Identification and analysis of Eimeria nieschulzi gametocyte genes reveal splicing events of gam genes and conserved motifs in the wall-forming proteins within the genus Eimeria (Coccidia, Apicomplexa)

Abstract

The genus Eimeria (Apicomplexa, Coccidia) provides a wide range of different species with different hosts to study common and variable features within the genus and its species. A common characteristic of all known Eimeria species is the oocyst, the infectious stage where its life cycle starts and ends. In our study, we utilized Eimeria nieschulzi as a model organism. This rat-specific parasite has complex oocyst morphology and can be transfected and even cultivated in vitro up to the oocyst stage. We wanted to elucidate how the known oocyst wall-forming proteins are preserved in this rodent Eimeria species compared to other Eimeria. In newly obtained genomics data, we were able to identify different gametocyte genes that are orthologous to already known gam genes involved in the oocyst wall formation of avian Eimeria species. These genes appeared putatively as single exon genes, but cDNA analysis showed alternative splicing events in the transcripts. The analysis of the translated sequence revealed different conserved motifs but also dissimilar regions in GAM proteins, as well as polymorphic regions. The occurrence of an underrepresented gam56 gene version suggests the existence of a second distinct E. nieschulzi genotype within the E. nieschulzi Landers isolate that we maintain.

Figure 1. Light micrographs of macrogametocytes of E. nieschulzi (149 h p.i.) after Percoll density-gradient centrifugation.

A Upper gradient fraction contained gametocytes (ga), rat small intestine cells (hc) and erythrocytes (e). B Discontinuous Percoll gradient. C Lower gradient fraction contained purified macrogametocytes (ga) and erythrocytes (e). e erythrocyte, ga gametocyte, hc host cell, m merozoite of fourth generation. Bar: 20 μm.

Figure 2. Assembly of gam contigs and expression analysis.

A) Graphical Illustration of BLAST results in the Eimeria nieschulzi genome data. Contig Enie_470 encodes the complete gam82 ortholog, whereas contigs Enie_5532, Enie_5533, and Enie_23299 encode only 5' parts and contig Enie_10086 encodes a 3' area of gam56. Dashed areas of the boxes indicate the missed sequences of the corresponding genes. Sequences were completed, ordered and manually assembled, resulting in the map shown in B2). B1-2) Mapping of contigs for final assembly. Based on the initial BLAST analysis (A) PCR experiments (B1) with potential primer pairs were performed on genomic DNA (1*-3*). 1*: PCR product (lane 2) at ca. 2,100 bp (primer #3/#7) show that contig Enie_5533 is adjacent to contig Enie­_16402. 2*: PCR products at 1,800 bp (primer #4/#5) and 2,500 bp (primer #4/#6) show vicinity of contig Enie_5532 and contig Enie_16403. 3*: PCR products at ∼8 kb (primer #8/#10 (lane 2), primer #8/#9 (lane 3) respectively, show interconnection between contig Enie_5532 and contig Enie_470 (B2). PCR products were verified by sequencing. Several other low-coverage contigs (Enie_23088 cov. 10.19; Enie_18448 cov. 13.62; Enie_17906 cov. 15.53; Enie_9794 cov. 15.48) map to different areas of the gam locus, predominantly outside of open reading frame-containing regions (B2). DNA size standard: 1 kb ladder. C) The expression of gam genes in gametocytes was confirmed by amplification from gametocyte cDNA with gene-specific primers (lane 2: reaction control (ribosomal subunit); lane 3: gam56_1; lane 4: gam56_2; lane 5: gam82; size standard lane 1: 1 kb ladder). Gam transcripts were absent in sporozoite cDNA (control).

Figure 3. Splicing of E. nieschulzi gam56 genes.

A) Detailed view on the splicing of Engam56_2. There are two alternative splice donor sites. Intron variants result in the occurrence of an alternative stop codon and a change of the encoded amino acid C-terminus. The intron of splicing variant 2 ranges from bp 1,329-1,473 and results in an alternative stop codon in the transcript, directing to an alternative C-terminal amino acid composition (→PIGGMPL). The intron of splicing variant 1 ranges from bp 1,363-1,473 and affects only the 3' UTR, but not the amino acid encoding region, which is consistent with the genomic open reading frame (→PIGGYGVYTWGPK). Splicing variant 2 was the predominant form found in the transcripts (ratio 4 to 1). B) Graphical overview of splicing variants of Engam56_2. Indicated are the GenBank accession numbers and amino acids at the C-terminus of both variants, as well as the region of the intron. C) Graphical overview of splicing of Engam56_1 shows predominantly a splicing variant, like shown for Engam56_2 splicing variant 2. The intron ranges from bp 1,368-1,512. No other splicing variant was found.

Figure 4. The gt2Engam56_2 (Enie_23299) and polymorphisms within GAM56 proteins.

A) The major differences for identification of the gt2Engam56_2 (partially encoded by contig Enie_23299 and complete sequenced after PCR amplification) and the representative Engam56_2 (GenBank AJG00896.1) are different restriction sites (shown in D) and few amino acid changes (AA) (see A) LL(15 AA)G) vs. L(15 AA)S). For full DNA and translated sequences (AA) of the gt2EnGAM56_2, see SF1A and B. B) Sequence analysis of Engam56_2 cDNA clones (translated sequence is shown) revealed a polymorph region (SYAYSYP motif) in some clones (C1-C4), which is present in both Engam56_2 versions. Both versions are also alternatively spliced. C) Sequence analysis of Engam56_1 cDNA clones (translated sequence is shown) revealed two versions (red background, see also SF1D and E). C1-C8 belongs to the high-coverage gam locus assembly, whereas C9-C12 is associated with the low-coverage contig Enie_23088, which maps to contig Enie_5532. Please note that both versions were found spliced and unspliced. D) The representative Engam56_2 and gt2Engam56_2 can be amplified by the same primer pair, but can be discriminated by restriction site analysis in plasmid DNA. The representative Engam56_2 (**) has two AvaI sites and harbours a HindIII and XhoI site, the gt2Engam56_2 (*) has only one AvaI site and no HindIII or XhoI site resulting in different fragment lengths in the agarose gel. The cloning vector pJET1.2 also contains these restriction sites at insert flanking positions.

Figure 5. Interspecific analysis of gam genes and GAM proteins

A) Clustered organisation of gam56 and gam82 orthologues. The gam genes are situated in a cluster in all investigated Eimeria species [GenBank ID: En (E. nieschulzi), ToxoDB-ID: Emax (E. maxima), Eacer (E. acervulina), Enec (E. necatrix), Efal (E. falciformis)]. B) Conservation within GAM proteins. The consensus structure of GAM56 and GAM82 proteins from different Eimeria species are based on the ClustalOmega alignment. RRL*: RRL motif is not conserved in Emax and Eacer GAM56_2, but in all other species and all GAM56_1 variants. (Complete alignment of GAM56 sequences in the SF1G, and H for the GAM82 alignment). C) Phylogenetic trees show distribution of GAM82 and GAM56 in particular branches of rodent and avian Eimeria species. GAM82 shows larger distances in the avian branch than in the rodent branch, which confirms the close relationship of E. falciformis and E. nieschulzi. GAM56 branches within the avian Eimeria into two groups, GAM56_1 and GAM56_2, indicating that the common ancestor of this Eimeria species already had two GAM56 versions that evolved further in the particular Eimeria species.