Comparative transcriptome profiling of Eimeria tenella in various developmental stages and functional analysis of an ApiAP2 transcription factor exclusively expressed during sporogony

Abstract

Background The apicomplexan parasites Eimeria spp. are the causative agents of coccidiosis, a disease with a significant global impact on the poultry industry. The complex life cycle of Eimeria spp. involves exogenous (sporogony) and endogenous (schizogony and gametogony) stages. Unfortunately, the genetic regulation of these highly dynamic processes, particularly for genes involved in specific developmental phases, is not well understood. Methods In this study, we used RNA sequencing (RNA-Seq) analysis to identify expressed genes and differentially expressed genes (DEGs) at seven time points representing different developmental stages of Eimeria tenella. We then performed K-means clustering along with co-expression analysis to identify functionally enriched gene clusters. Additionally, we predicted apicomplexan AP2 transcription factors in E. tenella using bioinformatics methods. Finally, we generated overexpression and knockout strains of ETH2_0411800 to observe its impact on E. tenella development. Results In total, we identified 7329 genes that are expressed during various developmental stages, with 3342 genes exhibiting differential expression during development. Using K-means clustering along with co-expression analysis, we identified clusters functionally enriched for oocyte meiosis, cell cycle, and signaling pathway. Among the 53 predicted ApiAP2 transcription factors, ETH2_0411800 was found to be exclusively expressed during sporogony. The ETH2_0411800 overexpression and knockout strains did not exhibit significant differences in oocyst size or output compared to the parental strain, while the resulting ETH2_0411800 knockout parasite showed a relatively small oocyst output. Conclusions The findings of our research suggest that ETH2_0411800 is not essential for the growth and development of E. tenella. Our study provides insights into the gene expression dynamics and is a valuable resource for exploring the roles of transcription factor genes in regulating the development of Eimeria parasites. Graphical Abstract

Molecular characterization and immune protective efficacy of 3 Eimeria tenella antigens

“…Three antigens of E. tenella were selected because of their relatively high expression in intracellular stages (sporozoite, merozoite, and gametocyte), including a rhoptry protein (Eten5-B, ETH2_0801700), a surface antigen (Et-SAG14, ETH2_0630800) and phosphoglucomutase 1 (GPM1, ETH2_1540300). The transcription levels of these genes were obtained from the RNA-seq data of Chen et al (2023) , which showed that Eten5-B was highly expressed in sporozoites, whereas Et-SAG14 and Et-PGM1 were highly expressed in merozoites and gametocytes, respectively ( Figure 2 ).

Figure 2 Transcription levels of Et-SAG14, Et-PGM1, and Eten5-B.

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Figure 2 Transcription levels of Et-SAG14, Et-PGM1, and Eten5-B. The data were obtained from RNA-seq data of Chen et al (2023) . FPKM: fragments per kilobase of transcript per million mapped fragments; USO: unsporulated oocyst; SO (8 h): sporulating oocyst for 8 h; SO: sporulated oocyst; SP: sporozoite; MZ (108 h): merozoites isolated at 108 h postinfection; MZ (120 h): merozoites isolated at 120 h postinfection; Gam: gametocyte.

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“…The nucleotide sequences Eten5-B(ETH2_0801700), Et-SAG14(ETH2_0630800), and Et-PGM1(ETH2_1540300) were downloaded from ToxoDB ( https://toxodb.org/toxo/ ). Transcript level data were obtained from the RNA-seq data of Chen et al (2023) . Signaling peptides as well as transmembrane structural domains were predicted using SMART ( http://smart.embl-heidelberg.de/ ).…”

Localization in vivo and in vitro confirms EnApiAP2 protein encoded by ENH_00027130 as a nuclear protein in Eimeria necatrix

“…feature a DNA binding domain known as AT-hook, a zinc finger domain, an Acyl-CoA-N-acetyltransferase domain, as well as a pentapeptide-repeat-like domain. Additionally, several P. falciparum ApiAP2 proteins contain an ACDC domain, primarily located at the C-terminus, referred to as the AP2-coincident domain ( Jeninga et al., 2019 ; Chen et al., 2023 ). Furthermore, it is noteworthy that ApiAP2 proteins exhibit minimal homology beyond the AP2 domain, and even the paralogous AP2 domains found in Plasmodium spp.…”

“…Apicomplexan AP2 family of protein (ApiAP2) is a single validated class of apicomplexan TFs, initially identified in the genera Cryptosporidium , Plasmodium and Theileria ( Balaji et al., 2005 ), and have been subsequently identified in all apicomplexan genomes analyzed to date ( Kim, 2018 ; Jeninga et al., 2019 ; Chen et al., 2023 ). In Eimeria , the number of genes containing AP2 domains was found to vary from 44 to 54 ( Reid et al., 2014 ).…”