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Despite their phylogenetic diversity, parasitic nematodes share attributes of longevity and developmental arrest (=hypobiosis) with free-living nematodes at key points in their life cycles, particularly in larval stages responsible for establishing infection in the host. Insulin-like signalling plays crucial roles in the regulation of life span and arrest (=dauer formation) in the free-living nematode, Caenorhabditis elegans. Insulin-like signalling in C. elegans negatively regulates the fork head boxO (FoxO) transcription factor encoded by daf-16, which is linked to initiating a dauer-specific pattern of gene expression. Orthologues of daf-16 have been identified in several species of parasitic nematode. Although function has been demonstrated for an orthologue from the parasitic nematode Strongyloides stercoralis (Rhabditida), the functional capabilities of homologues/orthologues in bursate nematodes (Strongylida) are unknown. In the present study, we used a genomic approach to determine the structures of two complete daf-16 orthologues (designated Hc-daf-16.1 and Hc-daf-16.2) and their transcripts in the parasitic nematode Haemonchus contortus, and assessed their function(s) using C. elegans as a genetic surrogate. Unlike the multiple isoforms of Ce-DAF-16 and Ss-DAF-16, which are encoded by a single gene and produced by alternative splicing, mRNAs encoding the proteins Hc-DAF-16.1 and Hc-DAF-16.2 are transcribed from separate and distinct loci. Both orthologues are transcribed in all developmental stages and both sexes of H. contortus, and the inferred proteins (603 and 556 amino acids) each contain a characteristic, highly conserved fork head domain. In spite of distinct differences in genomic organisation compared with orthologues in C. elegans and S. stercoralis, genetic complementation studies demonstrated here that Hc-daf-16.2, but not Hc-daf-16.1, could restore daf-16 function to a C. elegans strain carrying a null mutation at this locus. These findings are consistent with previous results for S. stercoralis and demonstrate functional conservation of the daf-16b orthologue between key parasitic nematodes from two different taxonomic orders and C. elegans. We conclude from these experiments that the fork head transcription factor DAF-16 and, by inference, other insulin-like signalling elements, are conserved in H. contortus, a parasitic nematode of paramount economic importance. We demonstrate that functionality is sufficiently conserved in Hc-DAF-16.2 that it can replace Ce-DAF-16 in promoting dauer arrest in C. elegans.
Fork head transcription factors are a large group of DNA-binding molecules that play key roles in the regulation of gene expression during embryogenesis, cell differentiation, development and/or ageing (Kaufmann and Knochel, 1996; Kaestner et al., 2000; Galbadage and Hartman, 2008). The first fork head transcription factor (designated FKH) was discovered in the terminal regions of early embryos of Drosophila melanogaster (see Weigel et al., 1989). At the time of its discovery, no known functional motifs were recognised in FKH. Shortly after this report, however, a mammalian fork head transcription factor, designated HNF-3A, was described and shown to contain a 160-amino acid region which is essential for DNA-binding and is structurally distinct from the binding domain of any known transcription factor (Lai et al., 1990). Comparison of the amino acid sequences of HNF-3A and FKH revealed a high degree of sequence identity in the DNA-binding domains (Weigel and Jackle, 1990). This domain, called the fork head/HNF-3 domain, was later identified in more than 100 molecules from a range of eukaryotes excepting plants (reviewed by Lai et al., 1993; Kaufmann and Knochel, 1996; Granadino et al., 2000; Kaestner et al., 2000).
Owing to the complexities of their names and classification, a new, unified nomenclature for these proteins as fork head box (Fox) transcription factors has been introduced and reflects the phylogenetic relationships of all known chordate Fox proteins (Kaestner et al., 2000). The subfamilies (A to O) of fork head transcription factors are presently designated based on amino acid sequence differences within the fork head domain. One of these subfamilies, FoxO, is considered to be particularly important in regulating the expression of genes involved in cell-cycle control, stress response, apoptosis, DNA damage repair, cell differentiation, ageing and tumour formation (e.g., Tran et al., 2003; Accili and Arden, 2004; Huang and Tindall, 2007).
In the free-living nematode Caenorhabditis elegans, the functions of the FoxO encoding gene, designated daf-16 (or Ce-daf-16 where necessary to distinguish it from its orthologues in other species) have been studied extensively (Murphy, 2006; Braeckman and Vanfleteren, 2007). The regulation of DAF-16 represents the key output of the insulin-like growth factor pathway in C. elegans (see Kenyon et al., 1993; Lin et al., 1997; Ogg et al., 1997). It plays critical roles in the regulation of life span and dauer formation, characterised by stress-resistant filariform morphology and arrested development (Kenyon et al., 1993; Lin et al., 1997; Ogg et al., 1997). Under conditions favouring growth and reproduction, DAF-16 is phosphorylated by the kinases from the insulin-like growth factor pathway and is transported to the cytoplasm, allowing the continuous development of C. elegans larvae to the adult stage. In contrast, under dauer-inducing conditions, such as starvation and/or overcrowding, insulin signalling ceases and unphosphorylated DAF-16 remains in the nucleus, binds to its response elements in the genome and brings about a pattern of gene expression, resulting in dauer-developmental arrest and its associated changes in morphology and life span. Recently, an orthologue of daf-16, originally called fktf-1, and now Ss-daf-16, was identified in the parasitic nematode Strongyloides stercoralis (see Massey et al., 2003. Comparison between Ss-daf-16 and Ce-daf-16 revealed similarities in inferred amino acid sequence and gene organisation. For example, both genes produce multiple transcripts via alternative splicing, and the highest levels of homology (79.5% identity in amino acid sequence) exists in the DNA-binding or “fork head” domain. Lower levels of sequence similarity are seen in the C-termini (31.4% identity) and N-termini (51.4% identity) of these proteins (Massey et al., 2003). Like Ce-daf-16, Ss-daf-16 is expressed at similar levels throughout development (Ogg et al., 1997; Massey et al., 2003). Besides these structural similarities, the ability of Ss-daf-16 to complement a null mutation in daf-16 also suggests that it has similar developmental regulatory capability to its C. elegans orthologue (Massey et al., 2006). These findings support the hypothesis that insulin-like signalling functions in S. stercoralis and that Ss-DAF-16 plays important roles in this pathway, possibly by regulating the formation of the infective L3 (iL3). While some information is now available for S. stercoralis, nothing is known about the functions of daf-16 orthologues in the vast majority of medically or economically important parasitic nematodes, such as those of the order Strongylida. Studying the structures and functions of daf-16-like transcription factors in these parasites will be important in gaining an understanding of their developmental biology, particularly as it relates to the infective process. Therefore, in the present study, we characterised the structures of the daf-16 orthologue in Haemonchus contortus (the barber's pole worm of small ruminants) and the DNA complementary to its transcripts.
Merino lambs (males; 8–12 weeks of age), maintained under helminth-free conditions, were infected intraruminally with 8000 iL3 of H. contortus. The patency of the infection (~24 days) was ascertained by the detection of strongylid eggs in the faeces using the McMaster flotation method (MAFF, 1977). L1, L2 and iL3 were collected after 1, 3 and 7 days of incubation of faeces at 28 °C, respectively, and purified by repeated sedimentation and migration through a nylon sieve (mesh size: 20 μm). For the collection of L4 and adults of H. contortus, infected lambs were euthanised with an overdose of pentobarbitone sodium (Lethobarb, Virbac Pty. Ltd.), administered i.v. 8 and 30 days p.i., respectively. Adult worms were collected from the abomasums at necropsy using fine forceps, washed extensively in chilled (4 °C) PBS, and males and females (adults) separated prior to snap-freezing in liquid nitrogen and subsequent storage at −70 °C. Animal ethics approval (AEC No. 0707528) was given by The University of Melbourne, and the care and maintenance of sheep followed this institution's guidelines.
Total genomic DNA was extracted from ~0.5 g of single-sex (male or female) adult worms using a small-scale SDS/proteinase K extraction procedure (Gasser et al., 1993), followed by mini-column (Wizard® Clean-Up, Promega) purification. Total RNA was extracted separately from different developmental stages (L2, L3, L4 or adults) or sexes of H. contortus (homogenised under liquid nitrogen using a mortar and pestle) employing the TriPure isolation reagent® (Roche Molecular Biochemicals). RNA yields were estimated spectrophotometrically, and the integrity of RNA was confirmed by detecting discrete 18S and 28S rRNA bands on ethidium bromide-stained gels. Each RNA sample (~10 μg) was treated with 2 U of DNase I (Promega) and incubated at 37 °C for 30 min prior to heat denaturation of the enzyme (75 °C for 5 min). Both DNA and RNA samples were stored at −70 °C.
Using the degenerate oligonucleotide primers DAF-16F100: 5′-CARGTNTAYGARTGGATGGT-3′ and DAF-16R100: 5′-CCNGCNCCYT CRTTYTG-3′, designed to a relatively conserved element (between nucleotide positions 679–698 and 805–821 with reference to the C. elegans gene; Accession No. NM_001026427), a portion of Hc-daf-16 was amplified by PCR from cDNA synthesised from total RNA extracted from adults of H. contortus. PCR products were cloned into the pGEM®-T-Easy vector (Promega) and sequenced. Based on these sequences (Accession No. FN433208), gene-specific primers Hc-daf16/1F: 5′-CAGGTGTACGAGTGGATGGTGCAG-3′; Hc-daf16/2R: 5′-GCTGAATGTAACGAGAGATTGTGCCGAA-3′; Hc-daf16/ 3F: 5′-GTGCCGTATTTCCGAGACAAGGGGCGA-3′ and Hc-daf16/4R: 5′-TCCGGCCCCTTCGTTTTGGATACGC-3′ were designed. Using pairs of gene-specific primers and primers specific to the nematode spliced leader 1 (SL1), two partially overlapping cDNA fragments were produced separately from total RNA from adult H. contortus using 5′- and 3′-rapid amplification of cDNA ends (RACE) (SMART™ RACE cDNA Amplification Kit, BD Biosciences). These cDNAs were ligated into the pGEM®-T-Easy vector. Escherichia coli (strain JM109) (108 colony forming U/μg) was transformed with recombinant plasmids via heat shock and grown overnight at 37 °C on Luria Bertani (LB) plates containing 10 mg/ml ampicillin, 0.5 mM isopropyl-β-d-thiogalactopyranoside (IPTG) and 80 μg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-galactosidase). Plasmid DNA was isolated from recombinant clones and column-purified (Wizard®, Promega) from overnight cultures, and inserts sequenced in both directions using vector oligonucleotide primers (M13 and SP6), employing Big Dye Terminator v.3.1 chemistry, in an automated ABI-PRISM sequencer (Applied Biosystems). Based on the resultant sequences, selected oligonucleotide primers were designed to amplify the full-length Hc-daf-16.
Four overlapping fragments representing the entire Hc-daf-16 gene were amplified by long-range PCR (Advantage 2 PCR Polymerase Kit, BD Bioscience) from ~100 ng of total genomic DNA from a single adult female of H. contortus, employing the primers designed based on the cDNA sequences determined (Accession Nos. FN432341 and FN432342) including Hc-daf16/9F: 5′-ACCGGTATG GCAAATCAGCTCTCGCAGACGC-3′, Hc-daf16/11F: 5′-ACCGGTATGA GCAGCCAAGTCACTGCC-3′; Hc-daf16/2R, Hc-daf16/3F, Hc-daf16/ 22R: 5′-GGAGTTGGCGGCGGCAGCGGCT-3′; Hc-daf16/5F: 5′-AGCCG CTGCCGCCGCCAACTCC-3′; Hc-daf16/16R: 5′-CACGCGCTGCACACCT ACTTGAG-3′. The cycling conditions in a 2400 thermal cycler (Applied Biosystems) were: 92 °C, 2 min (initial denaturation); then 92 °C, 10 s (denaturation); 60 °C, 30 s (annealing); 68 °C, 3 min (extension) for 35 cycles, and a final extension at 68 °C for 7 min. After the optimisation of conditions, the PCR yielded a single, abundant product, which was excised from the agarose gel (1%), purified over a mini-spin column (Wizard® PCR-Preps, Promega), cloned into the vector pGEM®-T-Easy and then used as a template for automated sequencing (as described in Section 2.3), employing (separately) vector primers T7 and SP6. The sequences obtained were assembled manually. The exon/intron boundaries of the full-length Hc-daf-16 were inferred based on the alignment of the cDNA and genomic DNA sequences of Hc-daf-16, following the AG-GT rule (Brethnach and Chambon, 1981).
Nucleotide sequences were assembled using the program EGassembler (http://egassembler.hgc.jp/) and compared with those in non-redundant databases (GenBank) using the BLAST v.2.0 (Altschul et al., 1997) suite of programs from the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/ BLAST), the Sanger Centre (www.sanger.ac.uk/Projects/Celegans/) and the Parasite Genome database (www.ebi.ac.uk/parasites/parasite_blast_server.html) as well as the genomic sequence data for H. contortus available at the Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/Projects/H_contortus/) to confirm the identity of the genes isolated. The conceptual translation of individual cDNAs into amino acid sequences was performed using the selection “translate”, available at http://bioinformatics.org/ sms/. Protein motifs were identified by scanning the databases PROSITE (Bairoch, 1993; www.expasy.ch/tools/scnpsit1.html) and Pfam (Bateman et al., 2000; www.sanger.ac.uk/Software/Pfam/). Amino acid sequence alignments were carried out using Clustal W (Thompson et al., 1994).
The amino acid sequences of fork head domains from 27 FoxOs representing various vertebrate and invertebrate taxa, including eight species of nematodes, were retrieved from NCBI GenBank databases and then aligned with the amino acid sequences inferred from the Hc-daf-16.1 and Hc-daf-16.2 transcripts from H. contortus. Phylogenetic analyses were conducted using maximum parsimony (MP) and neighbour-joining (NJ) methods, employing PAUP* v4.0b10 (Swofford, 1999), as described recently (Hu et al., 2005). Characters were weighted equally and treated as unordered. An heuristic search with tree bisection–reconnection (TBR) branch swapping was used to infer the shortest trees. The length, consistency index (C.I.), excluding uninformative characters, and retention index (R.I.) of the most parsimonious trees were recorded. A bootstrap analysis (using 1,000 replicates) was conducted using heuristic searches and TBR-branch swapping with the MulTrees option to determine the relative support for clades in the consensus tree.
Total RNA (1 μg) was used to synthesise first-strand cDNA by random priming using Superscript II reverse transcriptase (Cat. No. 18064-022, Invitrogen), following the manufacturer's instructions. Reverse transcription (RT) real-time PCR was used to analyse the transcriptional profiles among different developmental stages of H. contortus. Isoform-specific primers Hc-daf16/9F and Hc-daf16/18R: 5′-CATGTGGGCCGTCTGATTAGGC-3′ for Hc-daf-16.1; primers Hc-daf16/11F and Hc-daf16/20R: 5′-GACGAAGCACTTAGAGGTAG-3′ for Hc-daf-16.2, designed based on cDNA sequences (cf. Accession Nos. FN432341 and FN432342), were used in PCR. For each sample, 0.5 ng of cDNA was subjected to PCR (20 μl) using the SYBR Green-ER qPCR SuperMix Universal (Cat. No. 11762-100, Invitrogen) in a Rotor-Gene 3000 thermal cycler (Corbett Life Sciences) under the following conditions: one cycle of 50 °C for 5 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s, following by melting from 70 °C to 99 °C at 1 °C increments. Each sample was tested in triplicate, using a normaliser (i.e., a 364 bp region within the β-tubulin 8–9 gene, tub8–9) (Geary et al., 1992) using the primers tub8–9F3: 5′-GTTGTTCCATCACCCAAGGTA-3′ and tub8–9R4: 5′-TAAGCTCAGCAACTGTCGAA-3′ as well as positive and no-template controls. The specificity and identity of individual amplicons were verified by melting-curve analysis and direct, automated sequencing using the same primers employed for PCR. Relative transcriptional differences were calculated from normalised values using a well-accepted method (Livak and Schmittgen, 2001). Statistical analysis was conducted using a one-way ANOVA; P ≤ 0.05 was set as the criterion for significance.
The genetic stocks of C. elegans used were the same as described in a previous study (Massey et al., 2006). The strains were obtained from the Caenorhabditis Genetics Center (CGC) (University of Minnesota) and included the wild-type N2, mutants of daf-2 (e1370) and daf-2 (e1370); daf-16 (mg54). All known daf-16 alleles that affect dauer development in C. elegans, the phenotype of interest in the present study, map to the region of the gene encoding the daf-16a isoform, and the mg54 allele chosen for the present heterologous complementation study involves an amber stop mutation within the DNA-binding domain in this region (Ogg et al., 1997). The mg54 allele completely suppresses the dauer-constitutive phenotype exhibited by daf-2(e1370) mutants at elevated temperature, making it advantageous for the double mutant approach to daf-16 complementation described in Section 2.8. Worms from all genetic stocks as well as transgenic lines were maintained on nematode growth medium (NGM) agar plates containing E. coli OP50 lawns (cf. Lewis and Fleming, 1995.
For studies of cross-species complementation, the putative rescuing plasmid vectors pMH207-16a and pMH207-16b (see Fig. 1), containing the coding regions of Hc-daf-16.1 and Hc-daf-16.2, respectively, were made from vector pPV207, reported in the previous study (Massey et al., 2006). In brief, Ss-daf-16b was removed from vector pPV207 by restriction digestion with enzymes Agel (A) and NgoMIV (N) at sites located on the either end of Ss-daf-16b. The cDNAs of Hc-daf-16.1 and Hc-daf-16.2, with restriction sites A and N at either end were amplified from cloned full-length cDNAs of Hc-daf-16.1 and Hc-daf-16.2, digested with the enzymes and then cloned into vector pPV207 with Ss-daf-16 removed, creating vectors pMH207-16a and pMH207-16b (see Fig. 1). A previously synthesised “rescuing” plasmid vector, pPV200 (Massey et al., 2006), was also used in the cross-species complementation experiments.
The method used for establishing lines of C. elegans transformed with parasite sequences was the same as described previously (Massey et al., 2006). In brief, C. elegans of the daf-2; daf-16 double-mutant strain were transformed by microinjecting 80 ng/μl of plasmid pRF4, which contains the rol-6 mutation (Kramer et al., 1990), and 20 ng/μl of either pPV200 (Massey et al., 2006), pMH207-16a or pMH207-16b into the gonads of young hermaphrodites. Microinjected worms were then reared in isolation, and transformants were selected from their F1 progeny based on the “roller” phenotype and re-plated. The broods of individual micro-injected worms that transmitted transgenes to the F2 generation and beyond were propagated as transgenic lines. As in a previous functional study of Ss-daf-16 (originally designated fktf-1) (Massey et al., 2006), we used this double mutant approach to capitalise on the ability of particular mutations in Ce-daf-16 to suppress dauer-constitutive phenotypes associated with mutations in daf-2 as a “read-out” for heterologous complementation. We reasoned that any daf-16-like activity associated with a hypothetical parasite orthologue should reverse the genetic suppression occurring in a daf-2; daf-16 double mutant and give a significant increase in the percentage of dauer larvae arising under culture conditions of adequate nutrition. Such an approach offers the added advantage of obviating the application of an exogenous dauer pheromone, which would be necessary to undertake the complementation assay using daf-16 single mutants.
Expression of transgene-specific mRNA was verified in each of the transformed lines by qualitative RT-PCR (Massey et al., 2006). Worms were collected from OP50 plates and placed into PCR tubes containing 48 μl of worm lysis buffer (Williams et al., 1992). Tubes were placed at −80 °C overnight. To extract RNA from the worms, samples were thawed and 4 μl of RNAsin (New England Biolabs) added to each tube. The samples were incubated at 65 °C for 75 min and then at 95 °C for 15 min. RNA was treated with DNase I (Promega; according to the instructions) and then used as a template for RT-PCR, conducted using transgene-specific primers (Table 1) and Superscript II reverse transcriptase (Invitrogen; as specified in the instructions).
Lines with verified expression of transgene-specific mRNA were used in a dauer-switching assay to ascertain the capacity of Hc-daf-16 to complement the null mutation in C. elegans daf-16, carried out as described previously (Massey et al., 2006). In brief, 20–30 egg-laying transformant (roller) hermaphrodites were placed on 60 mm NGM agar plates with standard E. coli OP50 lawns, formed by drying 40 μl of an overnight LB broth culture. The hermaphrodites were allowed to produce eggs for 3–8 h and were then removed from the plates. The plates were then incubated at 25 °C for 65–70 h. At the end of this incubation, individual transformants, which were identified based on the right roller phenotype, were scored as non-dauer individuals (L4 or adult) or dauer larvae. Mean proportions of dauer and non-dauer development were calculated from at least four biological replicates of the assay. The total numbers of transformed worms observed per group in four replicates ranged from 61 in the Hc-daf-16.1 transgenics to 173 in the daf-2 mutants. Proportions of dauer development in transgenic lines were compared with those in the daf-2; daf-16 parental stock by χ2 analysis; P ≤ 0.05 was set as the criterion for significance.
The full-length cDNAs of two transcripts representing Hc-daf-16.1 and Hc-daf-16.2 were isolated by RACE from H. contortus. Both cDNAs were trans-spliced by SL1. The full-length cDNA of Hc-daf-16.1 (Accession No. FN432341) was 2,453 bp in length, including an open reading frame (ORF) of 1,812 bp, a 5′-untranslated region (UTR) of 191 bp, and a 3′-UTR of 428 bp. The full-length cDNA of Hc-daf-16.2 (Accession No. FN432342) was 2,247 bp in length, containing an ORF of 1,671 bp, a 5′-UTR of 126 bp and a 3′-UTR of 428 bp. The cDNAs of Hc-daf-16.1 and Hc-daf-16.2 encoded predicted proteins of 603 and 556 amino acids, respectively. An alignment of the two protein sequences revealed that the 447-amino acid C-terminal domains of both (positions 157–603 for Hc-DAF-16.1 and positions 110–556 for Hc-DAF-16.2) were almost identical (Table 2), except for amino acid substitutions at positions 54 and 138 at the C-terminal end. The predicted amino acid sequences of Hc-DAF-16.1 and Hc-DAF-16.2 were aligned with the DAF-16s of five other nematode species (see Fig. 2) for which full-length sequences are available in the GenBank database. The alignment showed that sequence identities at the N-terminal and C-terminal regions were low (3.3–97.5% and 21.4–99.2%, respectively), whereas the fork head domains were conserved (57–100%). The fork head DNA-binding domains of nematodes also had high similarity to human FoxO1 (see Fig. 2). Predicted kinase phosphorylation sites and 14-3-3 protein-binding sites were also identified in the alignment. The two fork head domains of H. contortus were identical to those proposed for Ancylostoma caninum and Ancylostoma ceylanicum (Accession Nos. ACD85815 and ACD85816, respectively; Gao et al., 2009) (Fig. 2). In addition, the C-termini of isoforms Ss-DAF-16A and Ss-DAF16B were identical over a stretch of 378 amino acids (positions 364–741 for Ss-DAF-16A and 189–566 for Ss-DAF-16B, respectively) as were those of isoforms Ce-DAF-16A1 and Ce-DAF-16B over 320 amino acids (positions 191–510 for Ce-DAF-16A1 and 211–530 for Ce-DAF-16B, respectively) (see Fig. 2 and Table 2).
The sequences of the fork head DNA-binding domains of nematode DAF-16s were also aligned with representatives of each subfamily of fork head transcription factors (A to O) from humans (cf. Kaestner et al., 2000) (Fig. 3). The alignment revealed that nematode fork head domains had similarities to human FoxO1 ranging from 5.8% to 65.8%, whereas they usually showed lower similarities (1.7–22.2%) to human fork head domains from other subfamilies. The most conserved region was in the H3 domain, which is the DNA-binding region (Clark et al., 1993). Although there is substantial amino acid sequence variation in the alignment, the two signatures of fork head domain in the PROSITE database: W(Q,K,R)(N,S)S(L,I,V)RH and (K,R)P(P,T,Q)(Y,F,L,V,Q,H)S(F,Y)xx (L,I,V,M)xxx-(x)(A,C)(I,L,M) were readily identified in the alignment for nematode fork head domains (see Fig. 3).
The amino acid sequences of fork head domains of Hc-DAF-16.1 and Hc-DAF-16.2 (inferred from the H. contortus genes Hc-daf-16.1 and Hc-daf-16.2, respectively), eight other nematode DAF-16s and 19 FoxOs from selected non-nematode species were aligned and subjected to phylogenetic analyses (Fig. 4). There was concordance between the MP and NJ trees. The fork head domains of all vertebrate FoxO1s included in the present analysis grouped together with strong bootstrap support (89–94%), as did those of all vertebrate FoxO3s (86–89%). Two fork head domains from the genus Drosophila clustered together with 100% bootstrap support. All FoxOs from vertebrates and invertebrate formed a group with 99% bootstrap support. The fork head domains of the isoforms Hc-DAF-16.1 and Hc-DAF-16.2 were identical and both clustered closely with those from A. caninum and A. ceylanicum (designated as Aca-DAF-16 and Ace-DAF-16, respectively) with 100% bootstrap support. All isoform A sequences formed a group with selected vertebrate and invertebrate FoxOs, with strong bootstrap support (93–94%), to the exclusion of DAF-16B isoforms of C. elegans, S. stercoralis (Ce-DAF-16B and Ss-DAF-16B) and Brugia malayi, which clustered together with strong bootstrap support (84–96%, Fig. 4).
Two full-length Hc-daf-16 gene sequences (from start codon to stop codon) were isolated from genomic DNA of H. contortus. The genomic sequences representing gene Hc-daf-16.1 (Accession No. FN432343) and Hc-daf-16.2 (Accession No. FN432344) were 3,106 bp and 2,965 bp in length, respectively. Both genes contained four exons of 61–1,380 bp and three introns of 326–568 bp (Fig. 5). Comparison of the two genes revealed that the sequence divergence was predominantly in the first 471 nucleotides in Hc-daf-16.1 and first 330 nucleotides in Hc-daf-16.2, respectively. The sequence identity in this region was 25.9%, whereas there was 99.6% of sequence identity between the 2,635 nucleotides downstream. In contrast to the situation in H. contortus, where the daf-16a and daf-16b transcripts are encoded by separate genes, the multiple isoforms of C. elegans DAF-16 and S. stercoralis Ss-DAF-16 are encoded by single genes and produced by alternative splicing (Fig. 5).
Real-time PCR analysis showed that Hc-daf-16.1 and Hc-daf-16.2 were transcribed in larval and adult developmental stages as well as both sexes of H. contortus examined (Fig. 6). There was no significant difference in the levels of transcription among different developmental stages for either isoform (P > 0.05).
In general, diagnostic RT-PCR confirmed the presence of appropriate transgene-specific mRNA in each of the transgenic daf-2; daf-16 lines (Fig. 7A). As expected, Ce-daf-16a message was detected in N2 (wild type) worms, daf-2 mutants and in the daf-2; daf-16 double mutants carrying the Ce-daf-16a transgene. Also, as expected, the Ce-daf-16a message was absent from daf-2; daf-16 double mutants carrying only the rol-6 marker. Relatively weak signals were detected in RT-PCR with Ce-daf-16a-specific primers and cDNAs derived from both Hc-daf-16.1 and Hc-daf-16.2 transgenics, which might be due to a degree of cross-hybridisation between the C. elegans-based primers and the Haemonchus cDNAs. An alignment revealing that the forward primer Ce-daf16aF is 45% similar to the corresponding Hc-daf-16 sequence and that the reverse primer is 65% similar supports this proposal. Reactions involving Hc-daf-16.1- and Hc-daf-16.2-specific primers detected transgene-specific message only in the respective transgenic line.
In dauer-switching assays conducted on well-fed larvae at 25 °C (Fig. 7B), N2 (wild type) worms developed uniformly to non-dauer individuals, as expected. These were mainly hermaphrodites with a minority of late L4. By contrast, 100% of daf-2 mutants developed to dauer L3, reflecting the expected pattern of development in worms carrying the temperature sensitive daf-2 (e1370) allele (Kimura et al., 1997). Ninety-nine percent of double mutants carrying both the daf-2 (e1370) and the daf-16 (mg54) alleles developed to non-dauer individuals (primarily hermaphrodites with a few L4), and one percent to dauer larvae. This pattern is consistent with the characterisation of mutations in daf-16 as genetic suppressors of daf-2 mutations and with the requirement for the DAF-16 transcription factor in the formation of normal dauers (Lin et al., 1997; Ogg et al., 1997). Given the distinct and clear-cut phenotypic differences between daf-2 single mutants and daf-2; daf-16 double mutants, we reasoned that the restoration of significant levels of dauer development to the double mutant worms by expression of Hc-daf-16 isoforms would be a straightforward indication of genetic complementation. This was clearly in evidence among daf-2; daf-16 individuals expressing the Ce-daf-16a-encoding transgene in plasmid pPV200 (Fig. 7B). As might be expected, this homologous-rescuing construct restored full daf-16 function in the double mutants, causing them to revert to the uniform dauer-constitutive phenotype of the daf-2 single mutant (χ2 = 162, P < 0.0001). Significantly, daf-2; daf-16 double mutant C. elegans expressing Hc-daf-16.2 from plasmid pMH207-16b also exhibited a complete phenotypic shift from the predominantly continuous pattern of development seen in untransformed worms of this genetic background to the dauer-constitutive pattern associated with daf-2 single mutants (χ2 = 183, P < 0.0001). By contrast, C. elegans daf-2; daf-16 double mutants expressing the Hc-daf-16.1 isoform from plasmid pMH207-16a did not show a significant departure from the phenotype seen in untransformed daf-2; daf-16 double mutants (χ2 = 4: P = 0.2854). It is emphasised that, as a control for any side effects of the rol-6 co-transformation marker, all genetic strains as well as the daf-16 transgenics were transformed with plasmid pRF4.
In the present study, we structurally and functionally characterised the daf-16 orthologue of H. contortus. The full-length cDNA sequences of two isoforms (Hc-daf-16.1 and Hc-daf-16.2) were isolated. Comparison of their predicted amino acid sequences revealed that they possess identical fork head domains, suggesting that they bind to the same DNA sequence in the promoter regions of their target genes. This finding is distinct from the results reported for Ss-DAF-16 of S. stercoralis and Ce-DAF-16 of C. elegans. The fork head domains from two isoforms of either transcription factor (Ss-DAF-16 or Ce-DAF-16) shared identical sequences only in the C-terminus of the domain (66 residues for Ss-DAF-16 and 67 residues of Ce-DAF-16; see Table 2). The fork head domain contains the crucial sequence-specific DNA-binding motif characteristic of fork head transcription factors (Granadino et al., 2000; Carlsson and Mahlapuu, 2002). Differences in the amino acid sequences of the isoforms A and B of Ss-DAF-16 and Ce-DAF-16 occur in the first half of the DNA-binding domain; most of these primary structural differences in these two isoforms are conserved between species (Lin et al., 1997; Ogg et al., 1997; Lee et al., 2001). This information suggests conserved differences in binding specificity of orthologous DAF-16 isoforms. In contrast to Ss-DAF-16 and Ce-DAF-16, the two isoforms of Hc-daf-16 have identical fork head domains. Interestingly, the fork head domain sequences of two hookworm species, A. caninum and A. ceylanicum, are identical to that of Hc-daf-16 (Figs. 2 and and3),3), suggesting that the fork head transcription factors of all three strongylids have similar DNA-binding characteristics and similar functions. In the present study, the sequences of the fork head domains of H. contortus, A. caninum and A. ceylanicum grouped with isoform A of DAF-16 from both S. stercoralis and C. elegans to the exclusion of isoform B of both genes. This finding suggests that fork head transcription factors of strongylid nematodes have a similar function to isoform A of Ce-DAF-16, which is responsible for the major genetic activity from the daf-16 locus for daf-2-mediated dauer arrest and longevity control (Lee et al., 2001). Further studies of the function of fork head transcription factors of strongylid nematodes (e.g., H. contortus) are required but could be challenging, given the limitations with maintaining live worms for longer periods in vitro.
In spite of their identical fork head domains, sequences of the two isoforms of Hc-daf-16 diverge in the N-terminal domains, which comprise 156 amino acid residues in Hc-DAF-16.1 and 109 amino acid residues in Hc-DAF-16.2 (Table 2 and Fig. 2). Comparison of this region of Hc-DAF-16.1 and Hc-DAF-16.2 with that of DAF-16s of other nematodes revealed that N-terminal region sequences of Aca-DAF-16 and Ace-DAF-16 were more similar to those of Hc-DAF-16.1 than to that of Hc-DAF-16.2. For example, an alignment (Fig. 2) showed that amino acid residues of Hc-DAF-16.1 at alignment positions 62–131 were almost identical to those of Aca-DAF-16 and Ace-DAF-16 between alignment positions 45–114, except for four amino acid substitutions. Sequence comparisons also reveal numerous insertions in the N-terminal domains of most parasite DAF-16 molecules compared with C. elegans DAF-16A or DAF-16B and S. stercoralis DAF-16B. This plasticity of structure in the N-terminal domain is likely related to functional divergence of these molecules. N-terminal domains in some fork head transcription factors, such as HNF-3β protein, are required for transcriptional activation (Pani et al., 1992; Qian and Costa, 1995). The difference in this region between the two isoforms reflects their possible difference in the transcriptional activation process.
In contrast to the high amino acid sequence similarity among fork head domains of fork head transcription factors of nematodes, the structures of the genes encoding these factors are distinctly different. Ss-daf-16 and Ce-daf-16 have similar gene organisations, although the numbers of exons and introns are distinct (Massey et al., 2003). Both genes are trans-spliced by SL1 and have alternative splicing, producing two isoforms. The promoter for isoform A (designated α for both C. elegans and S. stercoralis in Fig. 5) of either gene is located in the 5′-UTR upstream of the gene, respectively, whereas the promoter for isoform B (designated β for both species in Fig. 5) is in the large intron of each gene. This structural similarity suggests that the two genes might utilise similar transcriptional and/or splicing mechanisms. In H. contortus, genes encoding two distinct isoforms were identified. Both genes have three small introns and four exons. The main difference between these isoforms (at the nucleotide level) occurs at nucleotide sequence positions 1–471 for Hc-daf-16.1 and 1–330 for Hc-daf-16.2, both at the 5′-end. The rest of the gene sequences (positions 472–3,106 bp for Hc-daf-16.1 and 331–2,965 bp for Hc-daf-16b) share 99% sequence identity. All introns found within and upstream of the region encoding the fork head DNA-binding domain in Ce-daf-16 and Ss-daf-16 are absent from the H. contortus genes (Fig. 5). One of these missing introns contains the β-promoter and first exon of the daf-16b isoforms of C. elegans and S. stercoralis (see Ogg et al., 1997; Massey et al., 2003), suggesting that the loss of this isoform from H. contortus occurred as a result of “intron loss”. Although the presence of daf-16b isoforms in the Ancylostoma spp. could not be excluded by Gao et al. (2009), the substantial similarity of the encoded proteins and lack of daf-16b expressed sequence tags (ESTs) for these species suggests that an organisation similar to that in H. contortus might also occur in a range of Strongylida (clade V; Blaxter, 1998. Nevertheless, H. contortus has two forms of the daf-16 gene. Due to the close similarity of the amino acid sequences and identical genomic organisation of the Hc-daf-16.1 and Hc-daf-16.2 isoforms, the most parsimonious model for their evolution is by gene duplication after an intron loss and their subsequent divergence, particularly in the N-terminal domain. In this case, the Hc-daf-16.2 cannot be considered orthologous to either Ss-daf-16b or Ce-daf-16b, rather both Hc-daf-16.1 and Hc-daf-16.2 are interpreted to be paralogous descendents of an ancestral daf-16a duplicated after the daf-16b encoding intron had already been deleted.
Full complementation of the C. elegans daf-16 (mg54) mutation by Hc-daf-16.2 indicates that the parasite fork head transcription factor encoded therein is capable of fulfilling the dauer regulatory functions of DAF-16 in the C. elegans context. This finding is consistent with the previous observation that the Ce-daf-16b orthologue of another parasitic nematode, S. stercoralis, can also complement the daf-16 (mg54) mutation, albeit incompletely (Massey et al., 2006). The greater efficiency of complementation by Hc-daf-16.2 may reflect the closer phylogenetic relationship between C. elegans and H. contortus (both within clade V), compared with the relatively distant relationship of C. elegans and S. stercoralis (a member of clade IV (Blaxter et al., 1998). A similar correlation between cross-species complementation efficiency and phylogenetic relatedness is seen in the case of parasite orthologues of the heat shock protein 90 (HSP-90) encoded by C. elegans daf-21 (Gillan et al., 2009). These authors found that the H. contortus orthologue Hc-hsp-90 can partially complement a daf-21 mutation in C. elegans, whereas the orthologue Bm-hsp-90 from the filarial nematode, B. malayi (a member of nematode clade III (Blaxter et al., 1998) cannot.
Another similarity between the cross-species complementation studies conducted with daf-16 orthologues in H. contortus and S. stercoralis is the ability of one isoform of the daf-16 orthologue to complement the daf-16 (mg54) mutation in C. elegans and the failure of the second isoform to do so. In contrast, the functions of the isoforms Ce-DAF-16A and Ce-DAF-16B seem to be interchangeable in C. elegans when placed under the control of the daf-16α promoter (Lee et al., 2001) as they were in the present study. Therefore, it is puzzling that the Hc-daf-16.2 and Ss-daf-16b isoforms complement the Ce-daf-16a null mutation, whereas the Hc-daf-16.1 and Ss-daf-16a isoforms do not. One explanation might lie in differences between the free-living and parasitic life cycles. These differences must reflect differences in the regulatory circuitry of these organisms, likely including the associations of transcription factors, such as DAF-16, with other molecules of the regulatory network, including CREB (Nasrin et al., 2000) and nuclear hormone receptors, such as DAF-12 (Dieterich and Sommer, 2009; Wang et al., 2009). The complementation of Ce-daf-16 null mutations by Hc-daf-16.2 and Ss-daf-16b indicates that many of the functions of these molecules are conserved, but the lack of complementation by Hc-daf-16.1 and Ss-daf-16a may reflect independent divergence of these molecules, as these two taxa evolved independently to the parasitic state. In each case, these divergences have rendered these transcription factors unable to function in the genetic milieu of C. elegans. It would be interesting to know whether the paralogous DAF-16A molecules in H. contortus and S. stercoralis can complement each other. These experiments would necessarily focus on the N-terminal domains of these molecules, which have been under-investigated compared with the fork head and C-terminal domains.
Interest in daf-16 and other insulin-like signal transduction intermediates in parasitic nematodes stems, in part, from parallels drawn between the biologies of dauer larvae of C. elegans and the iL3 of parasitic nematodes. The role of insulin-like signalling, in general, and of daf-16, in particular, in regulating dauer development in C. elegans invites the hypothesis that similar mechanisms regulate development of parasitic L3. Complementation of mutations in C. elegans by insulin-like signalling intermediates from parasites, as shown here, constitutes a line of evidence supporting this hypothesis. However, viewed strictly, such findings only prove conservation of biochemical functionality in the C. elegans context, and functional homology in the parasite context awaits some method of altering the expression or function of a parasite gene of interest and assessing resultant phenotypes. Such direct functional genomic methods have been challenging to develop for parasitic nematodes. However, a recent study (Castelletto et al., 2009) shows that expressing mutant forms of FKTF-1/Ss-DAF-16 designed to exert a dominant negative effect on the endogenous transcription factor in transgenic S. stercoralis brings about a reversal of some dauer-like characteristics in post free-living larvae of this parasite and even induces an abnormal L3–L4 moult in some individuals in this phase of the life cycle. Together with functional data of this kind, cross-species complementation, as reported here, strongly supports the hypothesis of a similar developmental regulation of free-living dauer larvae and the iL3 of parasitic nematodes by insulin-like signal transduction.
In conclusion, we have identified genes encoding two isoforms of Hc-daf-16, an orthologue of C. elegans daf-16 from H. contortus. We also characterised the cDNA and genomic DNA structures of Hc-daf-16.1 and Hc-daf-16.2, ascertained their transcription in key developmental stages of H. contortus and demonstrated conservation of the regulatory function of dauer in Hc-daf-16.2 using C. elegans as a genetic surrogate. The findings from this study provide further evidence of the functional conservation of daf-16, and of insulin-like signalling, generally, between key parasitic nematodes and C. elegans. This study has important implications for understanding the developmental processes of parasitic nematodes, particularly those of the order Strongylida. Confirmation of the role of Hc-daf-16 in arrest and reactivation of the iL3 stage of H. contortus awaits direct functional assessment in the parasite itself.
This work was supported by grants from the Australian Research Council (ARC) (LP0667795 and LX0882231), Genetic Technologies Limited, Meat and Livestock Australia, the Australian Academy of Science, the Australian-American Fulbright Commission (R.B.G.), the National Institutes of Health (AI-50688 to J.B.L.) and The Ellison Medical Foundation (ID-IA-0037-02 to J.B.L.). The authors thank Ben Datu for sharing primers DAF-16F100 and DAF-16R100. The C. elegans strains used in this study were originally provided by the Caenorhabditis Genetics Center, University of Minnesota, which is funded by the NIH National Center for Research Resources.
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