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International Journal for Parasitology 38 (2008) 829–838 www.elsevier.com/locate/ijpara
Stage-specific gene expression in Teladorsagia circumcincta (Nematoda: Strongylida) infective larvae and early parasitic stages q Alasdair J. Nisbet a,*, Diane L. Redmond a, Jacqui B. Matthews a, Craig Watkins a, Raja Yaga a, John T. Jones b, Mintu Nath c, David P. Knox a a
c
Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, UK b Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK Biomathematics & Statistics Scotland, James Clerk Maxwell Building, The King’s Buildings, Edinburgh EH9 3JZ, UK Received 10 September 2007; received in revised form 10 October 2007; accepted 16 October 2007
Abstract Suppression subtractive hybridisation was used to enrich genes expressed in a stage-specific manner in infective, exsheathed L3s (xL3) versus early L4s of the ovine nematode, Teladorsagia circumcincta prior to gene expression profiling by microarray. The 769 cDNA sequences obtained from the xL3-enriched library contained 361 unique sequences, with 292 expressed sequence tags (ESTs) being represented once (‘‘singletons’’) and 69 sequences which were represented more than once (overlapping and non-overlapping ‘‘contigs’’). The L4-enriched EST dataset contained 472 unique sequences, with 314 singletons and 158 contigs. Of these 833 sequences, 85% of the xL3 sequences and 86% of the L4 sequences exhibited homology to known genes or ESTs derived from other species of nematode. Quantitative differential expression (P < 0.05) was demonstrated for 563 (68%) of the ESTs by microarray. Within the L3-specific dataset, more than 30% of the transcripts represented the enzyme, guanosine-5 0 -triphosphate (GTP)-cyclohydrolase, which is the first and rate-limiting enzyme of the tetrahydrobiopterin synthesis pathway and may be involved in critical elements of larval development. In L4s, proteolytic enzymes were highly up-regulated, as were collagens and a number of previously characterised secretory proteins, reflecting the rapid growth of these larvae in abomasal glands. 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Stage-specific; Teladorsagia; SSH; Microarray; GTP-cyclohydrolase
1. Introduction Dramatic physiological changes occur during the transition from the free-living to the parasitic phase of a nematode’s life cycle. These physiological changes are initiated, and accompanied by, up- and down-regulation in expression levels of suites of genes associated with changes in the nematode’s environment and with nutrition, develop-
q Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB databases under accession numbers AM743198–AM744942. * Corresponding author. Tel.: +44 (0)131 445 5111; fax: +44 (0)131 445 6235. E-mail address:
[email protected] (A.J. Nisbet).
ment, the onset of sexual maturation and establishment in the host (for recent reviews see Nisbet et al., 2004; Nikolaou and Gasser, 2006). A detailed understanding of these processes provides an opportunity to define drug and vaccine targets in parasitic helminths. Previous studies of the mechanisms underlying the transition from infective to parasitic stages of nematodes have used various molecular technologies, the more recent employing microarrays generated from pools of expressed sequence tags (ESTs) derived from cDNA libraries (e.g. Moser et al., 2005). By utilising clustering software prior to selection of ESTs to be printed on the microarrays, redundancy can be reduced, however the majority of genes represented by the ESTs on the microarray slides are unlikely to be differentially expressed between two test populations. For example, only
0020-7519/$34.00 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2007.10.016
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2.7% of the genes represented by 4191 ESTs derived from an Ancylostoma caninum L3 cDNA library were shown to be differentially regulated between infective and parasitic stages using this method (Moser et al., 2005). Stage-specific gene expression has been implied via the study of relative representation of ESTs in cDNA libraries synthesised from different developmental stages of nematodes (e.g. Hoekstra et al., 2000; Thompson et al., 2005) however doubts now exist over the merits of this approach of comparing differences in gene expression (Thompson et al., 2006). In the present study, we have used suppression subtractive hybridisation (SSH, Diatchenko et al., 1996) to allow the enrichment of genes expressed in a stage-specific manner between infective, exsheathed L3 (xL3) and early L4 Teladorsagia circumcincta prior to gene expression profiling by microarray. These technologies have enabled identification of suites of differentially expressed genes which, via bioinformatic analyses, have provided insights into the biochemical and physiological processes associated with both the free-living stages and their adaptation to a parasitic lifestyle. 2. Materials and methods 2.1. Parasite material Teladorsagia circumcincta L3s were harvested from coproculture and exsheathed by exposure to sodium hypochlorite as detailed in Jackson et al. (2004). L4s were collected at 8 days p.i. from the abomasal mucosa of sheep infected with 50,000 L3s using previously published methods (Knox and Jones, 1990). The worms were washed extensively in PBS, snap-frozen and stored in liquid nitrogen. 2.2. SSH Frozen nematodes were homogenised in liquid nitrogen using a pre-cooled mortar and pestle. TRIZOL reagent (Invitrogen) was added, and the nematodes were ground to a fine powder after solidification. Total RNA was isolated according to the manufacturer’s protocol. mRNA was purified from total RNA using the Poly(A)Purist kit (Ambion). The SSH was performed according to the manufacturer’s protocol (PCR-Select cDNA subtraction kit, Clontech) with minor modification. Briefly, this procedure involved the synthesis of cDNA from xL3 or L4 mRNA by reverse transcription, digestion of the cDNA with endonuclease RsaI and ligation of adapters to the digested fragments to produce ‘tester’ cDNA for each stage. The ‘‘xL3 tester cDNA’’ was then hybridised with an excess of L4 cDNA (‘driver’) to remove common cDNA species from the adapter-ligated, xL3 cDNA. The reciprocal process was performed using ‘‘L4 tester cDNA’’ and ‘‘xL3 driver cDNA’’. As controls, both xL3 and L4 testers were used in mock-hybridisations using distilled water instead of driver cDNA to yield ‘‘un-subtracted’’ cDNA
for each stage (Mak et al., 2001). Hybridised fractions were then subjected to PCR to amplify differentially expressed cDNAs. PCR-amplified, xL3- or L4-specific cDNA, produced by SSH, was ligated into the pGEM-T Easy vector (Promega) and transformed by electroporation into competent Escherichia coli (strain DH10B, Invitrogen). Individual positive clones were automatically picked (based on blue/white selection) using a QBot system (Genetix) and grown overnight at 37 C in Luria Bertani medium (LB) containing glycerol and 10 mg ml1 of ampicillin to give primary cultures of each bacterial/plasmid stock. 2.3. Confirmation of subtraction efficiency Sub-samples of each primary culture were arrayed on duplicate nylon membranes (Performa II, Genetix) using the QBot robotic platform, laid onto LB agar plates containing ampicillin (10 mg ml1) and cultured overnight. Colonies were then lysed and denatured by soaking the nylon membranes in 1% sodium dodecyl sulphate (SDS) for 5 min, followed by 1.5 M NaCl, 0.5 M NaOH for 5 min, 3 M NaCl, 0.5 M Tris, pH 7, for 5 min and finally rinsed in 2· SSC (0.3 M NaCl, 0.03 M C6H5O7Na3, pH 7) and cross-linked at 80 C for 2 h. The nylon membranes were then probed with [32P]-labelled, un-subtracted cDNA prepared from xL3 or with [32P]-labelled, un-subtracted cDNA prepared from L4 T. circumcincta. All labelling, hybridisation, washing and detection conditions were as described previously (Nisbet and Gasser, 2004). 2.4. Sequence analysis of subtracted cDNA libraries Five microlitres of each primary (see Section 2.2, above) culture were used to inoculate 1 ml of 2· LB containing 10 mg ml1 of ampicillin and grown for 18 h at 37 C with vigorous shaking. The culture was centrifuged (4000g for 10 min) and plasmids extracted by alkaline lysis. Automated sequencing (in a 96-well tray format) was performed using a T7 oligonucleotide primer and BigDye Terminator v3.1 chemistry (Applied Biosystems). All sequences representing each stage-specific dataset were aligned using the CAP3 assembler (Huang and Madan, 1999), employing a minimum sequence overlap length cut-off of 30 bases and an identity cut-off of 98%. Nucleotide sequences representing non-overlapping contigs subjected to this analysis were compared with those in public databases including the GenBank non-redundant database, using the Basic Local Alignment Search Tool (BLASTn and BLASTx) programme from the National Center for Biotechnology Information; WormBase (website: http://www.wormbase.org) and the Parasite Genomes WU-Blast2 Nematoda database (BLASTn and BLASTx), administered by the European Bioinformatics Institute. Alignments were determined to be statistically significant based on homology scores from alignment of translated sequence (P or E values, depending on the database interrogated of 60.001).
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2.5. Microarray analysis For each stage-specific library, ESTs representing each of the 833 contigs and singleton (occurring once) sequences were PCR-amplified using vector-specific primers. The amplicons were purified, desiccated, resuspended and then arrayed on CMT-GAPS aminosilane-coated glass slides (Corning) as three separate, replicate grids. Each slide also contained several positive control spots (spikes). The slides were then baked, cross-linked and hybridised with fluorescence-labelled cDNA. To generate these cDNAs, total RNA (8 lg) was purified from xL3 or L4 T. circumcincta and purity and quality were assessed using nanochips coupled with an Agilent 2100 Bioanalyzer platform. The RNA samples were then reverse transcribed to generate amino allyl-modified cDNA which was then coupled to Cy3 or Cy5 using the FairPlay III Microarray labelling kit (Stratagene). The labelled cDNAs were then purified using DyeEx columns (Qiagen) and the concentration of labelled cDNA (pmol of dye per ll) calculated using a Nanodrop 10,000 instrument (Nanodrop Technologies). Equal dye quantities from the two labelled samples were mixed and dried in a speedvac. The samples were then resuspended in 20 ll cDNA hybridisation solution (Pronto! Universal Hybridisation Kit, Corning), incubated at 95 C for 2 min then centrifuged at 13,500g for 2 min. Prior to hybridisation, each slide was placed in a pre-soak solution of 1:100 liquid borohydride for 20 min at 42 C followed by 2· 30 s washes in wash solution 2 (Pronto! Universal Hybridisation Kit, Corning). Each slide was then immersed in pre-hybridisation solution (Pronto! Universal Hybridisation Kit, Corning) for 20 min at 42 C, washed once in wash solution 2 for 60 s, twice in wash solution 3 for 30 s and once in nuclease-free water for 30 s. The slides were dried by centrifugation at 1600g for 2 min. Samples were introduced onto the microarrays by capillary action under a lifterslip (VWR) and slides transferred to a Slidebooster hybridisation station (Implen). Slides were incubated for 18 h at 42 C, then washed in the post-hybridisation washes 2 and 3 (Pronto! Universal Hybridisation Kit, Corning) according to the manufacturer’s instructions. The slides were then dried by centrifugation (1600g for 2 min) and scanned in a Genepix 4200AL scanner using the auto photo multiplier tube (PMT) gain algorithm at a resolution of 10 nm. Data were extracted from the images using Bluefuse (BlueGnome) software. A total of eight arrays were used in a simple dye-swap experiment. Labelled samples of xL3 and L4 were each measured four times on the green (Cy3) and red (Cy5) channels, respectively.
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malisation process and the spots were weighted according to the confidence assigned by the Bluefuse software. Poor quality spots (those which had both confidence less than or equal to 0.1 and zero quality as assigned by Bluefuse) were excluded from the analyses of the normalised data. The methods of Smyth (2004) were used to assess the significance of the expression ratios. Briefly, modified t-statistics, using an empirical Bayes approach, were calculated for each spot and the adjusted P-values were calculated using the false discovery rate approach. All the statistical analyses were carried out using the free open-source package ‘‘limma’’ which is implemented in the ‘‘Bioconductor’’ suite of programs (R Development Core team, http:// www.R-project.org). The analyses were carried out on an individual spot basis to check for consistency of responses. The expression level and statistical significance for each gene were evaluated based on the median of the fold changes (on a log2 scale) of the appropriate triplicate spots. Only genes that had a corresponding median adjusted P-value of less than 0.05 were considered to be differentially expressed. 2.7. Confirmatory stage-specific expression analysis by RT-PCR Single stranded cDNA (ss-cDNA) was synthesised from mRNA isolated from xL3s and L4s (harvested at 7 days p.i.) as described previously (Redmond et al., 2006). Approximately 5 ng ss-cDNA were used as template in semi-quantitative PCRs using internal specific primers for each of five xL3 or L4-enriched ESTs for which microarray analysis had been performed. Details of the primers are available on request from the authors. The following cycling conditions were used: 94 C for 5 min followed by 25–32 cycles of 94 C for 30 s, 55 C for 30 s, 72 C for 1 min with a final 7 min extension at 72 C. Samples were removed from each reaction at different cycle numbers to assess whether the PCR was in the exponential, linear amplification or plateau phase, to increase the sensitivity of detection of differentially expressed genes. Equal loading and integrity of each of the ss-cDNA preparations was verified by PCR using primers designed to amplify a 320 bp fragment of the T. circumcincta beta tubulin gene. Negative controls were performed where ss-cDNA was omitted from the reactions. Amplification products were separated on a 1.5% (w/v) agarose gel and visualised by staining with GelRed nucleic acid gel stain (Biotium, Hayward, USA). 3. Results 3.1. Suppression subtractive hybridisation (SSH)
2.6. Experimental design and data analysis Several alternative methods of normalisation were investigated and all gave similar results; only those from weighted print-tip loess are reported in the paper. Only cDNA spots and positive controls were included in the nor-
The analysis of subtraction efficiency, performed using 18,424 cloned plasmids (9212 from the xL3-specific library and 9212 from the L4-specific library) confirmed the high efficiency of the SSH procedure. Un-subtracted cDNA from xL3s hybridised almost exclusively to colonies gener-
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ated from the xL3-specific subtracted library (Fig. 1A) and un-subtracted cDNA from L4s hybridised almost exclusively to those colonies generated from the L4-specific subtracted library (Fig. 1B), demonstrating the removal of common genes from each of the stage-specific cDNA pools.
3.2. Sequence analysis of subtracted cDNA libraries In total, 1768 useable cDNA sequences (769 xL3 and 999 L4) were obtained from the stage-specific libraries. Individual sequences, edited to remove both vector and adaptor sequences, were subjected to analysis using the CAP3 programme (Huang and Madan, 1999) to align similar sequences and to establish the degree of redundancy
within each dataset. The xL3-specific EST dataset was shown to contain 361 unique sequences, with 292 ESTs singletons and 69 contigs. The L4-specific EST dataset contained 472 unique sequences, with 314 singletons and 158 contigs. Of these 833 sequences (361 from xL3; 472 from L4), 85% of the xL3 sequences and 86% of the L4 sequences had homology (P or E < 0.001) to known genes or ESTs derived from other species of nematode (see Supplementary Tables S1 and S2). Putative functions, based on homology, could be ascribed to 62% of the ESTs in the xL3-specific dataset and 69% of the L4-specific dataset (Fig. 2). Some sequences were very highly represented in each of the stage-specific datasets: In the xL3-specific EST dataset, 254 of the 769 sequences (33%) represented the enzyme guanosine-5 0 -triphosphate-cyclohydrolase (GTP-cyclohydrolase) and, in the L4-specific dataset, 107 of the 999 sequences (11%) encoded a secreted cysteine proteinase, cathepsin F (T. circumcincta cathepsin F-1, Tci-CF-1; Redmond et al., 2006). 3.3. Microarray analysis PCR amplicons representing each of the 833 ESTs (Section 3.2 above) were arrayed on microarray slides and hybridised with Cy3 and Cy5-labelled cDNA derived from xL3 or L4 as described in Section 2.5 (above). Overall, quantitative differential expression (P < 0.05) was demonstrated for 563 (68%) of these ESTs; 76% of the sequences representing the xL3 SSH library and 61% of those representing the L4 SSH library were shown to be differentially expressed between xL3 and L4 T. circumcincta (Fig. 3; Supplementary Tables S1 and S2). ESTs which were not shown to be differentially expressed represented genes with very low expression levels, genes for which PCR amplification had failed prior to microarray printing and some genes which were genuinely non-differentially expressed. Replicates of most of the top ranking differentially expressed genes ranked close to each other (data not shown) which indicated that differential expression of these genes was very similar in all three replicate spots on each microarray. 3.4. Confirmatory stage-specific expression analysis by RT-PCR
Fig. 1. High density filter array of Teladorsagia circumcincta infective L3 (xL3) and L4-specific subtracted cDNA library clones. Cloned plasmids [18,424 in total: 9212 from an xL3-specific library (solid outline, A); 9212 from an L4-specific library (dashed outline, B)] were grown on a nylon membrane as micro-colonies. The membranes were then probed with [32P]labelled L3 cDNA (A) or [32P]-labelled L4 cDNA (B).
The results of RT-PCR analysis reflected and verified the data collected by microarray analysis (Fig. 4). Genes which were highly differentially expressed, for example GTP-cyclohydrolase (xL3) or Tci-CF-1 (L4) possessing high expression ratio values were clearly differentially expressed in the RT-PCR experiments. Other ESTs, for example, one sequence representing a TLI-1 toll-interacting protein homologue (AM743887), were more ‘‘stageenriched’’ than truly stage-specific and this was reflected in both the expression ratio of the microarray data and in the RT-PCR experiments (Fig. 4 and Supplementary Tables S1 and S2).
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GTP cyclohydrolase Energy production and conversion Other metabolism Transport Neuropeptides Retinol and thyroxine-binding Heat shock proteins Signal transduction ASP-like Protein turnover Proteinase Ribosomal Others (knownfunction) Unknown function Hypothetical No homology
Tc-CF-1 Other protein ases ASP-like Collagen-like Globins Protein metabolism Lipid metablism Other metabolic Muscle and cytoskeletal ES proteins Lectins Cell:cell interactions Ribosomal Other proteins known function Other proteins,unknown function Hypothetical proteins No homology
Fig. 2. Functional classification of expressed sequence tags represented in stage-specific libraries of Teladorsagia circumcincta larvae. Sequences [833 in total: 361 from an infective L3 (xL3)-specific library (A), 472 from an L4-specific library (B)] representing contigs and singletons were assigned putative function based upon sequence homology to known genes in public databases including the GenBank non-redundant database, using the Basic Local Alignment Search Tool (BLASTn and BLASTx) programme from the National Center for Biotechnology Information; WormBase (website: http:// www.wormbase.org) and the Parasite Genomes WU-Blast2 Nematoda database (BLASTn and BLASTx), administered by the European Bioinformatics Institute.
4. Discussion Here, suppression subtractive hybridisation has proven to be a highly effective tool for enriching cDNA libraries for stage-specific and stage-enriched transcripts from xL3 and L4 T. circumcincta. Overall the ‘‘shift’’ of gene expression from L3 to L4 appears to reflect the changes in nutrition, aerobic/microaerobic metabolism and rapidity of growth (e.g. collagen synthesis in L4) associated with the transition to parasitic lifestyle. The transcript for one particular metabolic enzyme, GTP-CH, represented more than 30% of all ESTs in the xL3-specific cDNA library. GTP-CH transcripts have also
been found to be highly represented in the xL3 stage of the closely related cattle parasite, Ostertagia ostertagi (Moore et al., 2000) and in ESTs derived from L3 Haemonchus contortus (Hoekstra et al., 2000). Furthermore, expression of GTP-CH is limited to the L3 stage in both O. ostertagi and T. circumcincta. In the canine hookworm, A. caninum, GTP-CH is also expressed in L3 and, when A. caninum L3s were ‘‘activated’’ by exposure to host serum, GTP-CH expression was drastically reduced (Moser et al., 2005). Taken together, these observations suggest stage-specificity of expression of this enzyme across several species of parasitic nematodes indicating a potential conservation of function. GTP-CH catalyses the first, rate-limiting, step in the
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>2 fold difference
>2 fold difference
70
xL3
60
40 30 20 10
Frequency of observation
50
L4
-ve
Expression ratio*
βtubulin
1
GTP-CH
72
NPL-33
6
LEA-1
4
JC8.8
2
TLI-1
2
0 -10
0
8
Tc-CF-1
4180
Ratio of signal intensities (xL3/L4) Log2 transformed Fig. 3. Frequency distribution of log2 transformed signal intensity ratios (Cy5/Cy3) measured from Teladorsagia circumcincta expressed sequence tags (ESTs) on a microarray where infective L3 (xL3) and L4 cDNA were tagged with Cy3 and Cy5, respectively. The signal intensity when probed with Cy5-labelled cDNA derived from L4 nematodes was divided by that of the Cy3-labelled cDNA derived from xL3 and then Log2 transformed. Values on the x axis are thus ‘‘log fold ratios’’. Mean values for each EST were derived from three replicate measurements per slide. Data from a single slide is shown. Measurements with very low mean signal intensities [less than 200 u in both channels (Cy3 and Cy5)] were excluded as were measurements from known ‘‘control’’ spots in which signal intensities should have been equal in both channels.
de novo synthesis of tetrahydrobiopterin (BH4) from GTP (reviewed by Tho¨ny et al., 2000). BH4 is a co-factor in at least three known metabolic pathways: (i) The hydroxylation of aromatic amino acids by phenylalanine, tyrosine and tryptophan hydroxylases and thus the synthesis of biogenic amines such as serotonin (from tryptophan) and dopamine (from phenylalanine and tyrosine); (ii) Nitric oxide synthase function; (iii) Glyceryl-ether mono-oxygenase function. Moore et al. (2000) speculated that BH4 may be required in the enzymatic cross-linking of cuticular proteins to provide environmental resistance in O. ostertagi larvae and, in Caenorhabditis elegans, the gene encoding BH4-dependent phenylalanine hydroxylase (PheH), which converts phenylalanine to tyrosine, is localised in the hypodermal cells where it is proposed to function through the provision of tyrosine for cross-linking in the cuticle (Loer et al., 1999). Preliminary studies in our laboratory suggest that PheH is also up-regulated in xL3 T. circumcincta compared with L4, though transcripts for PheH were not detected in the SSH analysis (Nisbet et al., unpublished data). In addition, a transcript representing the enzyme aspartate/tyrosine/ aromatic aminotransferase (EC 2.6.1.1) which is involved in tyrosine biosynthesis was also up-regulated in the T. circumcincta L3-specific library (Supplementary Table S1) and, along with tyrosine phosphatase, was also highly rep-
Col-39
6
Gal-10
7
MEP-1b
2
VAP-1
5
Fig. 4. Confirmatory RT-PCR of transcripts identified, by microarray, as differentially and non-differentially expressed between Teladorsagia circumcincta infective L3 (xL3) and L4. Single stranded cDNA was used as a template in semi-quantitative PCRs using internal specific primers for each of five xL3 or L4-enriched expressed sequence tags for which microarray analysis had been performed. Equal loading and integrity of each of the single strand cDNA (ss-cDNA) preparations was verified by PCR using primers designed to amplify a 320 bp fragment of the T. circumcincta beta tubulin gene. Negative controls (‘‘ve’’) were performed where ss-cDNA was omitted from the reactions. GTP-CH, guanosine-5 0 -triphosphate cyclohydrolase AM743324; NPL-33, neuropeptide-like AM743896; LEA1, late embryogenesis abundant protein AM743625; JC8.8, transthyretinlike AM743905; TLI-1,toll-interacting protein AM743887; Tc-CF-1, cathepsin F AM743995; Col-39, collagen AM744550; Gal-10, galectin AM744026; MEP-1b, metalloproteinase AM744216; VAP-1, venom allergen-like AM744075. *Expression ratio is derived from microarray as described in text.
resented in ESTs derived from xL3s of Strongyloides stercoralis (Mitreva et al., 2004) adding further weight to the importance of tyrosine to this stage of development. Mutations in the gene cat-4, which encodes GTP-CH in the free-living nematode C. elegans, lead to drastic reductions in dopamine and serotonin biosynthesis (Sulston et al., 1975; Loer and Kenyon, 1993). In C. elegans, environmental stress on the first larval stage triggers an ‘‘alternative’’ developmental pathway and, after the second moult, a ‘‘dauer’’ or ‘‘arrested’’ larva is formed (Golden and Riddle, 1984). Two of the three signalling pathways which regulate this transition, the transforming growth factor b (TGFb) and the insulin-like pathway (Patterson and Padgett, 2000; Beall and Pearce, 2002) are influenced by serotonin levels and disruption of either of these pathways causes developmental arrest (Ren et al., 1996; Sze et al., 2000). Environmental stress on pre-parasitic (L1–L3) stages is also associated with developmental arrest at the
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L4 stage after entry into the host in some parasitic nematodes (most notably, O. ostertagi) and TGFb pathway molecules have also been described from some species of parasitic nematode (Gomez-Escobar et al., 1997, 2000; Crook et al., 2005). If a TGFb pathway regulates developmental events in parasitic nematodes, the high levels of GTP-CH transcripts in some parasitic species may also be associated with production of serotonin to regulate these processes in a similar way to C. elegans. An alternative, or additional, role for GTP-CH/BH4/ tyrosine could be in the synthesis of the catecholamine dopamine (via tyrosine). Many physiological processes in nematodes are regulated by biogenic amines because these parasites lack an autonomic nervous system and adrenergic receptors (reviewed by Komuniecki et al., 2004). Thus, the presence of high levels of dopamine may be appropriate as the neural network innervating the developing tissues of the reproductive systems (for example) starts to develop after the L3/L4 moult. Seventeen of the L3-library specific ESTs encoded neuropeptides or neuropeptide precursor molecules (see Supplementary Table S1). These intercellular signalling molecules, and in particular the FMRFamide-related peptides (FaRPs), have been most widely studied in Ascaris suum where they are present throughout the nervous system (for review see Maule et al., 2001). L3-specific neuropeptide-like proteins have also been shown to be present in O. ostertagi (Moore et al., 2000). The muscle-regulating actions of at least one FaRP, PF1 which was first isolated from the free-living nematode Panagrellus redivivus (see Geary et al., 1992), are mediated by nitric oxide (Bowman et al., 1995) which is synthesised from the terminal nitrogen atom of L-arginine by nitric oxide synthase, utilising BH4 as a co-factor. Transcripts encoding proteolytic enzymes accounted for 21% of the L4-specific genes and ESTs encoding a secretory cysteine proteinase, cathepsin F (T. circumcincta cathepsin F-1, Tci-CF-1; Redmond et al., 2006), accounted for 11% of all of the ESTs sequenced from L4 T. circumcincta in the present study. This proteinase was also the most abundant excretory/secretory (ES) molecule identified from cultures of L4 which had been maintained in vitro for 24–48 h following their removal from abomasal mucosa (Redmond et al., 2006). Tci-CF-1 was only expressed in the L4 and adult stages of the parasite and was recognised by the IgA component of gastric lymph from sheep which were immune to challenge infection, suggesting a potential use for this molecule as a vaccine candidate for teladorsagiosis (Redmond et al., 2006). In addition to Tci-CF-1, a cathepsin B-like (CBL) cysteine proteinase (L4contig103, Supplementary Table S2) with 58% amino acid identity (over 348 residues) to GCP7, a component of the immunoprotective excretory– secretory products of H. contortus (Yatsuda et al., 2006), was highly represented amongst the L4-specific transcripts. The theoretical gene product from this contig contains the motif 312YWTVANSWNTDWGE325 within
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the asparagine active site of the translated sequence. The presence of threonine at position 314 rather than the highly conserved leucine which is present in this haemoglobinase motif in the CBLs of number of other species of helminth bloodfeeders (Baig et al., 2002) may reflect either a difference in parasite diet or function of the enzyme. Teladorsagia circumcincta L4-specific ESTs also contained a number of sequences encoding aspartyl and metalloproteinases which are close homologues of components of the galactose-containing glycoprotein complex ‘‘H-gal-GP’’ – a highly immunoprotective complex extracted from the luminal surface of the gut of H. contortus (Smith et al., 1994, 1999). For example, L4contig123 (see Supplementary Table S2) encodes a protein with 57% amino acid identity (75% similarity) over 210 residues to PEP-1, one of the pepsinogen components of H-gal-GP (Longbottom et al., 1997). In addition, L4contig13 (see Supplementary Table S2) encodes a protein with 62% amino acid identity (75% similarity) over 223 residues to MEP1 (Redmond et al., 1997) and EST Tci_L4_01_01_C05 encodes an MEP2 homologue (Newlands et al., 2006). MEPs 1 and 2 are two of the four metalloproteinases identified from H-gal-GP (Redmond et al., 1997; Smith et al., 1999; Newlands et al., 2006). As well as H-gal-GP components, the T. circumcincta L4-specific library also contained a homologue (Tci_L4_01_04_A05, 82% amino acid identity over 92 residues; see Supplementary Table S2) of the protective antigen H11 of H. contortus (Smith et al., 1997). In comparison to the ‘‘digestion-associated’’ proteolytic enzymes present in the L4, the xL3-enriched dataset predominantly contained homologues of the Nematode Astacin family (NAS) proteinases (Mo¨hrlen et al., 2003). Astacin-like molecules in parasitic nematodes have been associated with moulting, tissue penetration and immunomodulation in addition to feeding (Zhan et al., 2002; Lun et al., 2003; De Maere et al., 2005; Williamson et al., 2006) and have also been proposed as promising vaccine candidates in parasitic nematodes (Hotez et al., 2003; Borchert et al., 2007). Globins, high affinity oxygen-binding proteins, are highly represented in the T. circumcincta L4-specific library (Supplementary Table S2). The ‘‘body’’ forms of these proteins are also expressed only upon invasion of the rodent host by the nematode parasite Nippostrongylus brasiliensis (Blaxter et al., 1994). The increase in production of globins by L4 T. circumcincta after entry into the host probably reflects the need to supply oxygen to tissues during a period of rapid growth in a microaerobic environment (Blaxter, 1993), a metabolic situation which is also reflected in the increased expression of glutamate dehydrogenase (Supplementary Table S2) (Skuce et al., 1999). Forty of the T. circumcincta L4 library EST clones sequenced had an O. ostertagi globin (Globin-like ES protein F6) as their closest homologue. This globin was identified in the ES material of L4 O. ostertagi (Vercauteren et al., 2003) and subsequent vaccination experiments using native
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globin-enriched protein fractions gave promising, though highly variable, levels of protection (Claerebout et al., 2005). Larval nematodes inhabiting the abomasa develop rapidly. This development is reflected in the large number of ESTs encoding collagen, the principal proteinaceous component of the nematode cuticle, in L4 T. circumcincta (Supplementary Table S2) and also in H. contortus which is known to possess at least 20 genes encoding collagens (Shamansky et al., 1989). Recently, in C. elegans, a protein disulphide isomerase (PDI-2) has been shown to be essential for the normal function of prolyl 4-hydroxylase, a key collagen-modifying enzyme during development (Winter et al., 2007) and the L4-specificity of at least two ESTs representing protein disulphide isomerase in T. circumcincta (Supplementary Table S2) may reflect similar functions. The transition from L3 to ‘‘activated’’ L3 hookworm larvae, which is achieved by exposing larvae to host serum, is accompanied by an increase in transcription levels of at least one member of the ‘‘Ancylostoma secreted protein’’ or ASP family of molecules (Moser et al., 2005). Homologues of these nematode-specific, cysteine-rich proteins were highly represented in the T. circumcincta L4-specific dataset, reflecting their proposed role in establishment and immune evasion (Hawdon et al., 1996). In addition to ASPs, a number of homologues of other ES products of parasitic nematodes were also highly represented in the L4-specific library. In particular, 16 L4-specific ESTs formed a contig representing a homologue of a 30-kDa antigenic glycoprotein secreted by the parasitic stages of Trichostrongylus colubriformis (Supplementary Table S2) which gave partial protection against the parasite in vaccination trials using a guinea pig model (Savin et al., 1990). Serum-stimulation of A. caninum L3s for 24 h resulted in up-regulation in expression of cysteine dioxygenase suggesting that this enzyme is necessary during the early physiological events surrounding establishment (Moser et al., 2005). In T. circumcincta cysteine dioxygenase is also upregulated in the L4s 8 days p.i. (Supplementary Table S2) indicating that enhanced L-cysteine metabolism is maintained during the parasitic lifestyle. This enzyme is expressed in the intestine and, to a lesser extent, in the hypodermal cells of C. elegans (McKay et al., 2003), though the precise function of L-cysteine metabolism in these tissues is unclear and RNA interference (RNAi) experiments have not shown any phenotypic effects. In conclusion, we have identified suites of genes which are differentially expressed between xL3 T. circumcincta, which represent nematodes at the cusp of entering a parasitic lifestyle, and L4 T. circumcincta which are fully established as parasites but have yet to begin their reproductive phase. Genes identified from both of these stages of nematode in the present study represent potential targets for control and future studies will further dissect the timing of the expression of these genes, and others, during the initial establishment of the nematodes and their early parasitic development.
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