Abstract
BackgroundThe family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can also be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Genome sequence analysis of Eimeria suggests that these proteins may also exist in this genus and led us to the assumption that these proteins may also play a role in oocyst wall formation.MethodsIn this study, COWP-like encoding sequences had been identified in Eimeria nieschulzi. The predicted gene sequences were subsequently utilized in reporter gene assays to observe time of expression and localisation of the reporter protein in vivo.ResultsBoth investigated proteins, EnOWP2 and EnOWP6, were expressed during sporulation. The EnOWP2-promoter driven mCherry was found in the cytoplasm and the EnOWP2, respectively EnOWP6, fused to mCherry was initially observed in the extracytoplasmatic space between sporoblast and oocyst wall. This, so far unnamed compartment was designated as circumplasm. Later, the mCherry reporter co-localised with the sporocyst wall of the sporulated oocysts. This observation had been confirmed by confocal microscopy, excystation experiments and IFA. Transcript analysis revealed the intron-exon structure of these genes and confirmed the expression of EnOWP2 and EnOWP6 during sporogony.ConclusionsOur results allow us to assume a role, of both investigated EnOWP proteins, in the sporocyst wall formation of E. nieschulzi. Data mining and sequence comparisons to T. gondii and other Eimeria species allow us to hypothesise a conserved process within the coccidia. A role in oocyst wall formation had not been observed in E. nieschulzi.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-015-0982-3) contains supplementary material, which is available to authorized users.
Highlights
The family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well
The identified, putative protein sequences were again analyzed by TBLASTN against predicted proteins in Eimeria species at GenBank and ToxoDB ([13] as well as in transcripts deposited at Eimeria Transcript database [14, 15])
Based on our data we suggest that COWP2 and COWP6 are expressed during sporulation and are exported and deposited in the extracytoplasmatic space between cytoplasm and oocyst wall
Summary
The family of cysteine rich proteins of the oocyst wall (COWPs) originally described in Cryptosporidium can be found in Toxoplasma gondii (TgOWPs) localised to the oocyst wall as well. Antigens of one or both wall forming bodies (WFB) were characterized by different studies [2,3,4], but only the amino acid composition of proteins of WFBII, and thereby of the inner oocyst wall, could be elucidated and are known as GAM-proteins. It can be assumed that these COWP, respectively TgOWP homologous proteins from Eimeria, play a role in the wall formation process of the oocyst and/or sporocyst wall in Eimeria. To test this hypothesis we utilized the parasite Eimeria nieschulzi and the transfection technology to follow the expression and dynamics of two COWP-like proteins in E. nieschulzi (EnOWP) via reporter gene assays. This is different to Toxoplasma gondii, where propagation and genetic modification of asexual stages in vitro is common, but experiments with cats, the definitive host of T. gondii, are subject to high requirements
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