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ORIGINAL RESEARCH article
Front. Vet. Sci.
Sec. Parasitology
Volume 11 - 2024 |
doi: 10.3389/fvets.2024.1445646
Molecular Characterization of cDNA Coding for 33.5 kDa and 41 kDa Oocyst and Sporocyst Proteins that are Differentially Regulated in Different Strains of Eimeria maxima
Provisionally accepted- 1 Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service (USDA), Beltsville, United States
- 2 Electron and Confocal Microscope Unit, Soybean Genomics and Improvement Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service (USDA), Beltsville, Maryland, United States
Eimeria maxima (APU1 and APU2) differ in virulence for chickens, due in part to the greater fecundity of the former. In a previous study, RNA-seq was used to identify a transcripts upregulated in E. maxima APU1 compared to E. maxima APU2. In this study, 2 of these upregulated genes (EMWEY 23530 and EMWEY 48910) were characterized by first confirming upregulation using quantitative RT-PCR. For both EMWEY 23530 and EMWEY 48910, RNA transcription was fairly consistent during sporulation. The extent of differential expression was about 2-fold log2 higher in APU-1 compared to APU-2 (peaking at 18 h for EMWEY 23530 and 0 h for EMWEY 48910). EMWEY 23530 and EMWEY 48910 cDNA were cloned and expressed as polyHis-fusion proteins in Escherichia coli. The observed size of recombinant EMWEY 23530 was 24 kDa; the observed size of recombinant EMWEY 48910 was 35 kDa, which are consistent with the predicted size based on the coding sequences. Immunostaining 2D gel blots of E. maxima APU1 and APU2 oocyst/sporocyst protein with antisera specific for EMWEY 23530 identified a 33.5 kDa protein with a pH 7.4 isoelectric point (Emax p33.5). Similar 2D gel blot analysis with EMWEY 48910 identified a 41 kDa protein with a pH 7.2 isoelectric point (Emax p41). The intensity of Emax p33.5 and Emax p41 was noticeably greater in oocyst/sporocyst proteins from E. maxima APU1 compared to E. maxima APU2. This was corroborated by ELISA wherein equal amounts of total E. maxima APU1 and APU2 protein were probed with serial dilutions of anti-rEmax p33.5 or anti-rEmax p41. Immunofluorescence (IFA) staining of permeabilized unsporulated E. maxima APU1 and APU2 oocysts revealed Emax p33.5 to be localized in one end of oocysts, while Emax p41 appeared on the surface of oocysts. After sporulation, the p33.5 and p41 antigens appeared loosely associated with sporocysts. Taken together, these data confirm excess expression of two proteins in the E. maxima strain characterized by greater fecundity and virulence, and may provide insight into basis for phenotypic differences among different E. maxima.
Keywords: Eimeria maxima, recombinant protein, 2D gel, immunofluorescence, Eimeria, RT-PCR, EMWEY 23530, EMWEY 48910
Received: 01 Jul 2024; Accepted: 26 Aug 2024.
Copyright: © 2024 Jenkins, Parker, Jansen, Papadopoulos and Tucker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence:
Mark Jenkins, Animal Parasitic Diseases Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service (USDA), Beltsville, United States
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