Characterisation of full-length cDNA sequences provides insights into the Eimeria tenella transcriptome
- Nadzirah Amiruddin†1, 2,
- Xin-Wei Lee†1,
- Damer P Blake3, 4,
- Yutaka Suzuki5,
- Yea-Ling Tay1, 2,
- Lik-Sin Lim1, 2,
- Fiona M Tomley3, 4,
- Junichi Watanabe^6,
- Chihiro Sugimoto7 and
- Kiew-Lian Wan1, 2Email author
© Amiruddin et al; licensee BioMed Central Ltd. 2012
Received: 22 June 2011
Accepted: 13 January 2012
Published: 13 January 2012
Eimeria tenella is an apicomplexan parasite that causes coccidiosis in the domestic fowl. Infection with this parasite is diagnosed frequently in intensively reared poultry and its control is usually accorded a high priority, especially in chickens raised for meat. Prophylactic chemotherapy has been the primary method used for the control of coccidiosis. However, drug efficacy can be compromised by drug-resistant parasites and the lack of new drugs highlights demands for alternative control strategies including vaccination. In the long term, sustainable control of coccidiosis will most likely be achieved through integrated drug and vaccination programmes. Characterisation of the E. tenella transcriptome may provide a better understanding of the biology of the parasite and aid in the development of a more effective control for coccidiosis.
More than 15,000 partial sequences were generated from the 5' and 3' ends of clones randomly selected from an E. tenella second generation merozoite full-length cDNA library. Clustering of these sequences produced 1,529 unique transcripts (UTs). Based on the transcript assembly and subsequently primer walking, 433 full-length cDNA sequences were successfully generated. These sequences varied in length, ranging from 441 bp to 3,083 bp, with an average size of 1,647 bp. Simple sequence repeat (SSR) analysis identified CAG as the most abundant trinucleotide motif, while codon usage analysis revealed that the ten most infrequently used codons in E. tenella are UAU, UGU, GUA, CAU, AUA, CGA, UUA, CUA, CGU and AGU. Subsequent analysis of the E. tenella complete coding sequences identified 25 putative secretory and 60 putative surface proteins, all of which are now rational candidates for development as recombinant vaccines or drug targets in the effort to control avian coccidiosis.
This paper describes the generation and characterisation of full-length cDNA sequences from E. tenella second generation merozoites and provides new insights into the E. tenella transcriptome. The data generated will be useful for the development and validation of diagnostic and control strategies for coccidiosis and will be of value in annotation of the E. tenella genome sequence.
Coccidiosis is an economically important intestinal disease of poultry caused by parasitic Eimeria species. The annual cost of coccidiosis to the poultry industry worldwide has been estimated to exceed £2 billion . Control of this disease in intensively reared poultry is accomplished principally by prophylactic chemotherapy with specific anticoccidial drugs, although drug-resistance is a serious problem that has to be constantly managed. No new drugs have been introduced in recent years and alternative methods of control are now required. Vaccination using live vaccines is a viable option, though it is hampered by the complexity and production constraints of live parasites. Thus, new approaches for control continue to be sought.
Eimeria tenella is widely considered to be the most economically relevant and well known of the seven Eimeria species that cause coccidiosis in chickens . The second generation merozoite of Eimeria is an interesting target for transcriptomic studies as it is the progeny derived from the most pathogenic endogenous stage of the E. tenella life cycle  and may contribute to the stimulation of the protective immune response in the host for at least some Eimeria species . In addition, it is among the most readily isolated stages of the life cycle . Detailed study of the merozoite stage will support the identification of proteins important to key biological processes in the parasite including host invasion, replication, pathogenicity and the stimulation of host immunity.
The availability of segments of sequences from randomly selected cDNA clones, known as expressed sequence tags (ESTs), has provided valuable resources for the identification and study of genes in E. tenella[6–8]. Sequencing of full-length cDNAs provides additional advantages including data derived from a single clone rather than an assembly of multiple ESTs, which can generate ambiguous contigs, and complete transcripts, which include open reading frames (ORFs) and untranslated regions (UTRs). Thus, a large collection of full-length cDNA sequences provides a set of protein coding sequences that facilitate the prediction of gene identity and function by comparison with other known protein coding genes .
In this study, partial sequences were generated from the 5' and 3' ends of randomly selected clones of an E. tenella second generation merozoite full-length cDNA library. These partial sequences were pre-processed and subsequent sequence clustering and primer walking generated full-length cDNA sequences. Characterisation of these full-length cDNA sequences included determination and analysis of ORFs and UTRs, Kozak sequence consensus, simple sequence repeats (SSRs) and codon usage. Analysis of the full-length cDNA sequences generated also identified candidate secretory and membrane proteins that may prove relevant in developing disease control strategies against avian coccidiosis.
Results and discussion
Generation of full-length cDNA sequences
A total of 9,024 clones were randomly selected for plasmid extraction and subsequent single-pass sequencing from the 5' and 3' ends. After eliminating low quality and vector contaminated sequences, 8,433 and 6,981 good quality sequences were obtained from the 5' and 3' ends respectively [dbEST: JK017416-JK032828, JK032875]. These partial sequences were clustered and resulted in the identification of 1,529 unique transcripts (UTs). Using the clustered sequences 81 full-length cDNA sequences were generated by aligning overlapping 5' and 3' end partial sequences. In addition, clones representing 586 consensus sequences with both 5' and 3' end partial sequences were randomly selected and subjected to complete sequencing by single-pass primer walking, generating a further 363 full-length cDNA sequences. Primary sequence analysis revealed the absence of in-frame start or stop codons in one and 10 clones respectively. Such sequences might represent non-coding RNAs, although they could also have been derived from contaminants or cloning artefacts and have been excluded from our subsequent analyses. Thus, a total of 433 full-length cDNA sequences were generated and analysed in this study [GenBank: JN987230-JN987662].
cDNA, ORF and UTR length distribution
The sequences were also analysed to predict ORF and UTR components following start and stop codon identification [Additional file 3]. Length distribution analysis of the 433 predicted ORFs showed that the majority were between 501 to 900 bp in length, with the average size being 867 bp [Figure 2]. Approximately 18.2% (79/433) of the ORFs were less than 500 bp, while only 1.4% (6/433) of the ORFs were more than 2,000 bp. The length distribution of the 5'UTRs showed that few exceeded 500 bp (20.3%; 88/433), with the average size of the 5'UTR being 342 bp. The length distribution of the 3'UTRs showed that 9.9% (43/433) were more than 800 bp long, with the average size of the 3'UTR being 438 bp.
Although the 5'UTR does not contribute directly to the encoded protein, the characterisation of 5'UTR features is important as this region is believed to be involved in the control of translation and transcription processes that subsequently reflect gene expression [13, 14]. Thus, data generated on these regions may reveal control elements and regulatory mechanisms of gene expression patterns in the parasite. In a previous study of apicomplexan full-length cDNA sequences, Wakaguri et al  reported that the average size of 5'UTRs was consistent amongst Plasmodium species, namely P. falciparum (303 bp), P. vivax (304 bp), P. yoelii (345 bp) and P. berghei (299 bp), but varied between genera with C. parvum and T. gondii presenting average 5'UTR lengths of 137 bp and 288 bp respectively. The average 5'UTR length was shortest for C. parvum, which may reflect the fact that both the genome size and the average gene size are the smallest in this species. This comparison has revealed longer 5'UTRs in the E. tenella genome than reported for most other apicomplexan parasites. While the significance of this finding is not yet clear the detection of numerous SSRs may once again be a contributory factor.
Genomic cDNA transcript mapping-E. tenella chromosome 1
Eimeria tenella is the first of the Eimeria species parasites to have been subjected to genome sequencing, although the draft assembly remains fragmented . In order to demonstrate the utility of the data generated here for gene prediction the 1,529 UTs and 433 full-length cDNA sequences were mapped onto the first sequenced E. tenella chromosome (chromosome 1), representing ~1.8% of the genome . Based on an overlap of at least 70% of the original transcript length, a total of 13 UTs were successfully mapped-seven to genes in the feature-poor 'P'-regions and the remaining six to genes in the feature-rich 'R'-regions of the chromosome [Additional file 4; Additional file 5]. Further analysis revealed that mapping of the UT sequences identified and resolved several inconsistencies with the previously predicted coding regions, indicating the usefulness of the transcript sequences in improving gene structure prediction on the E. tenella genome sequence. Two full-length cDNA transcripts were mapped to E. tenella chromosome 1 [Additional file 6] where the alignment of ln23_Etm023C06 showed consistency with the previously characterised 15-exon structure of the glucose-6-phosphate isomerase gene [16, 17].
SSR motif analysis
SSRs can be found in the genome of both prokaryotic and eukaryotic organisms [18, 19]. These repeats represent a rich source of hypervariable markers due to the constant allelic changes of array length caused by their high mutation rate [20, 21]. As a result, they have been widely used in the fields of linkage mapping [22, 23], population genetics  and phylogenetic or comparative genomic research [25, 26]. In addition, SSRs are believed to be important in genome evolution, stimulating the development of genetic variability  and influencing gene expression [28, 29].
SSR motif distribution in full-length cDNA sequences of Eimeria tenella
Codon usage analysis
Codon usage in full-length coding sequences of Eimeria tenella
Codon Usage Value*
Codon Usage Value*
Determination of consensus sequence of translational initiation sites from full-length cDNA sequences of E. tenella
Secretory and membrane protein prediction
Parasite secreted proteins commonly interact with host cells at the molecular level and are exposed to the host immune system. Parasite growth and invasion processes may be prevented once an essential secretory protein is inhibited. Therefore, many secretory proteins can be considered to be vaccine candidates or potential drug targets [38–41]. Prediction analysis using SignalP suggested that 19.6% (85/433) of the peptide sequences contain a signal peptide. Out of these 85 peptide sequences, 60 were predicted to contain one or more transmembrane domains and/or a GPI-anchor, leaving 25 as predicted unbound secretory proteins. Similarity searches based upon homology showed that a large proportion of these predicted secretory proteins could not be assigned a putative function as 24.0% (6/25) had matches with hypothetical proteins or proteins with unknown function, while 56.0% (14/25) had no significant similarity to any publicly available protein sequence [Additional file 10]. Intriguingly, although most of the putative secretory proteins identified were apparent homologues of apicomplexan genes no recognised apical organellar proteins were found.
Many apicomplexan surface proteins have been shown to play an important role in the pathogenicity of these parasites and a number of them are potential vaccine candidates or drug targets. Proteins that are attached via a GPI-anchor to the surface of protozoan parasites can induce a variety of host immunological responses [42, 43]. In this study, membrane proteins were predicted by identifying the presence of signal peptides, transmembrane domains and GPI-anchors. The prediction of transmembrane helices carried out using TMHMM revealed a total of 92 peptide sequences likely to contain at least a single transmembrane domain. GPI-anchor prediction analysis carried out using GPI-SOM, which detects both the N-terminal signal peptide and C-terminal GPI-anchor signal, suggested a total of 26 peptides with a GPI-anchor. Protein sequences that contain a signal peptide and a transmembrane domain or a GPI-anchor were predicted to be membrane proteins. Based on these criteria, 60 membrane proteins were predicted in this study. Database similarity searches showed that putative functions could not be assigned to most of the predicted membrane proteins as 5.0% (3/60) were most similar to hypothetical proteins, while 48.3% (29/60) had no significant similarity with sequences in the GenBank database [Additional file 11]. In total 31.7% (19/60) of the predicted membrane proteins had matches with E. tenella surface antigens (EtSAGs). Two proteins had a perfect match with members from the previously described A family (i.e. EtSAG4 and EtSAG6) . Interestingly, seven other predicted surface proteins showed between 45.8% and 95.5% similarity to the entire coding region length of the EtSAGs. Using multiple sequence alignment these sequences can be divided into two groups, representing the A and B families [Additional file 12]. The alignments show the presence of the six conserved cysteine residues in both families. Family A revealed a mosaic pattern with conserved and variable regions distributed throughout the alignment while family B exhibited a more biased pattern with variation predominantly in the N-terminal half of the alignment, consistent with the analysis described by Tabares et al. . This analysis strongly suggests that the surface antigens discovered in this study represent new members of the EtSAG families. Both of the previously annotated EtSAGs identified in this study had been reported to be expressed in second generation merozoites . Using GO many of the other putative membrane proteins were classified as involved in cellular and metabolic processes; for example identification of a putative longevity-assurance (LAG1) domain-containing protein. As described elsewhere such molecules can present opportunities to disrupt parasite infection and thus have the potential to become good targets for novel intervention strategies .
In this study, we generated and analysed 433 full-length cDNA sequences with complete coding regions derived from the E. tenella second generation merozoite transcriptome. These sequences provide access to a relatively large resource of nucleotide and amino acids sequences for E. tenella that will support a better understanding of the transcriptome of this economically relevant parasite. Moreover, in combination with other genomic resources including whole genome sequences and genome maps , these full-length cDNA sequences will offer new insights into the structure, composition and function of the E. tenella genome. We have also identified panels of 25 and 60 predicted secretory and membrane proteins, with potential for development as novel diagnostic and/or control strategies for E. tenella via molecular techniques.
Parasite passage and purification
The reference E. tenella Houghton strain was used throughout this study . The parasite was routinely propagated as described elsewhere  using specific pathogen free Light Sussex chickens produced and maintained at the Institute for Animal Health. Second generation merozoites were purified following the method of Prof. N. Smith as described elsewhere using several serial five minute incubation steps, each in fresh incubation medium . Only incubation medium washes lacking microscopically detectable red blood cells were processed for RNA extraction to limit host cell contamination.
Full-length cDNA library construction
RNA was extracted from E. tenella second generation merozoites using the TRIzol reagent as described by the manufacturer (Invitrogen, USA) and used in the construction of a full-length cDNA library by the oligo-capping method . In brief, RNAs were sequentially treated with bacterial alkaline phosphatase (BAP) and tobacco acid pyprophosphatase (TAP). The BAP-TAP treated RNAs were then ligated with 5' oligo-cap linker using RNA ligase. First strand cDNAs were synthesised with the oligo-capped mRNA as a template, followed by PCR using the oligo-cap linker sequence and oligo-dT-adapter as primers. The full-length cDNAs produced were then cloned into the pME18S-FL3 plasmid vector and subsequently transformed into Escherichia coli ElectroMAX DH10B cells (Invitrogen, USA).
Plasmid extraction and cDNA sequencing
Colonies were picked randomly and inoculated into individual wells of 96-deep well plates containing LB media, and subsequently grown overnight. Plasmid DNAs were extracted using the Montage™ Plasmid MiniPrep96 Kit (Milipore, USA) according to the manufacturer's instructions. The cDNA inserts were sequenced once from the 5' and 3' ends using the forward (5' GGA TGT TGC CTT TAC TTC TA 3') and reverse (5' TGT GGG AGG TTT TTT CTC TA 3') primers respectively, and the Big Dye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystem Inc., USA) on an ABI PRISM 3730×l DNA Analyzer (Applied Biosystem Inc., USA).
Generation of full-length cDNA sequences
The generated 5' and 3' end sequences were pre-processed using a Phred [48, 49] cut-off quality value of 20. The sequences were subsequently screened against the GenBank non-redundant nucleotide database, and specifically against chicken genome sequences. No sequences with more than 90% similarity to a known chicken genome sequence were identified. Clustering was then carried out using StackPACK version 2.2 [50, 51]. Consensus sequences with overlapping 5' and 3' end sequences were identified as representing full-length cDNA sequences, while those containing both the 5' and 3' end sequences that did not overlap were selected for single-pass primer walking to generate full-length cDNA sequences. Internal primers for primer walking were designed using Primer3 . The sequence reads generated were manually assembled to produce a consensus sequence with a coverage of at least one strand.
Functional annotation and mapping of transcript sequences
The consensus and full-length cDNA sequences were compared against the GenBank non-redundant database using BLASTX , and the assignment of GO terms was carried out using the BLAST2GO pipeline . Mapping of UTs and the full-length cDNA sequences to E. tenella chromosome 1  was carried out separately using ssahaEST  with the following parameters: kmer = 10, seeds = 3, skip = 10, cutp = 80, score = 40, depth = 50, memory = 40, array = 0, edge = 200, identity = 95. Each transcript aligned to the chromosome 1 sequence was required to include at least 70% of the original transcript sequence and mapped in a single contiguous sequence without non-intron/exon gaps. Single-exon alignments were required to include at least 50 bp, while in multi-exon alignments, each aligned exon was required to be longer than 10 bp, with introns between 5 bp to 5000 bp. The transcript mapping results were inspected manually using the Artemis genome browser .
Characterisation of ORFs and UTRs
The coding region in each full-length cDNA sequence was individually predicted using ORF Finder . Whenever possible, BLAST matches were used to confirm the reading frame, and in-frame start and stop codon positions. The determined ORFs and UTRs were analysed with MISA  to identify and localise SSRs. The coding regions were also submitted to CodonW  to generate a codon usage table. Kozak sequence consensus analysis was carried out by generating sequence logos using WebLogo .
Secretory and membrane protein prediction
Secretory and membrane proteins were predicted using SignalP 4.0  and TMHMM 2.0 . GPI-anchored proteins were predicted using GPI-SOM , which predicts both the N-terminal signal peptide and C-terminal GPI-anchor signal. Protein localisation analysis using WoLF PSORT  and BLAST matches were used to support each prediction.
The bioinformatic tools used in this study are summarised in Additional file 13.
We would like to acknowledge the technical assistance provided by Richard D. Oakes and Karen J. Billington in parasite material preparation, Kazumi Abe in full-length cDNA library construction, Halimah Alias in sequence generation and Mohd. Noor Mat Isa in bioinformatics analysis. The research described here has been supported in part by the Genomics and Molecular Biology Initiatives Programme of the Malaysia Genome Institute, MOSTI, Malaysia (grant number 07-05-16-MGI-GMB10) and the "Asia-Africa Science and Technology Strategic Cooperation Promotion Program" Special Coordination Funds for Promotion of Science and Technology, MEXT, Japan.
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