EaMIC3 inhibits cell apoptosis and facilitates Eimeria acervulina infection through interacting with EpCAM

EaMIC3 inhibits cell apoptosis and facilitates Eimeria acervulina infection through interacting with EpCAM | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article EaMIC3 inhibits cell apoptosis and facilitates Eimeria acervulina infection through interacting with EpCAM Yadong Zheng, wang pu, zhang renzhe, zeng huan, wanjing li, Changyong Cheng, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7302653/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Background Coccidiosis caused by Eimeria species is a deadly parasitic disease particularly affecting chicks, leading to huge economic losses to the global poultry industry. The mechanisms underlying parasite infection remain poorly understood. Results Here, we showed that Eimeria acervulina microneme protein 3 (EaMIC3) was involved in the apoptosis of infected host cells and parasite infection by interacting with epithelial cell adhesion molecule (EpCAM). The interaction was mediated by the binding of three microneme adhesive repeat regions (MAR) 1, 2, and 5 in EaMIC3 to EpEX, an extracellular domain cleaved from EpCAM. During infection, EaMIC3 regulated the expression of both EpCAM and EpEX, with EpEX in turn inhibiting apoptosis of infected duodenal epithelial cells through the EGFR/Akt/mTOR signaling pathway. Moreover, EpCAM knockdown inhibited sporozoite infection, while EpCAM overexpression promoted sporozoite infection. Conclusion Consistently, the administration of pooled EaMIC3 and EpCAM antisera conferred a good therapeutic effect on E. acervulina infection in chickens, with a maximum oocyst reduction rate of 67.86%. Overall, these findings reveal a mechanism by which EaMIC3 inhibits cell apoptosis and facilitates E. acervulina infection, shedding light on the prevention and control of coccidiosis in chickens. Health sciences/Pathogenesis/Infection Scientific community and society/Agriculture Eimeria acervulina chicken EaMIC3 EpCAM apoptosis infection EGFR/Akt/mTOR pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Coccidiosis in chickens leads to considerable economic losses worldwide, amounting to approximately $ 13.2 billion annually [ 1 ]. At present, the control of coccidiosis primarily relies on drugs and attenuated live vaccines. However, their application is limited due to drug resistance and the pathogenicity of live vaccines. The lack of effective control measures for coccidiosis is largely attributed to a poor understanding of the pathogenesis of Eimeria species. During the life cycle, when ingested by chickens, sporulated oocysts release sporozoites under the action of gastric acid and duodenal peristalsis [ 2 ]. These sporozoites invade the epithelial cells of the chicken duodenum and transform into schizonts, which subsequently undergo multiple fission to form merozoites [ 3 ]. Once matured, the merozoites are released from the cells and proceed to infect additional cells, leading to extensive duodenum infection. One of the main mechanisms by which Eimeria species mitigate host cell damage is the inhibition of apoptosis, thereby facilitating their own growth, development and reproduction [ 4 ]; however, the precise mechanisms are not yet fully understood. Microtubules are small secretory organelles that accumulate at the apex of Apicomplexan parasites and are mainly present in gliding or invading protozoa [ 5 ]. They secrete multiple microneme (MIC) proteins, and MIC2, MIC3, and MIC5 have so far been identified in Eimeria acervulina [ 6 ]. E. acervulina MIC3 (EaMIC3) serves as a key virulence factor and is characterized seven adhesive repeat motifs that are expressed in sporozoites and merozoites. At the early stage of infection, EaMIC3 is situated at the apex of the complex and specifically binds to host cells [ 7 ]. Therefore, it not only mediates the invasion of sporozoites into host cells, but also determines the specificity of binding. Moreover, it has been reported that the epidermal growth factor (EGF) motifs of Toxoplasma gondii MIC3 and MIC6 interact with the EGF receptor (EGFR) of host cells, thereby inhibiting cell apoptosis by activating the EGFR/Akt signaling pathway and ultimately promoting the survival of T. gondii in host cells [ 8 ]. Similarly, Neosporidium MIC3 is also capable of activating the EGFR signaling, thereby preventing host cell apoptosis [ 9 ]. Though EaMIC3 lacks EGF motifs, it has been shown to inhibit early apoptosis of infected host cells [ 10 ], yet the precise mechanism behind remains unclear. Results EpCAM interacts with EaMIC3 To determine the role of EaMIC3 in invasion, a yeast two-hybrid assay was used to identify the molecules that interact with EaMIC3. From initial screening, we identified 6 positive clones, of which only 3 were further confirmed to be true positives. BLAST analysis confirmed that 2 of these clones exhibited high sequence similarity to ubiquitin-conjugating enzyme E2F (UBE2F, NP_001006512.1) and epithelial cell adhesion molecule (EpCAM, NP_001012582.2), respectively, while the remaining clone could not be aligned to any known coding sequences (data not shown). To further investigate the interaction between EpCAM and EaMIC3, we first ruled out the possibilities of the toxicity and autoactivation activity of both the pGADT7-AD-EpCAM and pGBKT7-BD-EaMIC3 plasmids (Fig. S1 A and S1B). We then showed that the yeast cells co-transformed with the pGADT7-AD-EpCAM and pGBKT7-BD-EaMIC3 plasmids exhibited blue colonies on both the -Trp/-Leu/-x-α-gal and -Trp/-Leu/-His/-ADE/-x-α-gal plates (Fig. S1 C). Furthermore, we provided additional evidence that EpCAM interacted with EaMIC3 in the immuno-precipitation assay (Fig. 1 A) and was also co-localized with EaMIC3 (Fig. 1 B). Collectively, these findings suggest that EpCAM directly interacts with EaMIC3. EpEX domain and MARs 1, 2, and 5 are crucial to EpCAM-EaMIC3 interaction EpCAM is a transmembrane glycoprotein located on the surface of epithelial cells, consisting of intracellular (EpICD), transmembrane, and extracellular domains (EpEX) [ 15 , 16 ]. To determine the domain involved in the interaction, we first assessed the binding capacity of different truncated forms of EpCAM. The results showed that EaMIC3 bound to and was also co-localized with EpEX rather than EpICD (Fig. 1 C and 1 D). It has been documented that EaMIC3 has 7 adhesive repeat regions 1–7 (MAR 1–7, Fig. 2 A) [ 12 ]. We further defined the regions important to binding. The results indicated that EpEX interacted with MAR1, MAR2, and MAR5 (Fig. 2 B-H) and was also co-localized with these regions in the cytoplasm (Fig. 2 I-K). These results indicate that the EpEX domain and MARs 1, 2, and 5 are key determinants of the interaction between EpCAM and EaMIC3. Sporozoite infection promotes EpCAM expression and EpEX cleavage by EaMIC3 Due to the interaction between EaMIC3 and EpCAM, we next sought to determine whether EaMIC3 regulates the expression of EpCAM during parasite infection. The results revealed that the expression levels of EpCAM in the infected DF-1 cells and EpEX, an extracellular domain cleaved from EpCAM [ 17 ], in the cell culture supernatant, exhibited a significant increase in a time-dependent manner ( P <0.05, Fig. 3 A-C). However, the expression levels of both EpCAM and EpEX exhibited a significant reduction at 6 h after incubation with sporozoites and antibodies specifically against EaMIC3 (1−180aa; 485−576aa) compared to the control ( P <0.001, Fig. 3 D-F). In this scenario, the number of sporozoites present in the culture showed a significant increase ( P <0.001, Fig. 3 G), while the number of sporozoites within the DF-1 cells had a substantial decrease ( P <0.01, Fig. 3 H). These results indicate that sporozoite infection promotes EpCAM expression and EpEX cleavage, possibly induced by EaMIC3. EpCAM accelerates sporozoite infection As EpCAM was found to be upregulated at the early stage of the infection, we postulated that EpCAM, as a receptor, plays a role in sporozoite infection process. To test this, we assessed the infectivity of sporozoites to DF-1 cells with EpCAM knockdown, which was achieved through the delivery of siRNA E872 ( P <0.001, Fig. S2). The findings showed that, in comparison to the control group, EpCAM knockdown led to a notable decrease in the number of sporozoite-infected cells but a significant increase in the number of sporozoites present in the culture supernatant ( P <0.05, Fig. 4 A, 4 B and 4 C). Conversely, EpCAM overexpression resulted in a significant increase in the number of sporozoite-infected cells compared to the control ( P <0.001, Fig. 4 D). Consistently, the expression levels of EpCAM in the infected DF-1 cells as well as EpEX in the culture supernatant were significantly decreased in the E872 group in comparison to the mock group ( P <0.001, Fig. 4 E). These results imply that EpCAM promotes the infection of sporozoites into DF-1 cells. EpEX is involved in EaMIC3-induced apoptosis inhibition via the EGFR/Akt/mTOR signaling pathway As EaMIC3 inhibits early apoptosis of infected host cells [ 10 ], we further tested whether EpCAM is directly involved in this process. The results showed that the levels of cell apoptosis were significantly elevated in the E872 + Infection group compared to the NC + Infection and Infection groups at 4 h, 6 h, and 8 h after infection ( P <0.01, Fig. 5 A and 5 B). Moreover, the apoptosis levels were also significantly higher in the E872 + Infection + Act-D group than those in the NC + Infection + Act-D and Infection + Act-D groups at 4 h, 6 h, and 8 h after infection ( P <0.01, Fig. 5 A and 5 B). These results indicate that EaMIC3 inhibits early apoptosis of infected host cells by promoting EpCAM expression. It has been reported that the N-linked glycosylation of EpCAM induces its cleavage, resulting in the formation of EpEX that can activate the EGFR pathway in tumors [ 17 ]. Meanwhile, several protozoa are known to activate the EGFR/AKT signaling pathway during infection [ 18 , 19 ] and the infection of E. acervulina sporozoite has been shown to induce EpEX cleavage (Fig. 3 A and 3 C). Therefore, we hypothesized that EpEX may act as a suppressor of apoptosis through the EGFR/AKT signaling pathway during the infection. The results showed that EpEX directly bound to and was co-localized with the extracellular region (638-1050aa) of EGFR (Fig. 6 A). Additionally, the EGFR level showed a continuous and significant increase post infection; however, it was markedly lower in the E872 + Infection group compared to the infection group ( P <0.01, Fig. 6 B and 6 C). Furthermore, the levels of both p-Akt and p-Akt/Akt were significantly lower in the E872 + Infection group than those in the infection group ( P <0.001, Fig. 6 B, 6 D, and 6 E). To further confirm the aforementioned findings, we assessed the levels of Akt, p-Akt, mTOR, and p-mTOR in infected DF-1 cells with or without incubation with EaMIC3 (1−180aa; 485−576aa) and EpCAM (20−260aa) antibodies. After incubation, the levels of p-mTOR/mTOR, and p-PI3K/Akt were significantly lower in the anti-EpCAM + Infection and anti-EaMIC3 + Infection groups than those in the Infection group ( P <0.001, Fig. 7 A, 7 B, 7 C, and 7 D). Meanwhile, the levels of cell apoptosis were markedly increased in the anti-EaMIC3 + Infection and anti-EpCAM + Infection groups relative to the Infection group ( P <0.001, Fig. 7 E). These findings indicate that sporozoite infection inhibits early apoptosis of infected host cells by EpEX via the EGFR/AKT/mTOR signaling pathway. EaMIC3 is associated with duodenal epithelial cell apoptosis inhibition through the Akt/mTOR pathway during sporozoite infection To evaluate the effects of EaMIC3 on the apoptosis of duodenal epithelial cells during the infection, we accessed the apoptosis rate and the expression of apoptosis-associated proteins in challenged chicken immunized with EaMIC3 (Fig. 8 A). There were no obvious lesions in the duodenum in all the groups (Fig. 8 B). However, both early and late apoptosis rates in the EaMIC3 + Infection group was significantly higher than that in the Infection group but lower than those in the blank control ( P <0.05, Fig. 8 C and 8 D). Additionally, the ratio of p-Akt/Akt was elevated at 6 h but decreased at 8 h and 24 h in the EaMIC3 + Infection group compared to the Infection and GST + Infection groups ( P <0.05, Fig. 8 E and 8 F). Similarly, the ratio of p-mTOR/mTOR was significantly decreased at 8 h in the EaMIC3 + Infection group compared to the Infection and GST + Infection groups ( P <0.01, Fig. 8 E and 8 G). Collectively, these findings suggest that EaMIC3 is involved in the inhibition of duodenal epithelial cell apoptosis induced by E. acervulina infection through the Akt/mTOR signaling pathway. EaMIC3/EpCAM antisera confer a good therapeutic effect on E. acervulina infection Given the significant role of EaMIC3-EpCAM interaction in E. acervulina infection, we then evaluated the therapeutic effect of EaMIC3/EpCAM antisera (Fig. 9 A). The results showed that the duodenal intestinal bleeding was markedly alleviated in the EaMIC3 + Infection, EpCAM + Infection, EaMIC3/EpCAM + Infection groups in comparison to the Infection group (Fig. 9 B). Consistently, all the antiserum-treated groups exhibited significantly higher weight gain compared to the Infection group ( P < 0.01, Fig. 9 C). Similarly, all the treated groups also exhibited significantly lower intestinal lesion scores ( P < 0.01, Fig. 9 D). The highest oocyst reduction rate was 67.86% in the EaMIC3/EpCAM group, followed by 61.68% in the EaMIC3 group and 46.85% in the EpCAM group (Fig. 9 E). Moreover, all the treated groups showed a statistically significant decrease in oocyst shedding compared to the Infection group ( P < 0.001, Fig. 9 F). These results indicate that EaMIC3/EpCAM antisera confer a good therapeutic effect on E. acervulina infection. Discussion EpCAM is involved in various cellular processes, such as cell apoptosis, cell signaling, differentiation, proliferation, invasion, and migration [ 20 ]. In this study, we found that EpCAM low expression led to a significant reduction in the invasion and intracellular proliferation rates of E. acervulina , suggesting a key role during the infection. Meanwhile, we demonstrated that the extracellular domain of EpCAM, EpEX, interacted with the MARs 1, 2, and 5 of EaMIC3, suggesting that the interactions occur through multiple sites. Consistently, after being incubated with EaMIC3 (1−180aa; 485−576aa) antibodies, sporozoites exhibited a dramatically decrease in both the expression level of EpCAM and the infection rate. These results suggest that EaMIC3 promotes the infection of E. acervulina by interacting with EpCAM. However, the sites specific for the EaMIC3-EpCAM interaction remain unclear and need to be further explored. It was demonstrated that EpEX was involved in the apoptosis-related signaling pathways in human cancer cells [ 21 – 26 ]. Previously, EaMIC3 was also found to inhibit host cell apoptosis during the initial phase of infection [ 10 ]. Consistent with these observations, the cell apoptosis rate was significantly increased during E. acervulina infection following the knockdown of EpCAM, suggesting that EpCAM negatively regulates the inhibition of host cell apoptosis during the parasite infection. Parasitic protozoa maintain their intracellular survival by inhibiting host cell apoptosis through the PI3K/Akt and NF-κB signaling pathway at the early stages of infection. For instance, T. gondii inhibits apoptosis of T cells and macrophages through the PI3K/Akt signaling pathway [ 27 , 28 ], whereas Theileria, Cryptosporidium , and Neosporidium species inhibit host cell apoptosis by activating the NF-κB signaling pathway [ 29 – 31 ]. Moreover, it has been reported that E. tenella suppresses the apoptosis of cecal epithelial cells by activating the PI3K/Akt pathway [ 32 ]. The PI3K/Akt and NF-κB signaling pathways are closely associated with the functions of EGFR [ 33 , 34 ]. In the current study, we found that EpEX interacted with EGFR and the expression levels of EGFR, p-Akt, and p-mTOR were significantly decreased during the infection in the context of EpCAM low expression. These findings indicate that EpEX activates the EGFR/Akt/mTOR signaling pathway and inhibits cell apoptosis by interacting with EGFR during the infection. It was further shown that both the levels of EpEX in the culture supernatant and EpCAM in infected cells were significantly higher in the Infection group than those in the control. In contrast, following the incubation of sporozoites with EaMIC3 (1−180aa; 485−576aa) antibodies, there was a notable reduction in the levels of both EpEX and EpCAM during the infection process. These results suggest that EaMIC3 enhances the expression of EpCAM and facilitates its cleavage. Consistently, the levels of EGFR, p-Akt, and p-mTOR were markedly decreased and the rates of apoptosis were significantly increased following incubation with EpCAM antibodies, particularly EaMIC3 antibodies. In summary, this study reveals the underlying mechanism by which EaMIC3 inhibits apoptosis of host cells and facilitates E. acervulina infection through interacting with EpCAM. The findings obtained herein offer valuable insights for the development of drugs or vaccines for the control of E. acervulina infection. Materials and Methods Ethics approval The animal experiments in the study were approved by the Animal Care and Use Committee of Zhejiang A & F University. The protocols were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People’s Republic of China. Parasites E. acervulina , Jilin strain, was passaged and maintained in our lab. Sporozoites from E. acervulina oocysts were purified using DE-52 anion-exchange columns as previously described [ 6 , 11 ]. Cells and Yeast 293T and DF-1 cells (Fenghui Biotechnology, China) were cultured in Dulbecco's modified eagle medium (DMEM) medium containing 10% fetal bovine serum (FBS) at 37°C with 5% CO 2 . The chicken duodenal epithelial cells were isolated and collected from 18-day-old SPF chick embryos (Merial Vital Corp, China) as previously described [ 10 ]. The yeast strain Y2HGold (Clontech, USA) was cultured in YPDA medium at 30°C. Yeast two-hybrid test For yeast two-hybrid screening, the fragment of the EaMIC3 gene (1-2607 bp, AMN15064.1), which encodes seven repeating domains, was cloned into pGBKT7 and the positive plasmid, designated as pGBKT7-EaMIC3, was used as bait. Using a library construction and screening kit (Clontech, USA), an E. acervulina cDNA library was constructed using the pGADT7 vector and used as prey. The yeast strain Y187 was transformed with the bait plasmid pGBKT7-EaMIC3, and an α-galactosidase assay was performed to examine autoactivation. The yeast strain Y187 was transformed with the prey plasmid, which contained a GAL4 activation domain. For interaction mating, the library and bait strains were co-cultured at 30°C for 20 h. After mating, the culture was plated on selection medium lacking histidine, leucine, tryptophan, and adenine but containing 20 µg/mL X-α-gal (SD/-Ade/-His/-Leu/-Trp/X-α-gal). The resultant blue colonies were selected for further analysis. The cells transfected with the pGBKT7-53 and pGADT7-T plasmids were used as a positive control, while the cells transfected with the pGBKT7-Lam and pGADT7-T plasmids were used as a negative control. To further confirm the hits from the above screening, the positive prey plasmid pGADT7-EpCAM was co-transformed into the Y187 strain with the bait plasmid pGBKT7-EaMIC3 and plated on the selection medium. Co-immunoprecipitation and Western blotting The recombinant plasmids pCMV-myc-EaMIC3, pCMV-myc-EGFR, and pCMV-HA-EpCAM were constructed respectively as previously reported [ 12 ]. These recombinant plasmids were then extracted and co-transfected into 293T cells using the FuGENE HD transfection reagent (Roche, Switzerland). After 48 h, the transfected cells were harvested and then lysed with precooled lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 5% glycerol and a protease inhibitor cocktail (Roche, Switzerland)) for 30 min on ice, followed by incubation with protein A/G-conjugated Sepharose beads (Millipore, USA) at 4°C for 30 min. The supernatants were incubated with rabbit polyclonal antibodies against the Myc tag (Cell Signaling, USA) at 4°C overnight, followed by mixing with protein A/G-conjugated Sepharose beads at 4°C for 2 h. The mixture was centrifuged at 8,000 × g at 4°C for 5 s, followed by rinse with wash buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM EDTA, and 1% NP40) three times. The bound proteins were eluted with elution buffer (0.1 M glycine, pH 2.8), neutralized with neutralization buffer (1 M Tris-HCl, pH 9.5) and separated using 12% SDS-PAGE. Western blotting was performed to identify bound proteins using rabbit polyclonal antibodies against HA (Cell Signaling, USA) at 1: 1,000 and a mouse monoclonal antibody against Myc (Cell Signaling, USA) at 1: 1,000. Goat anti-rabbit IgG (1:3,000, TransGen Biotech, China) and goat anti-mouse IgG (1: 3,000, TransGen Biotech, China) HRP-conjugated antibodies were employed and then the results were detected by ECL (enhanced luminol-based chemiluminescent) Western blotting substrate kit (Bio-Rad, USA). Confocal immunofluorescence The pCMV-myc-EaMIC3 and pCMV-HA-EpCAM plasmids were co-transfected into 293T cells, and then the cells were fixed in 4% paraformaldehyde for 15 min at 25°C. The cells were dealt with 10 mM PBS containing 1% Triton X-100 at 37°C for 15 min, suspended in 10 mM PBS containing 5% BSA and then incubated at 37°C for 1 h. Immunofluorescence was performed using rabbit polyclonal antibodies against HA and Myc (Cell Signaling, USA) at 1:100. Goat anti-rabbit IgG (1:3,000, TransGen Biotech, China) HRP-conjugated antibodies were then employed. DAPI staining solution was added and incubated for 10 min, followed by observation under a confocal microscope (Olympus, Japan). Antibody blocking test Sterile purified sporozoites were co-incubated with EaMIC3 (1−180aa; 485−576aa) antibodies and rabbit negative serum (NC), respectively, as previously described [ 13 ]. DF-1 cells were cultivated until their density reached at 80% − 90%, followed by the addition of the purified sporozoites at a ratio of 2:1 (sporozoites: cells) and incubation for 1 h. The expression levels of EpCAM in the cells and EpEX in the culture supernatant were detected, and the number of sporozoites in the cells and the supernatant was determined, respectively. RNA interference Small interfering RNAs (siRNAs) targeting the epcam gene were designed and synthesized (OBIO, China). 2.2 pmol of EpCAM siRNAs were added into DF-1 cells (40–60% confluence) in 200 µL of DMEM medium without FBS, followed by the addition of 12 µL transfection reagent (Roche, Switzerland) and cultivation for 4 h. After the supernatant was removed, the cells were further cultured in DMEM medium with 15% FBS for 72 h as previously described and then the expression level of EpCAM was detected [ 14 ]. Apoptosis DF-1 cells were plated in 96-well plates at a density of 1.0×10 4 cells/well. After 48 h, the culture medium was then replaced with fresh medium supplemented with 20µM Act D, and the cells were further incubated for 4 h. Then the cells were harvested and analyzed using the Annexin V-FITC apoptosis detection kit (KeyGEN, China) as previously described [ 10 ]. In brief, 1 × 10 5 cells were resuspended in 500 µL of binding buffer and then labeled with Annexin V-FITC and propidium iodide (PI) for 15 min in the dark at room temperature. The cells were examined by flow cytometry (BD Biosciences, USA). Apoptosis of the duodenum in EaMIC3-immunized chickens A total of 20 one-day-old broiler chickens were obtained from a local hatchery (Hangzhou Xiaoshan Donghai Breeding, China) and raised in a temperature-controlled closed-house environment. The clean water and formulated feed were used for raising chickens. At 7 days of age, chickens were randomly divided into the 4 groups with 5 chickens of each group: Control group, E. acervulina infection group, E. acervulina infection group with immunization of EaMIC3, and E. acervulina infection group with immunization of GST. At 7 d up to 21 d, EaMIC3 was vaccinated (25 µg/chicken) to the chickens by intramuscular injection. At 28 d, all the groups except the control were orally challenged with freshly sporulated E. acervulina oocysts (5,000/chicken). All chickens were humanely executed at 8 h after infection, and the apoptosis rate and the expression of the apoptotic factors in the duodenum were analyzed. Therapeutic efficacy of anti-EaMIC3/EpCAM sera Both EaMIC3 and EpCAM rabbit antisera were prepared as previously reported [ 10 ]. Twenty-eight-day-old chickens with a similar body weight were randomly divided into 5 groups: no infection group (negative control), infection group (positive control), EaMIC3 antiserum-treated group, EpCAM antiserum-treated group, and mixed antisera-treated (EaMIC3 + EpCAM) group. Except no infection group, all the groups were orally challenged with 2×10 4 oocysts and then each chicken was administered with 0.2 mL corresponding antiserum for 7 consecutive days. The therapeutic efficacy was evaluated via weight gain, oocysts per gram (OPG) of feces, and intestinal lesion scores. Statistical analysis GraphPad Prism was used to conduct statistical analyses. Unpaired Student’s t -test and ANOVA were performed to compare the difference between two groups and three or more groups, respectively, with a P value less than 0.05 to be considered statistically significant. Declarations Competing interests The authors have declared that no competing interest exists. Acknowledgement The study was financially supported by the National Key Research and Development Program of China (2023YFD1801000), Zhejiang Provincial Natural Science Foundation of China (LZ24C180001), the Base and Talent Project of the Department of Science and Technology of the Tibet Autonomous Region (XZ202401JD0012), the NHC Key Laboratory of Echinococcosis Prevention and Control (2022WZK1004), and Key R&D and Conversion Plan of Qinghai Province (2025-QY-227). 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Supplementary Files Supplementaryfile.docx EaMIC3 inhibits cell apoptosis and facilitates Eimeria acervulina infection through interacting with EpCAM Cite Share Download PDF Status: Under Review Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7302653","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":501027928,"identity":"17a16ecb-2543-4c61-a4f2-eaed3a332c76","order_by":0,"name":"Yadong 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ying","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"wang","middleName":"","lastName":"ying","suffix":""},{"id":501027937,"identity":"5125998f-b44b-4a7a-91c0-1886ebcfcb57","order_by":9,"name":"Jing Zhang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Zhang","suffix":""},{"id":501027938,"identity":"e1c24aa5-4206-4f9a-afa7-66375d8a6f09","order_by":10,"name":"Pengfei Cai","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Pengfei","middleName":"","lastName":"Cai","suffix":""},{"id":501027939,"identity":"b84a0bfa-d3f7-47b6-8e44-dc60a9131c8e","order_by":11,"name":"William Cho","email":"","orcid":"https://orcid.org/0000-0003-4174-4586","institution":"Queen Elizabeth Hospital","correspondingAuthor":false,"prefix":"","firstName":"William","middleName":"","lastName":"Cho","suffix":""},{"id":501027940,"identity":"a0444edb-ea15-4e24-8d44-979cbda4031b","order_by":12,"name":"Xueyong Zhang","email":"","orcid":"","institution":"The Academy of Animal and Veterinary Sciences, Qinghai University","correspondingAuthor":false,"prefix":"","firstName":"Xueyong","middleName":"","lastName":"Zhang","suffix":""},{"id":501027941,"identity":"5553d267-a585-48ba-95ec-ee8b4db7f6e0","order_by":13,"name":"Houhui Song","email":"","orcid":"https://orcid.org/0000-0001-6530-5794","institution":"Zhejiang A\u0026F University","correspondingAuthor":false,"prefix":"","firstName":"Houhui","middleName":"","lastName":"Song","suffix":""}],"badges":[],"createdAt":"2025-08-05 16:15:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7302653/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7302653/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89819879,"identity":"0af6eca3-342d-46a1-8e23-e701ca62fe3f","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":174921,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the domain in EpCAM key to the EpCAM-EaMIC3 interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInteraction between EpCAM and EaMIC3 was validated by co-immunoprecipitation (A) and co-immunofluorescence assay (B). Interactions of EaMIC3 with EpEX (C) and EpICD (D) were identified by co-immunoprecipitation and/or co-immunofluorescence assay. EpCAM, epithelial cell adhesion molecule; EaMIC3, \u003cem\u003eE. acervulina\u003c/em\u003e microneme protein 3; EpEX, an extracellular domain of EpCAM; EpICD, an intracellular domain of EpCAM.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/402a63a53a9733f8a4c17ea6.jpeg"},{"id":89819878,"identity":"2fae3b41-f003-42f1-8f14-a0d8b9401341","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":288826,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of the regions in EaMIC3 key to the EpEX-EaMIC3 interaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe amino acid sequence of EaMIC3 was segmented into 7 regions (A). EpEX was shown to interact with MAR1 (B), MAR2 (C), and MAR5 (F), but not with MAR3 (D), MAR4 (E), MAR6 (G), and MAR7 (H) by co-immunoprecipitation. Moreover, it was further shown to be colocalized with MAR1 (I), MAR2 (J), and MAR5 (K) by co-immunofluorescence assay. EaMIC3, \u003cem\u003eE. acervulina\u003c/em\u003e microneme protein 3; EpEX, an extracellular domain of EpCAM; MAR, microneme adhesive repeat regions.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/b2502d9f760fe342908c14e7.jpeg"},{"id":89819880,"identity":"be3d1c0a-ccf4-488f-a54d-b8e9abb15e40","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":131449,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of\u003c/strong\u003e\u003cem\u003e\u003cstrong\u003e E. acervulina\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection on expression of EpCAM and EpEX and blocking \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. acervulina\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e infection by anti-EaMIC3 antibodies\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe levels of EpCAM and EpEX were analyzed at 0.5 h, 1 h, 2 h, 4 h, and 6 h after \u003cem\u003eE. acervulina \u003c/em\u003einfection without (A) or with (B) anti-EaMIC3 antibodies. The relative values of EpCAM/GAPDH and EpEX/GAPDH without (C and D) or with (E and F) antiserum treatment were calculated using ImageJ. Moreover, the numbers of sporozoites in the supernatant (G) and in cells (H) with antiserum treatment were counted. DF-1 cells without infection were used as blank control and DF-1 cells with infection of sporozoites incubated with healthy rabbit serum were used as negative control (NC). EpCAM, epithelial cell adhesion molecule; EaMIC3, \u003cem\u003eE. acervulina\u003c/em\u003e microneme protein 3; EpEX, an extracellular domain of EpCAM. **, \u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no significance.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/5bfde99c9c719f16621ba272.jpeg"},{"id":89820154,"identity":"2abad342-075d-4882-a78b-32600254fbd8","added_by":"auto","created_at":"2025-08-25 11:21:27","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":306646,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of EpCAM knockdown and overexpression on the infection of sporozoites\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDF-1 cells infected with sporozoites were detected by immunofluorescence assay (A) and the numbers of infected cells (B) and sporozoites in the culture supernatant (C) were counted in the context of EpCAM knockdown using specific siRNA E872. Moreover, the number of cells infected by sporozoites was also detected after EpCAM overexpression (D). The relative values of EpCAM/GAPDH and EpEX/GAPDH at 4 h, 10 h, and 20 h after transfection of E872 were also evaluated using ImageJ (E). Sporozoite-infected DF-1 cells without and with irrelevant siRNA (NC) were used as blank/mock control and NC control, respectively. EpCAM, epithelial cell adhesion molecule; EaMIC3, \u003cem\u003eE. acervulina\u003c/em\u003emicroneme protein 3; EpEX, an extracellular domain of EpCAM. ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no significance.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/725f2ea8baab3ac3a6a23b60.jpeg"},{"id":89819889,"identity":"d8173010-2e9d-45e4-8b9b-d8e9f49eb9dc","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":515207,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of EpCAM knockdown on apoptosis induced by sporozoite infection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe apoptosis rates of sporozoite-infected DF-1 cells were detected by Annexin V/PI assay (A) and further statistically analyzed (B). DF-1 cells without infection were used as control. NC, irrelevant siRNA; E872, specific siRNA against EpCAM; Act D, actinomycin D. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/5ba37aa24ff04faa1689eb8a.jpeg"},{"id":89819883,"identity":"365db580-ba88-45d3-bdce-e547dbaa96d3","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":201206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of EpCAM knockdown on the activation of the EGFR/Akt pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInteraction between EpEX and EGFR\u003csub\u003e638-1050aa\u003c/sub\u003e was identified by co-immunoprecipitation and immunofluorescence (A). The expression levels of EGFR, Akt and p-Akt in DF-1 cells transfected with irrelevant siRNA (mock) or E872 were analyzed using Western blotting (B) and the relative values of EGFR/GAPDH (C), p-Akt/GAPDH (D), and p-Akt/Akt (E) were analyzed using ImageJ. EpEX, an extracellular domain of EpCAM; E872, specific siRNA against EpCAM; EGFR, epidermal growth factor receptor. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/992dff2b38a9ea77e7e187f1.jpeg"},{"id":89819891,"identity":"93f6ac09-efe0-490c-8559-0b835e4c36f3","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":238114,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of anti-EaMIC3 and anti-EpEX antibodies on the activation of EGFR/Akt/mTOR pathway and apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe expression levels of EGFR, Akt, p-Akt, mTOR, and p-mTOR in infected DF-1 cells treated with anti-EaMIC3 or anti-EpEX antibodies were evaluated using Western blotting (A). Then the relative values of EGFR/GAPDH (B), p-Akt/Akt (C), and p-mTOR/mTOR (D) were analyzed using ImageJ. Moreover, the apoptosis rates of treated cells were detected by Annexin V/PI assay and statistically analyzed (E). DF-1 cells without infection were used as negative control (NC). EpEX, an extracellular domain of EpCAM; EaMIC3, \u003cem\u003eE. acervulina\u003c/em\u003e microneme protein 3; EGFR, epidermal growth factor receptor. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no significance.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/530ce200a440d1e4a7bc8090.jpeg"},{"id":89819887,"identity":"4ff0b24f-ca5e-4b1c-8650-bd1b14f65a4c","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":213091,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of immunization with EaMIC3 on the activation of EGFR/Akt/mTOR pathway and apoptosis in chickens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter immunization with EaMIC3, each chicken was challenged with 5,000 oocysts (A) and all the chickens were slaughtered to check lesions in the duodenum (B). The early (C) and late (D) apoptosis rates of duodenal epithelial cells were evaluated. Moreover, the expression levels of p-Akt, Akt, p-mTOR and mTOR were analyzed at 6 h, 8 h, and 24 h after challenge \u0026nbsp;by Western blotting (E). The relative values of p-Akt/Akt (F) and p-mTOR/mTOR (G) were calculated using ImageJ. GST protein was used as control. EaMIC3, \u003cem\u003eE. acervulina\u003c/em\u003e microneme protein 3. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no significance.\u003c/p\u003e","description":"","filename":"floatimage8.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/45b9a75f7f4aee0e8f9ae9d5.jpeg"},{"id":89819898,"identity":"8d56c136-f1a5-4cec-96b3-65c956ec932f","added_by":"auto","created_at":"2025-08-25 11:13:27","extension":"jpeg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":264760,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic effects of anti-EaMIC3/EpCAM antibodies on \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. acervulina \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003einfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing challenge with 2,000 oocysts, chickens were administrated with anti-EaMIC3 or/and -EpCAM antibodies for 6 consecutive days (A) and intestinal bleeding was checked (B). The weights (C), intestinal lesion scores (D), oocyst reduction rates (E), and oocyst excretion per gram of feces (F) among different groups were measured. EpCAM, epithelial cell adhesion molecule; EaMIC3, \u003cem\u003eE. acervulina\u003c/em\u003e microneme protein 3. **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001; ns, no significance.\u003c/p\u003e","description":"","filename":"floatimage9.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/202ca251c93cde68178de106.jpeg"},{"id":89821092,"identity":"370de137-3927-43fb-8a15-454f512e64b1","added_by":"auto","created_at":"2025-08-25 11:37:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3521898,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/23c465c0-38e5-4a5f-813b-a33239a4b822.pdf"},{"id":89820155,"identity":"ec4af8f9-14fd-45e2-8a5e-0a9d33c4a2b7","added_by":"auto","created_at":"2025-08-25 11:21:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":385544,"visible":true,"origin":"","legend":"EaMIC3 inhibits cell apoptosis and facilitates Eimeria acervulina infection through interacting with EpCAM","description":"","filename":"Supplementaryfile.docx","url":"https://assets-eu.researchsquare.com/files/rs-7302653/v1/239644fe2f36eb0c89813f5f.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"EaMIC3 inhibits cell apoptosis and facilitates Eimeria acervulina infection through interacting with EpCAM","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCoccidiosis in chickens leads to considerable economic losses worldwide, amounting to approximately \u003cspan\u003e$\u003c/span\u003e13.2\u0026nbsp;billion annually [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. At present, the control of coccidiosis primarily relies on drugs and attenuated live vaccines. However, their application is limited due to drug resistance and the pathogenicity of live vaccines. The lack of effective control measures for coccidiosis is largely attributed to a poor understanding of the pathogenesis of \u003cem\u003eEimeria\u003c/em\u003e species.\u003c/p\u003e\u003cp\u003eDuring the life cycle, when ingested by chickens, sporulated oocysts release sporozoites under the action of gastric acid and duodenal peristalsis [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. These sporozoites invade the epithelial cells of the chicken duodenum and transform into schizonts, which subsequently undergo multiple fission to form merozoites [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Once matured, the merozoites are released from the cells and proceed to infect additional cells, leading to extensive duodenum infection. One of the main mechanisms by which \u003cem\u003eEimeria\u003c/em\u003e species mitigate host cell damage is the inhibition of apoptosis, thereby facilitating their own growth, development and reproduction [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]; however, the precise mechanisms are not yet fully understood.\u003c/p\u003e\u003cp\u003eMicrotubules are small secretory organelles that accumulate at the apex of Apicomplexan parasites and are mainly present in gliding or invading protozoa [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. They secrete multiple microneme (MIC) proteins, and MIC2, MIC3, and MIC5 have so far been identified in \u003cem\u003eEimeria acervulina\u003c/em\u003e [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eE. acervulina\u003c/em\u003e MIC3 (EaMIC3) serves as a key virulence factor and is characterized seven adhesive repeat motifs that are expressed in sporozoites and merozoites. At the early stage of infection, EaMIC3 is situated at the apex of the complex and specifically binds to host cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Therefore, it not only mediates the invasion of sporozoites into host cells, but also determines the specificity of binding. Moreover, it has been reported that the epidermal growth factor (EGF) motifs of \u003cem\u003eToxoplasma gondii\u003c/em\u003e MIC3 and MIC6 interact with the EGF receptor (EGFR) of host cells, thereby inhibiting cell apoptosis by activating the EGFR/Akt signaling pathway and ultimately promoting the survival of \u003cem\u003eT. gondii\u003c/em\u003e in host cells [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Similarly, \u003cem\u003eNeosporidium\u003c/em\u003e MIC3 is also capable of activating the EGFR signaling, thereby preventing host cell apoptosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Though EaMIC3 lacks EGF motifs, it has been shown to inhibit early apoptosis of infected host cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], yet the precise mechanism behind remains unclear.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eEpCAM interacts with EaMIC3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo determine the role of EaMIC3 in invasion, a yeast two-hybrid assay was used to identify the molecules that interact with EaMIC3. From initial screening, we identified 6 positive clones, of which only 3 were further confirmed to be true positives. BLAST analysis confirmed that 2 of these clones exhibited high sequence similarity to ubiquitin-conjugating enzyme E2F (UBE2F, NP_001006512.1) and epithelial cell adhesion molecule (EpCAM, NP_001012582.2), respectively, while the remaining clone could not be aligned to any known coding sequences (data not shown).\u003c/p\u003e\u003cp\u003eTo further investigate the interaction between EpCAM and EaMIC3, we first ruled out the possibilities of the toxicity and autoactivation activity of both the pGADT7-AD-EpCAM and pGBKT7-BD-EaMIC3 plasmids (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B). We then showed that the yeast cells co-transformed with the pGADT7-AD-EpCAM and pGBKT7-BD-EaMIC3 plasmids exhibited blue colonies on both the -Trp/-Leu/-x-α-gal and -Trp/-Leu/-His/-ADE/-x-α-gal plates (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eC). Furthermore, we provided additional evidence that EpCAM interacted with EaMIC3 in the immuno-precipitation assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and was also co-localized with EaMIC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Collectively, these findings suggest that EpCAM directly interacts with EaMIC3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEpEX domain and MARs 1, 2, and 5 are crucial to EpCAM-EaMIC3 interaction\u003c/b\u003e\u003c/p\u003e\u003cp\u003eEpCAM is a transmembrane glycoprotein located on the surface of epithelial cells, consisting of intracellular (EpICD), transmembrane, and extracellular domains (EpEX) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. To determine the domain involved in the interaction, we first assessed the binding capacity of different truncated forms of EpCAM. The results showed that EaMIC3 bound to and was also co-localized with EpEX rather than EpICD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD). It has been documented that EaMIC3 has 7 adhesive repeat regions 1\u0026ndash;7 (MAR 1\u0026ndash;7, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. We further defined the regions important to binding. The results indicated that EpEX interacted with MAR1, MAR2, and MAR5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB-H) and was also co-localized with these regions in the cytoplasm (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-K). These results indicate that the EpEX domain and MARs 1, 2, and 5 are key determinants of the interaction between EpCAM and EaMIC3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eSporozoite infection promotes EpCAM expression and EpEX cleavage by EaMIC3\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDue to the interaction between EaMIC3 and EpCAM, we next sought to determine whether EaMIC3 regulates the expression of EpCAM during parasite infection. The results revealed that the expression levels of EpCAM in the infected DF-1 cells and EpEX, an extracellular domain cleaved from EpCAM [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], in the cell culture supernatant, exhibited a significant increase in a time-dependent manner (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). However, the expression levels of both EpCAM and EpEX exhibited a significant reduction at 6 h after incubation with sporozoites and antibodies specifically against EaMIC3\u003csub\u003e(1\u0026minus;180aa; 485\u0026minus;576aa)\u003c/sub\u003e compared to the control (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F). In this scenario, the number of sporozoites present in the culture showed a significant increase (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG), while the number of sporozoites within the DF-1 cells had a substantial decrease (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eH). These results indicate that sporozoite infection promotes EpCAM expression and EpEX cleavage, possibly induced by EaMIC3.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEpCAM accelerates sporozoite infection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs EpCAM was found to be upregulated at the early stage of the infection, we postulated that EpCAM, as a receptor, plays a role in sporozoite infection process. To test this, we assessed the infectivity of sporozoites to DF-1 cells with EpCAM knockdown, which was achieved through the delivery of siRNA E872 (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig. S2). The findings showed that, in comparison to the control group, EpCAM knockdown led to a notable decrease in the number of sporozoite-infected cells but a significant increase in the number of sporozoites present in the culture supernatant (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). Conversely, EpCAM overexpression resulted in a significant increase in the number of sporozoite-infected cells compared to the control (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). Consistently, the expression levels of EpCAM in the infected DF-1 cells as well as EpEX in the culture supernatant were significantly decreased in the E872 group in comparison to the mock group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). These results imply that EpCAM promotes the infection of sporozoites into DF-1 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEpEX is involved in EaMIC3-induced apoptosis inhibition via the EGFR/Akt/mTOR signaling pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs EaMIC3 inhibits early apoptosis of infected host cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], we further tested whether EpCAM is directly involved in this process. The results showed that the levels of cell apoptosis were significantly elevated in the E872\u0026thinsp;+\u0026thinsp;Infection group compared to the NC\u0026thinsp;+\u0026thinsp;Infection and Infection groups at 4 h, 6 h, and 8 h after infection (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Moreover, the apoptosis levels were also significantly higher in the E872\u0026thinsp;+\u0026thinsp;Infection\u0026thinsp;+\u0026thinsp;Act-D group than those in the NC\u0026thinsp;+\u0026thinsp;Infection\u0026thinsp;+\u0026thinsp;Act-D and Infection\u0026thinsp;+\u0026thinsp;Act-D groups at 4 h, 6 h, and 8 h after infection (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). These results indicate that EaMIC3 inhibits early apoptosis of infected host cells by promoting EpCAM expression.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIt has been reported that the N-linked glycosylation of EpCAM induces its cleavage, resulting in the formation of EpEX that can activate the EGFR pathway in tumors [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Meanwhile, several protozoa are known to activate the EGFR/AKT signaling pathway during infection [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and the infection of \u003cem\u003eE. acervulina\u003c/em\u003e sporozoite has been shown to induce EpEX cleavage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Therefore, we hypothesized that EpEX may act as a suppressor of apoptosis through the EGFR/AKT signaling pathway during the infection. The results showed that EpEX directly bound to and was co-localized with the extracellular region (638-1050aa) of EGFR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Additionally, the EGFR level showed a continuous and significant increase post infection; however, it was markedly lower in the E872\u0026thinsp;+\u0026thinsp;Infection group compared to the infection group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). Furthermore, the levels of both p-Akt and p-Akt/Akt were significantly lower in the E872\u0026thinsp;+\u0026thinsp;Infection group than those in the infection group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further confirm the aforementioned findings, we assessed the levels of Akt, p-Akt, mTOR, and p-mTOR in infected DF-1 cells with or without incubation with EaMIC3\u003csub\u003e(1\u0026minus;180aa; 485\u0026minus;576aa)\u003c/sub\u003e and EpCAM\u003csub\u003e(20\u0026minus;260aa)\u003c/sub\u003e antibodies. After incubation, the levels of p-mTOR/mTOR, and p-PI3K/Akt were significantly lower in the anti-EpCAM\u0026thinsp;+\u0026thinsp;Infection and anti-EaMIC3\u0026thinsp;+\u0026thinsp;Infection groups than those in the Infection group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Meanwhile, the levels of cell apoptosis were markedly increased in the anti-EaMIC3\u0026thinsp;+\u0026thinsp;Infection and anti-EpCAM\u0026thinsp;+\u0026thinsp;Infection groups relative to the Infection group (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). These findings indicate that sporozoite infection inhibits early apoptosis of infected host cells by EpEX via the EGFR/AKT/mTOR signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEaMIC3 is associated with duodenal epithelial cell apoptosis inhibition through the Akt/mTOR pathway during sporozoite infection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo evaluate the effects of EaMIC3 on the apoptosis of duodenal epithelial cells during the infection, we accessed the apoptosis rate and the expression of apoptosis-associated proteins in challenged chicken immunized with EaMIC3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). There were no obvious lesions in the duodenum in all the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). However, both early and late apoptosis rates in the EaMIC3\u0026thinsp;+\u0026thinsp;Infection group was significantly higher than that in the Infection group but lower than those in the blank control (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Additionally, the ratio of p-Akt/Akt was elevated at 6 h but decreased at 8 h and 24 h in the EaMIC3\u0026thinsp;+\u0026thinsp;Infection group compared to the Infection and GST\u0026thinsp;+\u0026thinsp;Infection groups (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). Similarly, the ratio of p-mTOR/mTOR was significantly decreased at 8 h in the EaMIC3\u0026thinsp;+\u0026thinsp;Infection group compared to the Infection and GST\u0026thinsp;+\u0026thinsp;Infection groups (\u003cem\u003eP\u003c/em\u003e\u0026lt;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG). Collectively, these findings suggest that EaMIC3 is involved in the inhibition of duodenal epithelial cell apoptosis induced by \u003cem\u003eE. acervulina\u003c/em\u003e infection through the Akt/mTOR signaling pathway.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEaMIC3/EpCAM antisera confer a good therapeutic effect on\u003c/b\u003e \u003cb\u003eE. acervulina\u003c/b\u003e \u003cb\u003einfection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eGiven the significant role of EaMIC3-EpCAM interaction in \u003cem\u003eE. acervulina\u003c/em\u003e infection, we then evaluated the therapeutic effect of EaMIC3/EpCAM antisera (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The results showed that the duodenal intestinal bleeding was markedly alleviated in the EaMIC3\u0026thinsp;+\u0026thinsp;Infection, EpCAM\u0026thinsp;+\u0026thinsp;Infection, EaMIC3/EpCAM\u0026thinsp;+\u0026thinsp;Infection groups in comparison to the Infection group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB). Consistently, all the antiserum-treated groups exhibited significantly higher weight gain compared to the Infection group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC). Similarly, all the treated groups also exhibited significantly lower intestinal lesion scores (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eD). The highest oocyst reduction rate was 67.86% in the EaMIC3/EpCAM group, followed by 61.68% in the EaMIC3 group and 46.85% in the EpCAM group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eE). Moreover, all the treated groups showed a statistically significant decrease in oocyst shedding compared to the Infection group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eF). These results indicate that EaMIC3/EpCAM antisera confer a good therapeutic effect on \u003cem\u003eE. acervulina\u003c/em\u003e infection.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eEpCAM is involved in various cellular processes, such as cell apoptosis, cell signaling, differentiation, proliferation, invasion, and migration [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In this study, we found that EpCAM low expression led to a significant reduction in the invasion and intracellular proliferation rates of \u003cem\u003eE. acervulina\u003c/em\u003e, suggesting a key role during the infection. Meanwhile, we demonstrated that the extracellular domain of EpCAM, EpEX, interacted with the MARs 1, 2, and 5 of EaMIC3, suggesting that the interactions occur through multiple sites. Consistently, after being incubated with EaMIC3\u003csub\u003e(1\u0026minus;180aa; 485\u0026minus;576aa)\u003c/sub\u003e antibodies, sporozoites exhibited a dramatically decrease in both the expression level of EpCAM and the infection rate. These results suggest that EaMIC3 promotes the infection of \u003cem\u003eE. acervulina\u003c/em\u003e by interacting with EpCAM. However, the sites specific for the EaMIC3-EpCAM interaction remain unclear and need to be further explored.\u003c/p\u003e\u003cp\u003eIt was demonstrated that EpEX was involved in the apoptosis-related signaling pathways in human cancer cells [\u003cspan additionalcitationids=\"CR22 CR23 CR24 CR25\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previously, EaMIC3 was also found to inhibit host cell apoptosis during the initial phase of infection [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Consistent with these observations, the cell apoptosis rate was significantly increased during \u003cem\u003eE. acervulina\u003c/em\u003e infection following the knockdown of EpCAM, suggesting that EpCAM negatively regulates the inhibition of host cell apoptosis during the parasite infection.\u003c/p\u003e\u003cp\u003eParasitic protozoa maintain their intracellular survival by inhibiting host cell apoptosis through the PI3K/Akt and NF-κB signaling pathway at the early stages of infection. For instance, \u003cem\u003eT. gondii\u003c/em\u003e inhibits apoptosis of T cells and macrophages through the PI3K/Akt signaling pathway [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], whereas \u003cem\u003eTheileria, Cryptosporidium\u003c/em\u003e, and \u003cem\u003eNeosporidium\u003c/em\u003e species inhibit host cell apoptosis by activating the NF-κB signaling pathway [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Moreover, it has been reported that \u003cem\u003eE. tenella\u003c/em\u003e suppresses the apoptosis of cecal epithelial cells by activating the PI3K/Akt pathway [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The PI3K/Akt and NF-κB signaling pathways are closely associated with the functions of EGFR [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the current study, we found that EpEX interacted with EGFR and the expression levels of EGFR, p-Akt, and p-mTOR were significantly decreased during the infection in the context of EpCAM low expression. These findings indicate that EpEX activates the EGFR/Akt/mTOR signaling pathway and inhibits cell apoptosis by interacting with EGFR during the infection. It was further shown that both the levels of EpEX in the culture supernatant and EpCAM in infected cells were significantly higher in the Infection group than those in the control. In contrast, following the incubation of sporozoites with EaMIC3\u003csub\u003e(1\u0026minus;180aa; 485\u0026minus;576aa)\u003c/sub\u003e antibodies, there was a notable reduction in the levels of both EpEX and EpCAM during the infection process. These results suggest that EaMIC3 enhances the expression of EpCAM and facilitates its cleavage. Consistently, the levels of EGFR, p-Akt, and p-mTOR were markedly decreased and the rates of apoptosis were significantly increased following incubation with EpCAM antibodies, particularly EaMIC3 antibodies.\u003c/p\u003e\u003cp\u003eIn summary, this study reveals the underlying mechanism by which EaMIC3 inhibits apoptosis of host cells and facilitates \u003cem\u003eE. acervulina\u003c/em\u003e infection through interacting with EpCAM. The findings obtained herein offer valuable insights for the development of drugs or vaccines for the control of \u003cem\u003eE. acervulina\u003c/em\u003e infection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003cp\u003eThe animal experiments in the study were approved by the Animal Care and Use Committee of Zhejiang A \u0026amp; F University. The protocols were performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals approved by the State Council of the People\u0026rsquo;s Republic of China.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eParasites\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eE. acervulina\u003c/em\u003e, Jilin strain, was passaged and maintained in our lab. Sporozoites from \u003cem\u003eE. acervulina\u003c/em\u003e oocysts were purified using DE-52 anion-exchange columns as previously described [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eCells and Yeast\u003c/b\u003e\u003c/p\u003e\u003cp\u003e293T and DF-1 cells (Fenghui Biotechnology, China) were cultured in Dulbecco's modified eagle medium (DMEM) medium containing 10% fetal bovine serum (FBS) at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The chicken duodenal epithelial cells were isolated and collected from 18-day-old SPF chick embryos (Merial Vital Corp, China) as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The yeast strain Y2HGold (Clontech, USA) was cultured in YPDA medium at 30\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cb\u003eYeast two-hybrid test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFor yeast two-hybrid screening, the fragment of the EaMIC3 gene (1-2607 bp, AMN15064.1), which encodes seven repeating domains, was cloned into pGBKT7 and the positive plasmid, designated as pGBKT7-EaMIC3, was used as bait. Using a library construction and screening kit (Clontech, USA), an \u003cem\u003eE. acervulina\u003c/em\u003e cDNA library was constructed using the pGADT7 vector and used as prey. The yeast strain Y187 was transformed with the bait plasmid pGBKT7-EaMIC3, and an α-galactosidase assay was performed to examine autoactivation. The yeast strain Y187 was transformed with the prey plasmid, which contained a GAL4 activation domain. For interaction mating, the library and bait strains were co-cultured at 30\u0026deg;C for 20 h. After mating, the culture was plated on selection medium lacking histidine, leucine, tryptophan, and adenine but containing 20 \u0026micro;g/mL X-α-gal (SD/-Ade/-His/-Leu/-Trp/X-α-gal). The resultant blue colonies were selected for further analysis. The cells transfected with the pGBKT7-53 and pGADT7-T plasmids were used as a positive control, while the cells transfected with the pGBKT7-Lam and pGADT7-T plasmids were used as a negative control. To further confirm the hits from the above screening, the positive prey plasmid pGADT7-EpCAM was co-transformed into the Y187 strain with the bait plasmid pGBKT7-EaMIC3 and plated on the selection medium.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCo-immunoprecipitation and Western blotting\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe recombinant plasmids pCMV-myc-EaMIC3, pCMV-myc-EGFR, and pCMV-HA-EpCAM were constructed respectively as previously reported [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. These recombinant plasmids were then extracted and co-transfected into 293T cells using the FuGENE HD transfection reagent (Roche, Switzerland). After 48 h, the transfected cells were harvested and then lysed with precooled lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 1% NP-40, 5% glycerol and a protease inhibitor cocktail (Roche, Switzerland)) for 30 min on ice, followed by incubation with protein A/G-conjugated Sepharose beads (Millipore, USA) at 4\u0026deg;C for 30 min. The supernatants were incubated with rabbit polyclonal antibodies against the Myc tag (Cell Signaling, USA) at 4\u0026deg;C overnight, followed by mixing with protein A/G-conjugated Sepharose beads at 4\u0026deg;C for 2 h. The mixture was centrifuged at 8,000 \u0026times; \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 5 s, followed by rinse with wash buffer (50 mM Tris-HCl, pH 8.0, 500 mM NaCl, 5 mM EDTA, and 1% NP40) three times. The bound proteins were eluted with elution buffer (0.1 M glycine, pH 2.8), neutralized with neutralization buffer (1 M Tris-HCl, pH 9.5) and separated using 12% SDS-PAGE.\u003c/p\u003e\u003cp\u003eWestern blotting was performed to identify bound proteins using rabbit polyclonal antibodies against HA (Cell Signaling, USA) at 1: 1,000 and a mouse monoclonal antibody against Myc (Cell Signaling, USA) at 1: 1,000. Goat anti-rabbit IgG (1:3,000, TransGen Biotech, China) and goat anti-mouse IgG (1: 3,000, TransGen Biotech, China) HRP-conjugated antibodies were employed and then the results were detected by ECL (enhanced luminol-based chemiluminescent) Western blotting substrate kit (Bio-Rad, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eConfocal immunofluorescence\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe pCMV-myc-EaMIC3 and pCMV-HA-EpCAM plasmids were co-transfected into 293T cells, and then the cells were fixed in 4% paraformaldehyde for 15 min at 25\u0026deg;C. The cells were dealt with 10 mM PBS containing 1% Triton X-100 at 37\u0026deg;C for 15 min, suspended in 10 mM PBS containing 5% BSA and then incubated at 37\u0026deg;C for 1 h. Immunofluorescence was performed using rabbit polyclonal antibodies against HA and Myc (Cell Signaling, USA) at 1:100. Goat anti-rabbit IgG (1:3,000, TransGen Biotech, China) HRP-conjugated antibodies were then employed. DAPI staining solution was added and incubated for 10 min, followed by observation under a confocal microscope (Olympus, Japan).\u003c/p\u003e\u003cp\u003e\u003cb\u003eAntibody blocking test\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSterile purified sporozoites were co-incubated with EaMIC3\u003csub\u003e(1\u0026minus;180aa; 485\u0026minus;576aa)\u003c/sub\u003e antibodies and rabbit negative serum (NC), respectively, as previously described [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. DF-1 cells were cultivated until their density reached at 80% \u0026minus;\u0026thinsp;90%, followed by the addition of the purified sporozoites at a ratio of 2:1 (sporozoites: cells) and incubation for 1 h. The expression levels of EpCAM in the cells and EpEX in the culture supernatant were detected, and the number of sporozoites in the cells and the supernatant was determined, respectively.\u003c/p\u003e\u003cp\u003e\u003cb\u003eRNA interference\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSmall interfering RNAs (siRNAs) targeting the \u003cem\u003eepcam\u003c/em\u003e gene were designed and synthesized (OBIO, China). 2.2 pmol of EpCAM siRNAs were added into DF-1 cells (40\u0026ndash;60% confluence) in 200 \u0026micro;L of DMEM medium without FBS, followed by the addition of 12 \u0026micro;L transfection reagent (Roche, Switzerland) and cultivation for 4 h. After the supernatant was removed, the cells were further cultured in DMEM medium with 15% FBS for 72 h as previously described and then the expression level of EpCAM was detected [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cb\u003eApoptosis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eDF-1 cells were plated in 96-well plates at a density of 1.0\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well. After 48 h, the culture medium was then replaced with fresh medium supplemented with 20\u0026micro;M Act D, and the cells were further incubated for 4 h. Then the cells were harvested and analyzed using the Annexin V-FITC apoptosis detection kit (KeyGEN, China) as previously described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In brief, 1 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells were resuspended in 500 \u0026micro;L of binding buffer and then labeled with Annexin V-FITC and propidium iodide (PI) for 15 min in the dark at room temperature. The cells were examined by flow cytometry (BD Biosciences, USA).\u003c/p\u003e\u003cp\u003e\u003cb\u003eApoptosis of the duodenum in EaMIC3-immunized chickens\u003c/b\u003e\u003c/p\u003e\u003cp\u003e A total of 20 one-day-old broiler chickens were obtained from a local hatchery (Hangzhou Xiaoshan Donghai Breeding, China) and raised in a temperature-controlled closed-house environment. The clean water and formulated feed were used for raising chickens. At 7 days of age, chickens were randomly divided into the 4 groups with 5 chickens of each group: Control group, \u003cem\u003eE. acervulina\u003c/em\u003e infection group, \u003cem\u003eE. acervulina\u003c/em\u003e infection group with immunization of EaMIC3, and \u003cem\u003eE. acervulina\u003c/em\u003e infection group with immunization of GST. At 7 d up to 21 d, EaMIC3 was vaccinated (25 \u0026micro;g/chicken) to the chickens by intramuscular injection. At 28 d, all the groups except the control were orally challenged with freshly sporulated \u003cem\u003eE. acervulina\u003c/em\u003e oocysts (5,000/chicken). All chickens were humanely executed at 8 h after infection, and the apoptosis rate and the expression of the apoptotic factors in the duodenum were analyzed.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTherapeutic efficacy of anti-EaMIC3/EpCAM sera\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBoth EaMIC3 and EpCAM rabbit antisera were prepared as previously reported [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Twenty-eight-day-old chickens with a similar body weight were randomly divided into 5 groups: no infection group (negative control), infection group (positive control), EaMIC3 antiserum-treated group, EpCAM antiserum-treated group, and mixed antisera-treated (EaMIC3\u0026thinsp;+\u0026thinsp;EpCAM) group. Except no infection group, all the groups were orally challenged with 2\u0026times;10\u003csup\u003e4\u003c/sup\u003e oocysts and then each chicken was administered with 0.2 mL corresponding antiserum for 7 consecutive days. The therapeutic efficacy was evaluated via weight gain, oocysts per gram (OPG) of feces, and intestinal lesion scores.\u003c/p\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eGraphPad Prism was used to conduct statistical analyses. Unpaired Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test and ANOVA were performed to compare the difference between two groups and three or more groups, respectively, with a \u003cem\u003eP\u003c/em\u003e value less than 0.05 to be considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting interests\u003c/h2\u003e\u003cp\u003eThe authors have declared that no competing interest exists.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe study was financially supported by the National Key Research and Development Program of China (2023YFD1801000), Zhejiang Provincial Natural Science Foundation of China (LZ24C180001), the Base and Talent Project of the Department of Science and Technology of the Tibet Autonomous Region (XZ202401JD0012), the NHC Key Laboratory of Echinococcosis Prevention and Control (2022WZK1004), and Key R\u0026amp;D and Conversion Plan of Qinghai Province (2025-QY-227).\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eAll study data are included in the article and supporting information.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eTrujillo-Peralta, C., Ashcraft, A., Senas-Cuesta, R., Coles, M., Hernandez-Velasco, X., Selby, C., Forga, A., Tellez-Isaias, G., Vuong, C., Bielke, L., et al.: Research Note: Isolation, speciation, and anticoccidial sensitivity of Eimeria spp. recovered from wild turkey feces in the United States. 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J. phytotherapy phytopharmacology. \u003cb\u003e116\u003c/b\u003e, 154860 (2023)\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Eimeria acervulina, chicken, EaMIC3, EpCAM, apoptosis, infection, EGFR/Akt/mTOR pathway","lastPublishedDoi":"10.21203/rs.3.rs-7302653/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7302653/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eCoccidiosis caused by \u003cem\u003eEimeria\u003c/em\u003e species is a deadly parasitic disease particularly affecting chicks, leading to huge economic losses to the global poultry industry. The mechanisms underlying parasite infection remain poorly understood.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eHere, we showed that \u003cem\u003eEimeria acervulina\u003c/em\u003e microneme protein 3 (EaMIC3) was involved in the apoptosis of infected host cells and parasite infection by interacting with epithelial cell adhesion molecule (EpCAM). The interaction was mediated by the binding of three microneme adhesive repeat regions (MAR) 1, 2, and 5 in EaMIC3 to EpEX, an extracellular domain cleaved from EpCAM. During infection, EaMIC3 regulated the expression of both EpCAM and EpEX, with EpEX in turn inhibiting apoptosis of infected duodenal epithelial cells through the EGFR/Akt/mTOR signaling pathway. Moreover, EpCAM knockdown inhibited sporozoite infection, while EpCAM overexpression promoted sporozoite infection.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eConsistently, the administration of pooled EaMIC3 and EpCAM antisera conferred a good therapeutic effect on \u003cem\u003eE. acervulina\u003c/em\u003e infection in chickens, with a maximum oocyst reduction rate of 67.86%. Overall, these findings reveal a mechanism by which EaMIC3 inhibits cell apoptosis and facilitates \u003cem\u003eE. acervulina\u003c/em\u003e infection, shedding light on the prevention and control of coccidiosis in chickens.\u003c/p\u003e","manuscriptTitle":"EaMIC3 inhibits cell apoptosis and facilitates Eimeria acervulina infection through interacting with EpCAM","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-25 11:13:23","doi":"10.21203/rs.3.rs-7302653/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-biology","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commsbio","sideBox":"Learn more about [Communications Biology](http://www.nature.com/commsbio/)","snPcode":"","submissionUrl":"","title":"Communications Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cd523201-59b9-4052-8fbb-1c32941093dd","owner":[],"postedDate":"August 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53225108,"name":"Health sciences/Pathogenesis/Infection"},{"id":53225109,"name":"Scientific community and society/Agriculture"}],"tags":[],"updatedAt":"2026-05-01T09:20:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-25 11:13:23","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7302653","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7302653","identity":"rs-7302653","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}