation and development, were assigned to 37 GO terms and 5 KEGG pathways. Fifty-six genes underwent significant upregulation in mature oocysts, each contributing at least 1,000 TPM. Of these, 40 were annotated by 215 GO assignments and 9 were associated with 18 KEGG pathways, encoding products involved in respiration, carbon fixation, energy utilization, invasion, motility, and stress and detoxification responses. Sporulation orchestrates coordinated changes in the expression of many genes, most especially those governing metabolic activity. Establishing the long-term fate of these transcripts in sporulated oocysts and in senescent and deceased oocysts will further elucidate the biology of coccidian development, and may provide tools to assay infectiousness of parasite cohorts. Moreover, because many of these genes have homologues in C . cayetanensis , they may prove useful as biomarkers for risk.
... Almost all of them have been shown to decrease the oocyst shedding of birds significantly [6][7][8][9][10][11][12]. The sporozoite stage plays a critical role in invasion and likely represents the most amenable target for the host immune response, since in immune birds, they undergo a very restricted development or even fail to penetrate cells in the intestinal tract [8,13,14]. The surface antigens are present on the sporozoite and some of these molecules have roles in host-parasite interactions, probably because these are naturally exposed during parasite recognition and invasion of the intestinal cells [8,[14][15][16]. ...
... The sporozoite stage plays a critical role in invasion and likely represents the most amenable target for the host immune response, since in immune birds, they undergo a very restricted development or even fail to penetrate cells in the intestinal tract [8,13,14]. The surface antigens are present on the sporozoite and some of these molecules have roles in host-parasite interactions, probably because these are naturally exposed during parasite recognition and invasion of the intestinal cells [8,[14][15][16]. Until now, it has been difficult to find a single target antigen that can elicit sterilizing immunity against coccidiosis [3,4,13]. ...
... Therefore, a mixture of antigens, which can act synergistically, might reduce oocyst output. The screening of sporozoites that highly express secreted antigens or surface antigens is crucial for the development of anticoccidial vaccines [5,14]. Immune responses to Eimeria involve many facets of innate and adaptive/acquired immunity, the latter encompassing both cellular and humoral immune mechanisms [2,9,17,18]. ...
This study investigated protection against Eimeria tenella following the vaccination of chicks with 5.3 × 106 E. tenella whole-sporozoites emulsified in the nanoparticle adjuvant IMS 1313 N VG Montanide™ (EtSz-IMS1313). One-day-old specific pathogen-free (SPF) chicks were subcutaneously injected in the neck with EtSz-IMS1313 on the 1st and 10th days of age. Acquired immunity was assayed through a challenge with 3 × 104 homologous sporulated oocysts at 21 days of age. The anticoccidial index (ACI) calculated for every group showed the effectiveness of EtSz-IMS1313 as a vaccine with an ACI of 186; the mock-injected control showed an ACI of 18 and the unimmunized, challenged control showed an ACI of −28. In a comparison assay, antibodies from rabbits and SPF birds immunized with EtSz-IMS1313 recognized almost the same polypeptides in the blotting of E. tenella sporozoites and merozoites. However, rabbit antisera showed the clearest recognition pattern. Polypeptides of 120, 105, 94, 70, 38, and 19 kDa from both E. tenella life cycle stages were the most strongly recognized by both animal species. The E. tenella zoite-specific IgG antibodies from the rabbits demonstrated the feasibility for successful B cell antigen identification.
... For exploiting DCs in vaccine development, vaccine antigens must induce DCs' maturation because functions of DCs are highly dependent on the level of maturation (Pulendran, 2004). E. maxima comprise numerous immunogenic antigens that play a crucial role in hostparasite interaction by modulating the immune response of the host against Eimeria infection (Blake et al., 2017;Huang et al., 2018). Hence, identification and screening of potential antigens from the complex biology of the parasite is always a priority for the development of novel vaccines. ...
Eimeria maxima possesses integral families of immunogenic constituents that promote differentiation of immune cells during host-parasite interactions. Dendritic cells (DCs) have an irreplaceable role in the modulation of the host immunity. However, the selection of superlative antigen with immune stimu-latory efficacies on host DCs is lacking. In this study, 5 recombinant proteins of E. maxima (Em), including Em14-3-3, rhomboid family domain containing proteins (ROM) EmROM1 and EmROM2, microneme protein 2 (EmMIC2), and Em8 were identified to stimulate chicken splenic derived DCs in vitro. The cultured populations were incubated with recombinant proteins, and typical morphologies of stimulated DCs were obtained. DC-associated markers major histocompatibility complex class II, CD86, CD11c, and CD1.1, showed upre-gulatory expressions by flow cytometry assay. Immunofluorescence assay revealed that recombinant proteins could bind with the surface of chicken splenic derived DCs. Moreover, quantitative real-time PCR results showed that distinct gene expressions of Toll-like receptors and Wnt signaling pathway were upregulated after the coincubation of recombinant proteins with DCs. The ELISA results indicated that the DCs produced a significant higher level of interleukin (IL)-12 and interferon-g secretions after incubation with recombi-nant proteins. While transforming growth factor-b was significantly increased with rEmROM1, rEmROM2, and rEmMIC2 as compared to control groups, and IL-10 did not show significant alteration. Taken together, these results concluded that among 5 potential recombinant antigens, rEm14-3-3 could promote immunogenic functions of chicken splenic derived DCs more efficiently, which might represent an effective molecule for inducing the host Th1-mediated immune response against Eime-ria infection.
... Although many invasion-related molecules of Eimeria have been studied [13,25], there are only a few reports concerning sporozoite receptors on host epithelia. In the current study, a cDNA library of chicken duodenal epithelial cells was constructed and screened for EaMIC3 receptor molecules by YTH. ...
Eimeria acervulina is one of seven Eimeria spp. that can infect chicken duodenal epithelial cells. Eimeria microneme protein 3 (MIC3) plays a vital role in the invasion of host epithelial tissue by the parasite. In this study, we found that chicken (Gallus gallus) ubiquitin conjugating enzyme E2F (UBE2F) could bind to the MIC3 protein of E. acervulina (EaMIC3), as screened using the yeast two-hybrid system, and that it might be the putative receptor protein of EaMIC3. The UBE2F gene was cloned from chicken duodenal epithelial cells. The recombinant protein of UBE2F (rUBE2F) was expressed in E. coli and the reactogenicity of rUBE2F was analyzed by Western blot. Gene sequencing revealed that the opening reading frame (ORF) of UBE2F was 558 base pairs and encoded a protein of 186 amino acids with a molecular weight of 20.46 kDa. The predicted UBE2F protein did not contain signal peptides or a transmembrane region, but had multiple O-glycosylation and phosphorylation sites. A phylogenetic analysis showed that the chicken UBE2F protein is closely related to those of quail and pigeon (Coturnix japonica and Columba livia). A sporozoite invasion-blocking assay showed that antisera against rUBE2F significantly inhibited the invasion of E. acervulina sporozoites in vitro. Animal experiments indicated that the antisera could significantly enhance average body weight gains and reduce mean lesion scores following a challenge with E. acervulina. These results therefore imply that the chicken UBE2F protein might be the target receptor molecule of EaMIC3 that is involved in E. acervulina invasion.
© Z. Zhang et al., published by EDP Sciences, 2020.
... The mass spectrometer was operated in MS/MS mode scanning from 380 to 1800 amu. The top 20 multiply charged ions were selected from each scan for MS/MS analysis ( [36] #1323). Then, the MS/ MS spectra were searched using MASCOT 2.2 (Matrix Science, London, UK), and the protein was identified by searching the B. tequilensis database in UniProt (https://www.uniprot.org/). ...
Background:
Tobacco stalk (TS), a major agricultural waste abundant in pectin, has resulted in concerns about the need for its reuse. The nicotine in TS is considered a chemical that is to\xic and hazardous to the environment.
Results:
In this study, Bacillus tequilensis CAS-MEI-2-33 was isolated from cigar wrappers to produce alkaline pectinase using TS. Subsequently, the medium and fermentation conditions for the production of pectinase by B. tequilensis CAS-MEI-2-33 were optimized. The optimal fermentation period, pH of the initial fermentation medium, concentration of TS, and inoculum amount for B. tequilensis CAS-MEI-2-33 were 40 h, 40 g/L, 7.0, and 3%, respectively. Under optimal conditions, the pectinase activity was 1370 U/mL. Then, the enzymatic properties, such as the optimum pH, reaction temperature, temperature stability, and effects of metal ions, were studied. The optimal pH was determined to be 10.0, indicating that the enzyme was an alkaline pectinase. The optimal temperature was 40 °C, and pectinase activity was stable at 40 °C. The Ag+ metal ions were shown to remarkably promote enzyme activity. The pectinase was partly purified by ammonium sulfate precipitation, ion exchange chromatography, and Sephacryl S-100 chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and LC-MS/MS analyses were utilized to analyze the pectinase.
Conclusions:
This study provided a new alkaline pectinase candidate and a new strategy for the use of TS.
... Microneme organelle proteins (MICs) are crucial for parasite host invasion and motility . MICs are secreted in the early stages of invasion and aid in the attachment of the parasite to the host cells and subsequent formation of the parasite actinomyosin system creating a platform for the invasion (Huang et al. 2018a). Nine MICs have been reported to date, MICs 1-7 and apical membrane antigens (AMA) 1 and 2 ( Barta et al. 2018). ...
Coccidiosis is a major poultry disease which compromises animal welfare and costs the global chicken industry a huge economic loss. As a result, research entailing coccidial control measures is crucial. Coccidiosis is caused by Eimeria parasites that are highly immunogenic. Consequently, a low dosage of the Eimeria parasite supplied by a vaccine will enable the host organism to develop an innate immune response towards the pathogen. The production of traditional live anticoccidial vaccines is limited by their low reproductive index and high production costs, among other factors. Recombinant vaccines overcome these limitations by eliciting undesired contaminants and prevent the reversal of toxoids back to their original toxigenic form. Recombinant vaccines are produced using defined Eimeria antigens and harmless adjuvants. Thus, studies regarding the identification of potent novel Eimeria antigens which stimulate both cell-mediated and humoral immune responses in chickens are essential. Although the prevalence and risk posed by Eimeria have been well established, there is a dearth of information on genetic and antigenic diversity within the field. Therefore, this paper discusses the potential and efficiency of recombinant vaccines as an anticoccidial control measure. Novel protective Eimeria antigens and their antigenic diversity for the production of cheap, easily accessible recombinant vaccines are also reviewed.
