Metazoan parasites have to survive in many different niches in order to complete their life-cycles. In the absence of reliable methods to manipulate parasite genomes and/or proteomes, identification of the molecules critical for parasite survival within these niches has largely depended on comparative transcriptomic and proteomic analyses of different developmental stages of the parasite; however, changes may reflect differences associated with transition between developmental stages rather than specific adaptations to a particular niche. In this study, we compared the transcriptome of two fourth-stage larval populations of the nematode parasite, Teladorsagia circumcincta, which were of the same developmental stage but differed in their location within the abomasum, being either mucosal-dwelling (MD) or lumen-dwelling (LD). Using RNAseq, we identified 57 transcripts which were significantly differentially expressed between MD and LD larvae. Of these transcripts, the majority (54/57) were up-regulated in MD larvae, one of which encoded for an ShKT-domain containing protein, Tck6, capable of modulating ovine T cell cytokine responses. Other differentially expressed transcripts included homologues of ASP-like proteins, proteases, or excretory-secretory proteins of unknown function. Our study demonstrates the utility of niche- rather than stage-specific analysis of parasite transcriptomes to identify parasite molecules of potential importance for survival within the host.
Many species of parasites have evolved complex and intricate lifecycles involving intimate associations with their intermediate, definitive, paratenic and, in some cases, vector hosts. Even within directly-transmitted species, such as the Strongylid nematodes which infect ruminants and other mammalian hosts, successive developmental stages are subject to a plethora of different environmental pressures and stimuli and their adaptations to these environmental factors are reflected in the diversity of their transcriptomic responses at each life stage throughout development (e.g. refs 1,2,3,4). In these nematodes, the transition to a parasitic existence, when the infective larval stage is consumed by the definitive host, represents a sea-change in which a free-living, non-feeding organism exposed to fluctuating temperatures and weather conditions becomes a parasitic organism living in darkness, feeding on protein, growing rapidly and, crucially, negotiating and subverting the host’s immune responses5. In the economically-important strongylid nematode Teladorsagia circumcincta, this transition from the infective third larval stage (L3) on pasture to the fourth larval stage (L4) which resides in the abomasum (true stomach) of sheep, is associated with the upregulation of suites of genes involved in nutrition, growth, host immunomodulation and respiration in a micro-aerobic environment6.
Although the paradigm for the initial parasitic development of T. circumcincta is often simplified to describe the L3 entering the gastric gland, moulting to L4 and emerging into the abomasal lumen as an immature adult, early descriptions of the lifecycle describe three populations of L4 worms in the abomasum7, 8: Thus, after entry of the iL3 into the gastric pits and glands and moulting to L4, the larvae either, (i) fail to develop further; (ii) emerge into the lumen immediately after moulting or, (iii) remain in the mucosa for a longer period, emerging into the lumen as late L4 or early adults7. The dynamics and biology of the latter two populations is of great interest from a biological, parasitological and immunological point of view as these worms represent niche-specific variants of the same age and stage of the species9. As a consequence, differences with the trancriptome and proteome between these two populations are more likely to represent transient adaptations to mucosal or luminal environments, rather than reflecting the more substantial differences associated with transition between individual developmental stages of the parasite lifecycle. One particularly intriguing aspect of the discrimination between populations of T. circumcincta L4 which inhabit the abomasal lumen or are intimately associated with the abomasal mucosa is their different requirements to immunomodulate the host dependent on niche, and this may be highly relevant to the development of novel parasite control strategies10. Indeed, immunomodulation by T. circumcincta L4 is likely to be critical for survival of the parasite within the host as immune responses directed against this stage of the parasite are highly correlated with protection11, 12.
The aim of the work presented here is therefore to discover which genes are differentially transcribed between the populations of worms which are in intimate contact with the host abomasal mucosa and those worms which exist in the less immunologically hostile environment of the abomasal lumen.
Gross morphology of lumen dwelling and mucosal dwelling T. circumcincta L4
Gross morphological data is shown in Fig. 1. There was no significant difference in worm lengths, sex (including no site × sex interaction), or stage of development between luminal dwelling (LD) and mucosal dwelling (MD) larvae. The mean stage of development was phase 6 for both LD and MD larvae8, indicating that both populations were mid-L4 stage.
Assembly and analysis of the T. circumcincta transcriptome
Assembly of the collated T. circumcincta sequencing data resulted in the generation of 28,155 contigs/isotigs and 67,445 singletons. The N50 value of the contigs >100 bp was 731 bp, maximum contig length 6481 bp and the number of contigs >1 kb was 4340. Following filtering for contig sequences of <100 bp, 28,143 contig sequences were retained for downstream analyses. Completeness of the T. circumcincta transcriptome assembly was assessed via Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis13 using the Nematoda lineage dataset as a reference. This was also compared to the current transcript predictions for the T. circumcincta genome available within Wormbase-Parasite (http://parasite.wormbase.org/Teladorsagia_circumcincta_prjna72569/Info/Index/). The analysis demonstrated that, as observed with other nematode transcriptomes the completeness level as assessed by BUSCO was relatively low (23% for our T. circumcincta assembly and 38% for the Wormbase-Parasite T. circumcincta predicted transcripts (BioProject: PRJNA72569)) however the level of completeness for each transcriptome was comparable. Of the 28,143 retained contig sequences, BLASTx analysis identified 17,863 (63.4%) with significant (<1e−02) homology to sequences deposited in the NCBI nr database (25th July 2014 release). The remaining 10,280 contigs had either no or low homology (E-value >1e−02) to sequences within the NCBI nr database, potentially representing T. circumcincta transcripts of, as yet, undefined function.
Determination of differentially expressed transcripts between lumen dwelling and mucosal dwelling larvae
The total number of Hi-Seq reads for each of the six RNA sample sets mapping to each contig/isotig of the T. circumcincta transcriptome is described in Table 1. RNA-Seq obtained approximately 28 to 34 million raw sequence reads for each of the six libraries (three each from luminal and mucosal isolated larvae). Trimmed reads were then aligned to the T. circumcincta transcriptome using the CLC Genomics Workbench (Version 8, Qiagen Ltd) and normalised read count tables generated for assessment of differential gene expression. Differential expression analysis using DESeq. 2 (Version 1.8.1) based on a fold change cut-off ≥±1.5 between LD and MD larvae samples and an FDR corrected p-value of ≤0.05, showed that 57 transcripts were significantly differentially expressed between LD and MD larvae. Of these differentially expressed transcripts, 54 were up-regulated and three were down-regulated in MD larvae, and Fig. 2 shows a volcano plot of log2 (fold change) against −log10 (p-value) for the entire T. circumcincta dataset, highlighting the bias towards increased gene expression within the MD larvae samples.
Of the differentially expressed transcripts, 23/54 of the MD up-regulated and 3/3 MD down-regulated transcripts had significant BLASTx hits (Table 2). In addition, 43 of the differentially expressed transcripts were homologues of genes present within the T. circumcincta genome assembly, PRJNA7256 (Table 2 and Supplementary Table S1). Up-regulated transcripts in MD larvae included five isotigs with homology to venom allergen/ASP-like proteins, five isotigs with homology to parasite proteases, one isotig (20922) with homology to a Stichodactyla helianthus toxin (ShKT)-related protein from the salmon louse Lepeophtheirus salmonis, and 12 isotigs with homology to parasite proteins of unknown function, of which nine were homologues of proteins previously identified in ruminant nematode excretory-secretory (ES) products: five were homologues of a putative L3 ES protein from Ostertagia ostertagi (accession no. CAH23216.1) and four were homologues of a previously identified 15 kDa excretory/secretory protein of Haemonchus contortus (accession no. CDJ91363.1). Thirty-four (60.0%) of the differentially expressed isotigs possessed a signal peptide sequence.
Relationships amongst those differentially-expressed molecules which had significant homologies to other molecules in public databases were inferred by alignment of the nucleotide sequences using ClustalX version 2.0.10, employing Neighbour-joining analysis and the resulting relationship tree constructed using TreeView is shown in Supplementary Fig. S1. This shows distinct groupings of related differentially-expressed, functionally related genes and that differentially regulated venom allergen/ASP-like transcripts segregate into four distinct clusters. Details of transcripts with no known homology are shown in Supplementary Table S1.
Stage-specific expression of transcripts upregulated in MD T. circumcincta L4
To validate the RNA-Seq analysis, we selected four isotigs for validation by qPCR. These represented three isotigs which were up-regulated in MD larvae: isotigs 05835, 20922 and 15536, representing Tci-ASP2, TcK6 and Tci-ES14-1, and one isotig (02529) which was homologous to Tci-CF-1 (accession no. ABA01328.1) and was not differentially expressed between MD and LD larvae. Isotigs for qPCR validation were selected to represent venom allergen/ASP-like proteins (Tci-ASP2) which are thought to play a key role in the parasitic stages of nematodes14, a ShK-domain containing protein (Tck6) with putative immunomodulatory activity15, and a representative of the novel family of transcripts (Tci-ES14-1) which were upregulated in MD larvae in this study. Levels of Tci-ASP2, TcK6 and Tci-ES14-1 transcripts were significantly upregulated in MD larvae compared to LD larvae, whereas Tci-CF-1 transcripts were similar in the two larval populations (Fig. 3A). Additional qPCR analysis was performed to determine the expression profile of the three MD up-regulated transcripts across the different lifecycle stages of T. circumcincta. The expression levels of all three transcripts (Tci-ASP2, TcK6 and Tci-ES14-1) were considerably higher in L4 larvae compared to L3, exsheathed L3 and adult parasites, further supporting a role for these transcripts in the mucosal stage of the parasite lifecycle (Fig. 3B).
Sequence analysis of T. circumcincta ShK-like peptides
ShKT-domain containing proteins from parasitic nematodes have recently been shown to possess immunomodulatory activity via blockage of voltage-gated potassium (Kv) 1.3 channels on human effector memory T cells (TEM)15. As the TcK6 isotig contained a ShKT domain, this was selected for further analysis. Full length coding sequence (CDS) for Tck6 was obtained and the transcribed sequence is shown in Fig. 4A. This encoded a predicted 12.7 kDa protein with a signal peptide and a C-terminal ShKT domain. A manual alignment of the ShKT-domain of TcK6 with ShKT-domains from the archetypal ShK protein from the Caribbean sea anemone, Stichodactyla helianthus (ShK) and ShKT proteins from Ancyclostoma caninum (AcK1) and Brugia malayi (BmK1), all of which possess immunomodulatory activity15, and previously identified ShKT-containing transcripts from an adult T. circumcincta cDNA library (TcK1-5) is shown in Fig. 4B. Isotigs representing TcK1-5 were present in the transcriptomes of both MD and LD larvae within this study but were not differentially expressed between the two populations. All sequences contained the six conserved cysteine residues typical of the ShKT-domain. TcK1 and TcK6 also contained a positively charged residue (Arg or Lys) and an adjacent aromatic/hydrophobic reside (Phe or Tyr) between the third and fourth cysteine residues which is necessary for potent Kv1.3 channel blockade in other ShKT-like proteins15,16,17.
To further explore the relationship between TcK6 and other previously identified ShKT-domain containing proteins, the 35–40 amino acid residues forming the ShKT domains (IPR003582, PF01549) of S. helianthus ShK and a selection of parasitic eukaryotes were derived from EST or protein depositions in public databases and aligned with TcK6 using ClustalX (version 2.0.10). Relationships amongst the molecules were inferred by Neighbour-joining analysis and the resulting dendogram was constructed using TreeView (Fig. 4C). In agreement with the results of BLASTx analysis of the CDS of TcK6, the ShKT domain of the molecule grouped closely with those from the archetypal ShKT from S. helianthus and the sea louse protein ZK643.6.
Immunomodulatory activity of Tck6
To test the immunomodulatory properties of TcK6, full length TcK6 protein without the signal peptide was synthesised and tested for the ability to modulate cytokine production by activated ovine PBMC. Thapsigargin, a SERCA pump inhibitor, was used to induce cytokine production by T cells18, 19. As shown in Fig. 5, nanomolar concentrations of TcK6 suppressed thapsigargin-stimulated IFN-γ production by ovine PBMC (p < 0.05) but had no effect on IL-17A or IL-4 production.
We have demonstrated here, for the first time, that the expression of a suite of genes is differentially regulated in a niche-specific manner between two populations of a single developmental stage of a parasitic nematode species within it’s host. The functions of several of the molecules that are up-regulated in mucosal-dwelling T. circumcincta L4 worms remain obscure beyond their identification as probable excretory/ secretory molecules which occur in expanded families. For other differentially-expressed molecules, the proposed functions are clearer.
Tck6, upregulated in MD larvae, belongs to a family of ShKT-related proteins. First described in the sea anenome S. helianthus, it is now apparent that ShKT-like proteins are produced by a large number of parasitic nematodes, including the economically important ruminant parasites T. circumcincta 15, Haemonchus contortus 15, Trichostrongylus colubriformis 20 and Ostertagia ostertagi 3, where they can contain single or multiple ShKT domains. These proteins are commonly found in nematode secretomes21,22,23 and can be upregulated during parasitic stages of the nematode life-cycle3, suggesting a role in the host:parasite interaction. Recently a role for ShKT-like proteins in parasite-mediated immune modulation has been described15. This immunomodulatory activity is related to ShK-toxin mediated blockage of the voltage-gated potassium (Kv) 1.3 channel which plays a critical role in activation of TEM cells by regulating membrane potential and Ca2+ signaling during T cell activation18. Other T cell subsets are refractory to Kv1.3 blockade as they use a different potassium channel, namely the calcium-activated potassium channel KCa3.1, during the activation process24. TEM cells are thought to confer immediate protection following re-exposure to pathogens by surveying mucosal barriers or diseased tissues and displaying immediate effector function upon recognition of their cognate antigens25, 26. Thus the targeting of TEM by the parasitic stages most closely associated with the mucosa would appear to be a useful strategy for immune evasion by the parasite.
In this study we have shown that TcK6 contains key residues within its single ShKT-domain predictive of functional Kv1.3 channel blockade, shared a high sequence similarity to the archetypal ShKT protein ShK from S. helianthus, and was capable of suppressing IFN-γ production by thapsigargin-activated ovine T cells in a similar manner to that seen with the parasite-derived ShKT proteins, AcK1 (from the hookworms Ancyclostoma caninum and A. celynum) and BmK1 (from Brugia malayi). The lack of suppression of IL-17A and IL-4 production by TcK6 is consistent with previous studies which found that S. helianthus ShK was more effective at suppressing production of TH1-associated compared to TH2 or TH17 cytokines18, 27. As thapsigargin results in T cell activation without upregulating KCa3.1 channels18, the assays employed in this study did not specifically interrogate the effects of TcK6 on TEM cells, as other T cell subsets within the PBMC population would be equally susceptible to the effects of Kv1.3 blockade. Nevertheless, these results suggest that TcK6 may play a role in immune evasion by T. circumcincta L4, particularly as a prominent role for mucosal T cells in immunity against this parasite has been described28. The additional observation that TcK6 transcripts are significantly upregulated in L4 compared to L3 and adult stage T. circumcincta, which are less closely associated with the abomasal mucosa, provides further evidence for a role of Tck6 in modulating the local mucosal environment.
Several of the transcripts identified as up-regulated in the mucosal-dwelling larvae were classified as activation-associated secretory proteins (ASPs). These nematode-specific proteins are members of the SCP/Tpx-1/Ag5/PR-1/Sc7 family, a diverse, evolutionarily-related, group of secreted proteins identified in a wide range of organisms14. ASPs are abundant in the transcriptomes and ES material of L4 T. circumcincta 6, 29 and, although their function(s) are likely to be diverse, it has been suggested that they are key to the transition of nematodes from the free-living to the parasitic state30, 31, having a role in maintenance and/or exit from arrested development32, in modulation of the host immune response33 and in maintenance of the parasites within their niche in the host34, 35. Recently a specific immunomodulatory function was demonstrated for Na-ASP-2 from the human hookworm Necator americanus: this ASP bound CD79A, a component of the B-cell antigen receptor complex, on protein microarrays and specifically bound ex-vivo human B cells36. In the latter, the binding of Na-ASP-2 led to the down-regulation of nearly 1000 transcripts in human B cells, including critical components of B cell receptor signaling pathways36. Although homology-searching using Blastp identified Tci-ES14 as a homologue of a putative L3 ES protein of unknown function from O. ostertagi, structural and functional prediction using I-TASSER37 demonstrated the highest structural match for Tci-ES14 to be Na-ASP-1 (TM score 0.715), further demonstrating the importance of SCP/Tpx-1/Ag5/PR-1/Sc7 family and similar proteins in host parasite interaction for T. circumcincta.
Nematode ASPs have previously shown great promise as vaccine candidates against parasitic hookworms in humans, though induction of generalized urticaria following immunisation of pre-exposed individuals has now halted the development of a vaccine based on these molecules38. From a veterinary perspective, immunisation of sheep with a preparation from H. contortus enriched for a C-type, single-domain ASP, “Hc24”, as well as a non-ASP 15 kDa protein, gave reductions of over 70% in both mean worm burdens and faecal egg counts39, 40. The N-type single domain ASPs, Oo-ASP-1 and Oo-ASP-2, are the principal components of an ASP-enriched native extract of adult O. ostertagi which has been used with success in vaccination trials in cattle41,42,43 and, in T. circumcincta, a homologue of Oo-ASP-1 is a component of a recombinant vaccine cocktail which has convincingly provided protection in repeated vaccine trials in sheep44.
Studies in which sheep have been infected with bolus doses or trickle-infections of T. circumcincta and subsequently euthanased at different time-points have shown that some worms are intimately associated with the mucosa whereas others are present in the lumen (e.g. refs 7, 9 and 44). These studies show only snapshots of what may be a more complex scenario: It may be the case that worms do not necessarily populate one niche or the other but in fact move between the two niches regularly, turning on the expression of suites of mucosal-associated genes at the appropriate time and then downregulating these transcripts when they re-emerge into the lumen: the observation that both mucosal and luminal dwelling larvae in this study were of the same developmental stage would support this hypothesis. Such movement of worms between different niches may also be true for other parasitic nematodes which share a luminal and tissue dwelling stages and warrants further investigation.
The niche-specific transcriptomic analysis described in this study allows the investigation of a relatively small number of transcripts which may be of very high value in answering basic biological questions about host-parasite interactions. For many parasitic nematodes it is not possible to answer these basic biological questions by using functional tools, such as genome-wide RNA interference studies, that are employed in many other eukaryote systems45. However, the reductionist approach of studying niche-specific transcripts, demonstrated here distils the list of molecules for functional analysis such that more expensive, but potentially effective, methodologies such as the use of phosphorodiamidate morpholino oligomers (PPMOs)46, which demonstrate gene function by translational silencing of the gene products, becomes realistic. The assignation of gene function becomes even more challenging when either no homology exists to molecules in public databases (as discussed in ref. 47) (which was the case for 54% of the transcripts with enriched expression in MD larvae here) or where no function has been ascribed to the homologue. For both of these categories of molecule (no homology/unknown function) it is important to start to build a profile to understand gene function; niche specific expression is a very good starting point and can then be augmented with technologies to demonstrate tissue specificity (single cell transcriptomics, immunolocalisation, in situ hybridisation) and gene function analysis (e.g. PPMO) to illuminate gene function in host:parasite interactions and thus potential for rational intervention.
In conclusion, this study has identified a subset of niche- rather than developmental stage-specific transcripts of T. circumcincta larvae. Most transcripts were up-regulated in mucosal- compared to lumen-dwelling larvae, suggesting that they encode for proteins which are important for survival of the parasite within the mucosal environment. In support of this, one of the upregulated transcripts encoded for a novel immunomodulatory protein, TcK6, which may be involved in regulating local T cell memory responses. Niche-specific analysis of parasites represents a promising approach to identify genes and molecules essential for within-host survival, particularly where parasites of identical or similar developmental stages exist in different host niches.
All experimental procedures described here were approved by the Moredun Research Institute Experiments and Ethics Committee and were conducted under the legislation of a UK Home Office License (reference PPL 60/03899) in accordance with the Animals (Scientific Procedures) Act of 1986.
Experimental T. circumcincta challenge studies and isolation of T. circumcincta fourth stage larvae (L4)
Six Texel cross lambs of three months of age reared under conditions to preclude accidental exposure to helminth parasites were challenged per os with 150,000 infective T. circumcincta larvae (isolate MTci-2_CVL). All of the animals were necropsied seven days post-challenge; the methods used to euthanase the animals and remove their organs for worm burden recovery were as described previously48. The abomasum was split along the greater curvature, contents emptied into a labeled container, and washed in physiological saline (PS; 0.85% NaCl w/v). For each individual animal, the abomasal contents and washings were combined together and processed as described below, to generate a ‘lumen dwelling (LD)’ population of worms: In brief, the combined contents and washings were allowed to settle and the supernatant discarded; the resultant digesta was encapsulated within a double layer of surgical muslin, suspended in PS and incubated at 37 °C for four hours. The worms migrating through the muslin were collected from the PS and this constituted the LD worms from each animal.
The ‘mucosal dwelling (MD)’ population was generated by pinning out the washed abomasum on to a polystyrene board, suspending it in PS and incubated at 37 °C for four hours. The migrating worms were collected from the PS.
Fifty individual juvenile worms were randomly selected from each lamb within a group, mounted in 2% formalin (v/v) on a microscope slide, examined for sex, stage of development assessment8, and photographed (Nikon D70), at magnification (x40) on a compound microscope to enable body length to be determined. Where multiple images were captured, the files were compiled to form a single composite using an image editing program (Adobe Photoshop Elements 8.0). Worm lengths were estimated using image processing software (ImageJ 1.46r).
Generation of the T. circumcincta transcriptome
In order to generate the T. circumcincta transcriptomic database, raw read data was obtained from the following resources as described in ref. 49 and consisted of a total of 1,188,329 sequence reads from five independent in-house databases (detailed in Table 3) including: (A) T. circumcincta EST database generated by suppressive subtractive hybridisation (SSH)6; (B) T. circumcincta L4 larvae EST database generated by 454 next generation sequencing (NGS) (Unpublished); (C) T. circumcincta L3 larvae EST database generated by 454 NGS50; (D&E) T. circumcincta adult EST databases generated by 454 NGS51, 52. Assembly of raw reads was performed using Newbler 2.9 (Roche 454) sequence analysis software. The level of completeness of the T. circumcincta transcriptome assembly was assessed via Benchmarking Universal Single-Copy Orthologs (BUSCO) analysis13 using the Nematoda lineage dataset as a reference. The T. circumcincta transcriptomic database used for this study is freely available via the Moredun Research Institute website (http://www.moredun.org.uk/research/teladorsagia-circumcincta-transcriptome) or via the corresponding author on request.
Illumina Solexa RNA-seq (Hi-Seq) analysis of niche-specific gene expression in T. circumcincta L4
RNA was extracted from three separate samples (biological replicates) of luminal or mucosal isolated T. circumcincta larvae using Trizol reagent (Life Technologies, UK) using a using a pestle and mortar on dry ice. RNA quality was assessed on a spectrophotometer (Nanodrop, Thermo Scientific Ltd, UK) with OD260/280 ranging from 1.89–2.1. Samples of sufficient quality for subsequent analysis were obtained from three paired MD and LD populations from three individual sheep. RNA (5 µg of each sample) was processed by Edinburgh Genomics sequencing service, University of Edinburgh for Illumina Solexa Hi-Seq sequencing following the manufacturer’s protocol53. Base calls were made using the Illumina CASAVA 1.8 pipeline. Post-sequencing, read quality of raw FASTQ files was checked with FastQC v0.10.154. The CLC Genomics Workbench (Version 8, Qiagen Ltd) was then used for adapter, quality, ambiguity, and length trimming; alignment of the RNA-seq data with the T. circumcincta transcriptome and generation and normalisation of the reads per kilobase per million mapped reads55. Allowing the total number of reads for each of the six RNA sample sets, which mapped to each contig/isotig of the T. circumcincta transcriptome to be determined.
Quantitative PCR (qPCR) of T. circumcincta transcript expression
Oligonucleotide primers were designed to amplify regions of 169–474 bp, dependent on transcript, for each of Tci-asp-2, Tci-es14, Tck6, Tci-tub-1 (β tubulin, accession number Z69258.1) and Tci-cf-1 (Cathepsin F, accession number DQ133568.1). The sequences for these primers are available from the authors on request. Using 0.8 μg of total RNA extracted from L4 T. circumcincta derived from the mucosal subset recovered from lamb number 1369 as a template, reverse transcription PCR (RT-PCR) was performed to amplify cDNA representing each of the transcripts. The SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase (Invitrogen) was employed, following manufacturer’s instructions. PCR-amplified cDNA representing each of the transcripts was ligated into the pGEM®-T Easy vector (Promega) and transformed by heat shock into competent Escherichia coli (strain JM109, Promega). Plasmids derived from cultures of these transformed cells were sequenced (eurofins genomics) and used as standards in qPCR employing nested gene-specific primers.
cDNA was generated from RNA samples used for previous Illumina Solexa Hi-Seq sequencing, plus an additional 3 samples from MD larvae and 1 sample from LD larvae (representing a total of 6 MD and 4 LD populations), using SuperScript® II Reverse Transcriptase (Invitrogen) and oligo(dT)23 primer (Sigma) according to manufacturer’s instructions. Real-time PCR amplification was performed using ABI PRISM® 7000 Sequence Detection System, and SYBR® GreenER™ qPCR SuperMix (Invitrogen), in 25 μl volumes using 1 μl cDNA and 0.2 μM of each primer. For all primer sets detailed, cycling conditions were as follows; 50 °C for 2 mins, 95 °C for 10 mins, 40 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s. Fluorescence was detected at the end of each cycle and following completion of 40 cycles, product dissociation performed by one cycle of 95 °C for 15 s, 60 °C for 20 s and 95 °C for 15 s. For each gene analysed, a standard curve was performed using appropriate plasmids, previously constructed and detailed earlier, from 108 to 101 copies per μl of plasmid. The number of copies per μl of cDNA was then normalized using results for β-tubulin (Tci-tub-1), as a housekeeping gene.
Validation of Illumina Solexa Hi-Seq sequencing was performed using Tci-cf-1, 05838, 15536 and 20922 genes. These genes were also assessed for stage specificity using cDNA (kindly supplied by Thomas Tzelos, Moredun Research Institute) which were from L3, exsheathed L3 (NaOCl), exsheathed L3 (CO2), L4 and Adult cDNA samples56.
Synthesis of Tck6
Full length coding sequence (CDS) for Tck6 was determined using a PCR amplification strategy: Gene specific primers for Tck6 were designed, based on the available contig sequence for this transcript, to amplify the 5′ and 3′ termini of cDNA representing the transcript when used in conjunction with vector-specific primers and employing a cDNA library [constructed using T. circumcincta L4 (8 days post infection, dpi) RNA in λTriplEx2] as template57 and Platinum® Taq DNA Polymerase (Invitrogen). Analysis of the resulting CDS showed the presence of a putative signal peptide (predicted using SignalP 4.058, which was omitted during the synthesis of the remaining polypeptide (Activotec Ltd, UK).
Cytokine production by Tck6 stimulated ovine peripheral blood mononuclear cells (PBMC)
PBMC were isolated from four adult helminth-free Texel cross sheep using a Ficoll-Paque™ PLUS density gradient (GE Healthcare, Little Chalfont, UK) as previously described59 and assays performed in triplicate for each source animal. PBMC were cultured in 96-well round-bottomed plates at 5 × 105 cells/well in RPMI 1640 (Invitrogen) containing 10% foetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin and 50 μM 2-mercaptoethanol at 37 °C 5% CO2. Cells were pre-incubated for 18 h with 0.1 µM Tck6 polypeptide, 0.01 µM Tck6 polypeptide, or media alone prior to stimulation with 0.4 µM Thapsigargin (Sigma-Aldrich) for 48 h. Cells which had not been incubated with Tck6 or Thapsigargin acted as unstimulated controls. As both TcK6 and Thapsigargin were initially solubilised in DMSO before dilution in culture medium, an equivalent concentration of DMSO in culture medium was added to the relevant unstimulated control wells. Cell-free culture supernatants were harvested and stored at −70 °C prior to analysis. Levels of IFN-γ, interleukin (IL)-4 and IL-17A within the supernatants were quantified using a commercial ELISA kit according to the manufacturer’s instructions (MABTECH AB, Augustendalsvägen, SE, Sweden for IFN-γ and IL-4; Kingfisher Biotech for IL-17A) and expressed as pg/mL.
The statistical package DESeq. 2 (Version 1.8.1), within the R software suite (Version 3.1)60 was used to analyse the RNA-seq (Illumina Hi-Seq) data and to identify transcripts significantly differentially expressed between LD and MD T. circumcincta larvae using a model based on a negative binomial distribution on variance estimated and size factor normalized data taking into account the 3 biological replicates for each of the two conditions61. Read count data for each replicate of each condition were normalised using RPKM (reads per kilobase per million mapped reads) as described previously in ref. 55. A minimum RPKM value of ≥1 was required for further analysis. Significantly differentially expressed transcripts were classified as those having a fold change ≥±1.5 between luminal and mucosal larvae samples and a False Discovery Rate (FDR) corrected p-value of ≤0.0562. Putative functions were assigned to the differentially expressed transcripts following homology searches using the NCBI nr database (25th July 2014 release) and motif identification using IPS within the Blast2GO pipeline. In addition, differentially expressed transcripts were mapped onto the T. circumcincta genome assembly (PRJNA72569): http://parasite.wormbase.org/Teladorsagia_circumcincta_prjna72569. The NGS data from this study is fully compliant with the MINSEQE guidelines and has been deposited in the publically available NCBI SRA Database under the following accession number: SRP096620.
Parasitological and cytokine-release data was analysed using GraphPad Prism version 6.05 for Windows (GraphPad Software, La Jolla California USA, www.graphpad.com). Worm length data (which was normally distributed) was analysed using 2-way ANOVA with site and sex as factors. Larval male:female ratios in MD and LD populations (also normally distributed) were analysed using a students t-test. Stage of larval development, qPCR data and cytokine release data, which were not normally distributed, were analysed using a two-tailed Mann-Whitney U -test. P values of <0.05 were considered significant.
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This work was funded by the Rural & Environment Science & Analytical Services Division of the Scottish Government. We thank Moredun Bioservices Unit for expert care of the animals, and Prof. George Chandy, Nanyang Technological University, for advice on in vitro immune assays.
The authors declare that they have no competing interests.
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McNeilly, T.N., Frew, D., Burgess, S.T.G. et al. Niche-specific gene expression in a parasitic nematode; increased expression of immunomodulators in Teladorsagia circumcincta larvae derived from host mucosa. Sci Rep 7, 7214 (2017). https://doi.org/10.1038/s41598-017-07092-0
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