Functional characterization of a surface antigen suppressing ApiAP2 in Eimeria tenella

Apicomplexa parasite Eimeria is the causative agent of coccidiosis, which leads to enteritis in animals and causes huge economic burden to the farm industry. The Apicomplexan Apetala2/ERF (ApiAP2) transcription factors have proved to play key roles in various processes in other Apicomplexa parasites. However, little is known about the function of ApiAP2s in Eimeria species. In this study, we functionally characterized an ApiAP2 by direct knockout mediated by CRISPR-Cas9. Our results showed that the invasion efficiency, total oocyst output and virulence to the host were significantly impaired after EtAP2-S1 depletion. RNA-Seq and CUT&Tag analyze showed that EtAP2-S1 targets on the promoters of numerous genes and its knockout results in upregulation of 59 sag genes. Additionally, the knockout strain exhibits significantly reduced virulence but provides excellent immune protection, which makes it a good vaccine candidate. This study demonstrates that EtAP2-S1 is a fitness coffering gene that suppress the expression of sag genes in E. tenella, and this is the first practice in developing gene knockout vaccine for the control of coccidiosis. Total RNAs from the sporozoites of EtHCYA and AP2-S1-KO strains were isolated for RNA-SEQ. Three biological replicates were employed. To further characterize the direct target genes of AP2-S1, CUT&Tag experiments were performed using the AP2-S1 overexpression strain.