AUTHOR=Cai Weimin , Feng Qianqian , Wang Liyue , Su Shijie , Hou Zhaofeng , Liu Dandan , Kang Xilong , Xu Jinjun , Pan Zhiming , Tao Jianping 

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

JOURNAL=Frontiers in Cellular and Infection Microbiology

VOLUME=13

YEAR=2023

URL=https://www.frontiersin.org/journals/cellular-and-infection-microbiology/articles/10.3389/fcimb.2023.1305727

DOI=10.3389/fcimb.2023.1305727

ISSN=2235-2988

ABSTRACT=<sec><title>Introduction</title><p>Apicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in <italic>Eimeria</italic> spp. <italic>ENH_00027130</italic> sequence is predicted to encode a <italic>Eimeria necatrix</italic> ApiAP2 protein (EnApiAP2).</p></sec><sec><title>Methods</title><p>The cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in <italic>Escherichia coli</italic> BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in <italic>E. necatrix</italic>, respectively. Finally, the recombinant pEGFP-C1-ΔNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein.</p></sec><sec><title>Results</title><p>The En<italic>ApiAP2</italic> gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an α-helix and three antiparallel β-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6×His tag mAb and the serum of convalescent chickens after infection with <italic>E. necatrix</italic>, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of En<italic>ApiAP2</italic> was significantly higher in SZ than in MZ-2, third generation merozoites (MZ-3) and gametocytes (<italic>P</italic>&lt;0.01). EnApiAP2 protein was localized in the nuclei of SZ, MZ-2 and MZ-3 of <italic>E. necatrix</italic>. The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the ΔNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein.</p></sec><sec><title>Discussion</title><p>EnApiAP2 protein encoded by <italic>ENH_00027130</italic> sequence was localized in the nucleus of <italic>E. necatrix</italic> parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study.</p></sec>