Abstract
Chicken coccidiosis inflicts severe economic losses on the poultry industry. The mechanisms underlying Eimeria tenella (E. tenella) invasion, colonization, and damage to host cells remain incompletely elucidated. This study was designed to elucidate EtMIC2-interacting proteins and the mechanism that EtMIC2 regulates E. tenella invasion and host cell apoptosis. His pull-down-MS and KEGG enrichment results showed apoptosis-related proteins including ITGAV (Integrin alpha-V), PlexinB2, MAP2K6, GNA11, TRK, CASP7, PAK1, and RAP1b. Only the interaction model between EtMIC2 and ITGAV met the AlphaFold2 thresholds (pLDDT > 70, pTM+ipTM > 0.75). The CO-IP results showed that in the primary host cells (CECs) of E. tenella, protein bands corresponding to EtMIC2 (50 kDa) and ITGAV (135 kDa) were successfully detected in the IP group. Blue colony growth on SD/-Ade,-His,-Leu,-Trp/X-α-Gal plates for the group pGBKT7-EtMIC2 or pGBKT7-EtMIC2-3 (153-1029 bp) or pGBKT7-EtMIC2-5 (855-1029 bp) co-transformed with pGADT7-ITGAV in Yeast Two-Hybrid assay. Green fluorescence was observed in DF-1 cells pBiFC-EtMIC2-VN173 or pBiFC-EtMIC2-3-VN173 or pBiFC-EtMIC2-5-VN173 were co-transfected with pBiFC-ITGAV-VC155 in Bimolecular fluorescence complementation. The addition of EtMIC2 protein significantly increased (P < 0.05) E. tenella infection rate, gene expression and protein activity of ITGAV, FAK, PLC, PKC, p65, PI3K, Akt, ERK, and Bcl2, and reduced host cell Caspase 3 activity, apoptosis rate, gene expression and protein activity of JNK, p38, Bax, and Caspase 3. Knockdown of ITGAV produced the opposite effects. The function of EtMIC2 was prevented following the knockdown of ITGAV. The above finding indicate that the EtMIC2 855-1029 bp (285-342 aa) region interacts with ITGAV, leading to the upregulation of ITGAV, FAK, PLC, PKC, p65, PI3K, Akt, ERK, and Bcl2, while downregulating JNK, p38, Bax, and Caspase 3, thereby promoting E. tenella invasion and inhibiting host cell apoptosis.