Skip to main content
Fig. 1 | Parasites & Vectors

Fig. 1

From: Discovery of a tyrosine-rich sporocyst wall protein in Eimeria tenella

Fig. 1

Identification of a tyrosine-rich protein transcribed specifically in sporulated oocysts of Eimeria tenella. a An integrative search for E. tenella genes with increased expression during sporulation was carried out using the New Search option available at www.toxodb.org, following the path New Search > Search for Genes > Transcript Expression > RNA Seq Evidence. The data set “Life Cycle Stage Transcriptomes (Reid)” was selected for E. tenella strain Houghton, using the FC (fold-change) option. The search identified 17 protein-coding genes that are upregulated with a fold change of ≥8 in the sporulated oocyst sample compared with the maximum expression value recorded for any of the reference samples, including unsporulated oocyst, sporozoite and 2nd generation merozoite. b Brightfield and c autofluorescence (UV excitation wavelength = 385 nm) microscopy of a bleached unsporulated (Un. O; top right) and a bleached sporulated (Sp. O; bottom left) oocyst of E. tenella indicates the possible presence of dityrosine bonds in the inner wall (bleaching removes the outer wall) of both the unsporulated and sporulated oocyst (white arrow-heads) and in the sporocyst walls (white arrows), including the stieda bodies (yellow arrows). Microscopy was done on a Zeiss Axiovert 200 microscope equipped with the Apotome imaging system. Images were generated and analysed using the Axiovision Software (Carl Zeiss SA). d The identification of potential ETH_00000115 (NCBI Reference Sequence: XP_013233236.1) homologues was carried out using BLASTP on the non-redundant NCBI database or on www.toxodb.org and alignments generated using the CLUSTAL O (1.2.1) multiple sequence alignment tool at http://www.ebi.ac.uk/Tools/msa/clustalo/. Conventional BLASTP analysis revealed only a single, highly conserved protein (96 % identity with ETH_00000115) in Eimeria necatrix (ENH_00020450, NCBI Reference Sequence: XP_013439901.1); however, searching for the KY-rich sequence, YKCKKAKGKGKYYKK, uncovered a further putative homologue in Eimeria brunetti (EBH_0074250, GenBank Reference Sequence: CDJ54052.1; 48 % identity with ETH_00000115), the ED-rich region and extended C-terminal of which are interspersed with several poly-alanine (A) sequences. The putative signal peptides for the three proteins are underlined. Residues of aspartic acid are highlighted by , glutamic acid by , lysine by , tyrosine by and cysteine by . Amino acid residues that are conserved across all three species are indicated by *. e A graphic depiction of the predicted ETH_00000115 protein highlighting a lysine (K) and tyrosine (Y)-rich region flanked by a negatively-charged, aspartic acid (D) and glutamic acid (E)-rich region, and a weak repeat sequence. f Quantitative reverse-transcriptase PCR carried out on different developmental stages of E. tenella [6] confirms the sporulated oocyst-specific expression of ETH_00000115. The relative transcript abundance of ETH-00000115 was determined relative to the et18s small subunit ribosomal RNA for each developmental stage (M = merozoites, G = gametocytes, U = unsporulated oocysts, S = sporulated oocysts). **** Indicates a statistically significant difference for sporulated oocysts vs every other stage at P < 0.001 (n = 3 samples per developmental stage; one-way ANOVA and Bonferroni multiple comparison post-hoc tests using GraphPad Prism® Version 6.03, GraphPad Software Inc., USA)

Back to article page