Cloning and characterisation of genes encoding two transforming growth factor-β-like ligands from the hookworm, Ancylostoma caninum

International Journal for Parasitology 35 (2005) 1477–1487 www.elsevier.com/locate/ijpara

Cloning and characterisation of genes encoding two transforming growth factor-b-like ligands from the hookworm, Ancylostoma caninum* Tori C. Freitas, Prema Arasu* Department of Molecular Biomedical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA Received 13 May 2005; received in revised form 24 June 2005; accepted 25 July 2005

Abstract To elucidate the role of transforming growth factor b (TGF-b) signalling in the arrest/reactivation pathway of the Ancylostoma caninum hookworm, two parasite-encoded TGF-b-like ligands were cloned and characterised. Ac-dbl-1 showed 60% amino acid identity to the Caenorhabditis elegans dbl-1 gene, which regulates growth while Ac-daf-7 showed 46% amino acid identity to Ce-daf-7 which regulates arrested development. Exon/intron organisation of the genes for Ac-dbl-1 and Ac-daf-7 were different from that of the corresponding C. elegans genes with nine and 10 exons, respectively, and introns ranging in size from 56 to 2556 bp. Based on real-time reverse transcriptase (RT)-PCR, Ac-dbl-1 and Ac-daf-7 were expressed in all stages tested, i.e. egg, first/second stage larvae (L1/L2), infective third stage larvae (iL3), serum-stimulated third stage larvae (ssL3), and male and female adult worms. Expression of Ac-dbl-1 peaked in the adult male stage suggesting a similar role to Ce-dbl-1 in regulating male tail patterning. Ac-daf-7 expression was at a maximum in the arrested iL3 and reactivated ssL3 stages, which differs from that of Ce-daf-7 expression and may be unique to parasitic nematodes that have an obligate requirement to undergo developmental arrest. In support of the PCR results, antibodies to the A. caninum TGF-b-like ligands detected proteins in iL3, ssL3, and adult worm extracts. Immunofluorescent studies showed that Ac-daf-7 is expressed in the anterior region of the iL3 similar to Ce-daf-7, which is localised to the ASI chemosensory neurons. q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Ancylostoma caninum; TGF-b; dbl-1, daf-7; Development

1. Introduction Members of the transforming growth factor b (TGF-b) superfamily of cytokines regulate a diverse set of cellular processes including cell proliferation, differentiation, and death (Massague et al., 2000). This highly conserved class of intercellular signalling molecules is found in all metazoans including five different genes in Caenorhabditis elegans, nine in Drosophila melanogaster, and 42 in mammals (Lander et al., 2001). TGF-b cytokines fall into two subfamilies based on sequence similarity and the different signalling pathways they activate, namely, * Nucleotide sequence data reported in this paper are available in the GenBank database under accession numbers Ac-dbl-1 (AY942843), Acdaf-7 (AY942844), Ac-dbl-1 gene (AY942845), Ac-daf-7 gene 5 0 (AY942846), and Ac-daf-7 gene 3 0 (AY942847). * Corresponding author. Tel.: C1 919 513 6530; fax: C1 919 513 6455. E-mail address: [email protected] (P. Arasu).

the TGF-b/activin/nodal subfamily and the BMP/GDF/ MIS (bone morphogenetic protein/growth and differentiation factor/Muellerian inhibiting substance) subfamily. All TGF-b proteins share several common characteristics. The functional molecule exists as a homodimer produced through the cleavage of the larger polypeptide releasing the C-terminal active domain (Massague et al., 1992). This dimer is held stable through hydrophobic interactions and the formation of a ‘cysteine knot’ utilising six of the seven conserved C-terminal cysteine residues in three disulfide bridges (Sun and Davies, 1995). Three of the five TGF-b genes found in the C. elegans genome have been well characterised. One homolog, dbl-1 (also called cet-1), is involved in regulating body size and male tail patterning and is a member of the BMP/GDF/MIS subfamily (Suzuki et al., 1999). A second, daf-7, is involved in controlling entry into and exit from the developmentally arrested dauer stage of C. elegans and is a member of

0020-7519/$30.00 q 2005 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2005.07.005

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the TGF-b/activin/nodal subfamily (Ren et al., 1996). By sensing environmental cues, including temperature, population density, and food availability, C. elegans uses at least three signalling pathways to determine whether or not to enter the dauer state of developmental arrest including a G-protein pathway, an insulin-like signalling pathway, and a TGF-b pathway (Ren et al., 1996; Riddle and Albert, 1997; Birnby et al., 2000; Tissenbaum et al., 2000). In the TGF-b pathway, the expression of daf-7 early in development prevents dauer entry and null mutants of this gene result in a constitutive dauer phenotype (Ren et al., 1996). A third homolog to be characterised in the C. elegans genome, unc-129, is involved in guiding axon growth (Colavita et al., 1998), while the remaining two, tig-2 and CE27471, have yet to be characterised. Members of the TGF-b family have also been identified in the Brugia sp. filarial parasitic nematodes and are suggested to be involved in regulating growth of the worm or modulating the host immune response by interacting with host TGF-b signalling (Gomez-Escobar et al., 1998; 2000). Virtually all parasitic nematodes appear to have an obligate stage in their life cycles involving developmental arrest. The life cycle of the hookworm Ancylostoma caninum contains two stages where the larvae could be considered developmentally arrested, the pre-parasitic infective L3 in the environment and the tissue-arrested L3 within the infected host. The tissue-arrested L3s are a subpopulation of larvae that halts maturation to bloodfeeding intestinal adults, instead becoming quiescent in the somatic tissues. These latent parasites are relatively resistant to anthelminthics typically used to eliminate intestinal adult worms (Arasu, 1998). Furthermore, tissuearrested L3s display an interesting propensity to reactivate during pregnancy, resulting in their transmission via milk to nursing puppies. Previous in vitro studies have shown that recombinant mammalian TGF-b stimulates feeding and the reactivation of tissue-arrested A. caninum L3s (Arasu, 2001). To investigate the potential role of parasite-encoded TGF-b in the reactivation process, we cloned and characterised genes encoding two A. caninum TGF-b-like ligands and monitored the transcription and translation of these gene products in various developmental stages.

2. Materials and methods 2.1. Parasites and animals Infective A. caninum L3s (iL3) were harvested from 7-day-old co-cultures of charcoal and feces from laboratory Beagle dogs (Marshal Farms, North Rose, NY, USA) infected with a North Carolina (Wake County) strain of hookworm (Arasu and Kwak, 1999). The use of dogs in this study was approved by the Institutional Animal Care and Use Committee at North Carolina State University. To generate serum-stimulated L3s (ssL3s), iL3s (5000 per

500 ml) were induced to feed in vitro by incubating at 378 C, 5% CO2 for 20–24 h in the presence of 5% normal dog serum in individual wells of a 24-well plate (Hawdon and Schad, 1990). Larvae were scored as feeding based on ingestion of FITC-conjugated bovine serum albumin (Sigma, St Louis, MO, USA) and presence of fluorescent intestinal tracts by UV microscopic examination; the pool of ‘activated’, feeding ssL3s showed O80% positive tracts. A. caninum adults were collected from the intestines of euthanised dogs from an animal shelter in Wake County, North Carolina; typically, there is a greater percentage of the larger female worms relative to male worms. Eggs were collected via sucrose flotation (Nolan et al., 1994). Mixed first- and second-stage larvae (L1/L2) were collected by hatching eggs on 1.5% agar plates for 24–36 h; unhatched eggs were removed and the L1/L2 were rinsed with PBS containing 20 mg/ml gentamicin and lincomycin before freezing at K70 8C. 2.2. Ac-dbl-1 isolation Degenerate primers within the conserved regions of the TGF-b sequence were used in PCR with 200 ng of A. caninum genomic DNA template as previously reported (Gomez-Escobar et al., 1998): TGF-A (forward) 5 0 TGGCANGAYTGGATHRTNGCNCC-3 0 and TGF-C (reverse) 5 0 -GGNACRCARCANGGNGGNGG-3 0 . Reactions were run on a MJR PTC-200 DNA Engine at 1 cycle of 94 8C for 5 min, 40 cycles of 94 8C for 1 min, 56 8C for 1 min and 72 8C for 3 min, followed by 1 cycle of 72 8C for 3 min. The resulting 300 bp product had sequence homology to C. elegans dbl-1 and was used as a probe to screen an A. caninum Bam HI genomic DNA lGem11 (Promega, Madison, WI, USA) library using low stringency conditions including hybridisation at 37 8C and room temperature washes with 5!SSC. A 2.5 kb Sal I fragment from a 10 kb insert genomic clone which hybridised to the 300 bp PCR product was sub-cloned and sequenced (GenBank accession number AY942845). To obtain the cDNA, RT-PCR was performed using a 5 0 primer to the conserved 22 nucleotide nematode splice leader sequence (SL1; Blaxter and Liu, 1996) and 3 0 RACE (SMART RACE kit, BD Biosciences Clontech, Palo Alto, CA, USA). The primers used in the SL1 PCR were (forward) 5 0 -G GTTTAATTACCCAAGTTTGAG-3 0 and (reverse) 5 0 CATGGTTGGTTGCGTTCA-3 0 using the same cycling conditions as above and 250 ng adult cDNA as template (Section 2.4). The gene specific primer for 3 0 rapid amplification of cDNA ends (RACE) was 5 0 -CGCGACCGAAAGAACAA-3 0 and PCR was performed as per manufacture’s protocol. The overlapping SL1 primed fragment and the 3 0 RACE fragment were each cloned into pT7-Blue3 and sequenced (GenBank accession number AY942843).

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2.3. Ac-daf-7 isolation Serum stimulated A. caninum L3s provided from our laboratory to the Washington University, Genome Sequencing Center, Parasitic Nematode Sequencing Project (www. nematode.net), produced an expressed sequence tag (EST), pb34h07.y1, GenBank accession number BQ125337, which showed sequence homology to the 5 0 end of C. elegans daf7. Sequencing of the entire clone confirmed that it encoded the full-length cDNA (GenBank accession number AY942844). Bam HI and Sau 3A lGem11 genomic clones, each with approximately 10 kb inserts, were isolated using the radiolabeled cDNA as a probe. By Southern analysis, the Bam HI genomic clone was shown to encode the 5 0 end of the gene (GenBank accession number AY942846), while the Sau 3A clone encoded the 3 0 end of the gene (GenBank accession number AY942847). The gene specific primers 5 0 -TTCACAGCATTGACGATG-3 0 and 5 0 -AGAGCAGGAACATTTGCG-3 0 were used to prime the initial sequencing reaction for the respective genomic clones followed by primer walking. 2.4. RNA extraction and real-time reverse transcriptase (RT)-PCR Total RNA from all stages of A. caninum were extracted using RNA-STAT60 (Tel-Test, Inc., Friendswood, TX, USA). First strand cDNA was synthesised using 10 mg of DNaseI treated total RNA, SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA), and an oligo dT primer in a total of 40 ml (250 ng/ml). To confirm absence of contaminating genomic DNA, RT-minus cDNA reactions were analysed by conventional PCR for each primer set. A second confirmation (data not shown) was performed using intron-spanning A. caninum actin primers (forward 5 0 TCCCATCAATAGTTGGACGACC-3 0 and reverse 5 0 GTATGATGCCAGATCTTCTCCAT-3 0 ). Ac-dbl-1 and Ac-daf-7 transcript levels for egg, L1/L2, iL3, ssL3, and adult male and female stages were quantified relative to the A. caninum 60S acidic ribosomal protein (GenBank accession number BF250585) based on previously conducted reference gene validation studies (Trivedi and Arasu, 2005). Real-time reverse transcriptase (RT)-PCR was performed on a Bio-Rad iCycler iQ Detection System using SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA). Primer sequences were, respectively: Ac-dbl-1 forward 5 0 -AAGGTTTCCTCCGTC GATTT-3 0 and reverse 5 0 -CCATCCTCACTTGCTTCCTC3 0 ; Ac-daf-7 forward 5 0 -CTCACCGTACATTCGAAAAC3 0 and reverse 5 0 -CATGCCCATTGACGATAACA-3 0 ; Ac60S forward 5 0 -GTCGGAATCGTCGGAAAGTA-3 0 and reverse 5 0 -GTCTTGTTGCATTTCGAGCA-3 0 ; and cAMP dependent protein kinase A, Ac-pka forward 5 0 -ATGGGAGAATCCAGCAGA-3 0 and reverse 5 0 -TCCAAAATCTTCATGGCAAA-3 0 (Hawdon et al., 1995). Total reaction volume was 25 ml including 12.5 ml SYBR Green Supermix,

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300 nM forward and reverse primers, and 1 ml cDNA (250 ng). The thermal cycler program was 1 cycle of 95 8C for 3 min, followed by 45 cycles of 95 8C for 1 min, 55 8C for 30 s and 72 8C for 30 s. Fluorescence was measured at the end of each cycle during the 72 8C step. A melting curve was generated after the 45 cycles by increasing the temperature from 55 to 100 8C at 0.5 8C increments for 90 cycles of 5 s each with fluorescence detected at the end of each cycle. PCR efficiency was determined for each set of primers in every reaction by including a 10-fold serial dilution of iL3 cDNA (250–0.025 ng). To determine PCR efficiency (E), cycle thresholds from the serial dilutions were plotted and the slope was used in the equation EZ 10(K1/slope); fluorescence readings were taken using the ‘PCR Base Line Subtracted Curve Fit’ method (Pfaffl, 2001). Quantification of Ac-dbl-1 and Ac-daf-7 transcript levels relative to Ac-60S were calculated using the following equation: ratio Z(EAcK60S)CT/(EAc-TG)CT where EAc-60S is the PCR efficiency of the reference gene Ac-60S, EAc-TG is the PCR efficiency of target genes Ac-dbl-1 or Acdaf-7, and CT is the cycle threshold. Given that cycle threshold and cDNA levels are inversely related, the calculated ratio is equal to the ratio of Ac-dbl-1 and Acdaf-7 cDNA relative to the Ac-60S cDNA (Salmon et al., 2004) with larger ratios indicating presence of more A. caninum transcript at a particular stage. In addition to meltcurve analysis, real-time PCR products were run on 2% ethidium bromide stained agarose gels at the end of the cycling program (45 cycle) to verify the presence of a single amplicon and further demonstrate the presence of transcript for both Ac-dbl-1 and Ac-daf-7 at each of the stages tested. Each experiment was repeated either two or three times. 2.5. Recombinant protein expression and antisera production Bam HI (forward) and Eco RI (reverse) tagged primers were designed to amplify the full-length Ac-dbl-1 coding sequence, minus the hydrophobic signal peptide, and the C-terminal Ac-daf-7 coding region. For amplifying fulllength Ac-dbl-1, the forward primer was 5 0 CCCGGATCCCGATTTGGTTTCCCCCGT-3 0 and reverse primer was 5 0 -CCCGAATTCGCGACACCCG0 CAAGCTTC-3 . C-terminal Ac-daf-7 was amplified using the forward primer 5 0 -CCCGGATCCAGTCCGGCCG TCTGTATGCCG-3 0 and reverse primer 5 0 -CCGAATTCAGAGCAGGAACATTTGCG-3 0 . The cycling parameters were 1 cycle of 94 8C for 2 min, followed by 30 cycles of 94 8C for 30 s, 65 8C for 30 s and 72 8C for 1 min, ending with 1 cycle of 72 8C for 10 min using PfuTurbo DNA Polymerase (Stratagene, La Jolla, CA, USA). The amplified products were cloned into the expression vector pET28a (Novagen, Madison, WI, USA) and sequence analysis confirmed that both constructs contained the six-histidine tag at the N-terminus. The C-terminal portion of Ac-daf-7 was free of mutations while the Ac-dbl-1 construct

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contained a thymidine insertion (likely due to the PCR amplification) resulting in an early stop codon and a truncation of 23 amino acids. The recombinant proteins were induced in Escherichia coli BL21 (DE3) and purified using the HIS-bind resin batch method as per manufacturer’s instructions and concentrated in PBS containing 6 M urea (Novagen, Madison, WI, USA). Antibodies to Ac-dbl-1 and Ac-daf-7 were generated by Cocalico Biologicals, Inc., PA, USA. Rabbits were pre-bled followed by subcutaneous inoculation of 100 mg of protein in Freund’s complete adjuvant. Subsequent boosts using 50 mg of protein in Freund’s incomplete adjuvant occurred on days 14, 21, and 49, followed by exsanguination on day 64. 2.6. Library construction Genomic DNA was extracted from adult A. caninum as previously described (Arasu et al., 1994) and restricted with either Sau 3A or Bam HI (Promega, Madison, WI, USA). Purified products in the 10–15 kb range were ligated into the lGem11 Bam HI bacteriophage vector (Promega, Madison, WI, USA) as per manufacturer’s instructions, resulting in two different genomic DNA libraries. 2.7. DNA sequencing and analysis Sequencing reactions were performed by the DNA Sequencing Core Lab, University of Florida, Gainesville, FL, USA. The BLAST algorithm (Altschul et al., 1997) was used for homology searches at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The Clustal W algorithm was used for sequence alignments. The unrooted radial tree was drawn using deduced amino acid sequences within the C-terminal domain and distances were calculated using the Dayhoff Pam matrix and neighbour-joining algorithm in the PHYLIP software package developed by Felsenstein, University of Washington, Seattle, WA, USA (http://evolution.genetics.washington.edu/phylip.html); percentages at branch points are based on 1000 bootstrap runs. 2.8. Western analysis Total antigen extracts from iL3, ssL3, and adult hookworms were prepared by grinding worms in liquid nitrogen and dissolving in 2!SDS buffer (100 mM Tris– HCl pH 6.8, 200 mM dithiothreitol, 4% SDS). The extracts were boiled at 95 8C for 5 min, sonicated, and spun at 12, 000 g to remove the residual debris. Extracts were separated on 12% SDS–PAGE gels and electroblotted onto NitroPure membranes (Osmonics, Inc., Minnetonka, MN, USA) at w3 V/cm for 16-18 h. Native Ac-dbl-1 and Ac-daf-7 were detected using the corresponding polyclonal rabbit anti-sera at 1:10,000 and the ECL Western blotting system (Amersham, Buckinghamshire, UK). Blots probed with pre-immunisation rabbit sera served as negative controls.

To verify antibody specificity, anti-Ac-dbl-1 and anti-Acdaf-7 sera were each preabsorbed to their corresponding recombinant proteins blotted onto NitroPure membranes as described above; on subsequent Western blot analyses, the preabsorbed sera showed no reactivity to native proteins in the 20 kD range (data not shown). 2.9. Immunofluorescent localisation of native proteins Immunofluorescent localisation was conducted as previously described (Goverse et al., 1994; de Boer et al., 1999). Briefly, iL3s were fixed overnight in 2% paraformaldehyde/M9 buffer and sectioned with a vibrating razor blade. The worm sections were digested with 0.5 mg/ml proteinase K followed by freeze-treatment with ice/ methanol and acetone. The worms were washed with PBS/0.5% Triton X (PBST) and blocked with 10% goat serum and a cocktail of protease inhibitors (iodoacetamide, leupeptin, phenylmethylsulfonyl fluoride and pepstatin A) to retain antigen integrity. Worms were then incubated with rabbit anti-Ac-daf-7, rabbit anti-Ac-dbl-1, sera from A. caninum iL3 infected mice (positive control), and rabbit anti-Heterodera glycines cellulase (plant parasitic nematode Hg-ENG-1 negative control; Wang et al., 1999) at 1:500 followed by 1:500 FITC conjugated goat-anti-rabbit IgG (Sigma, St Louis, MO, USA) or FITC conjugated goat-antimouse IgG (Sigma, St Louis, MO, USA). The worms were washed three times for 10 min each with PBST, resuspended in distilled water, and examined by fluorescence microscopy.

3. Results 3.1. Cloning and sequence analysis of Ac-dbl-1 and Ac-daf-7 cDNAs Completion of the C. elegans genome sequence suggested the presence of five TGF-b homologs. The first A. caninum TGF-b homolog was cloned through degenerate PCR using genomic DNA as template and primers previously used to identify Bm-tgh-1 from Brugia malayi (Gomez-Escobar et al., 1998). Sequence analysis of the resulting 288 bp amplicon contained 117 bp of coding sequence showing homology to the TGF-b superfamily, with most significant similarity to C. elegans dbl-1, and was named Ac-dbl-1. Further attempts by PCR using degenerate primers or non-stringent library screenings with the 288 bp product or the C. elegans daf-7 cDNA failed to identify other TGF-b-like ligands in A. caninum. The second A. caninum TGF-b homolog, Ac-daf-7, was identified from ssL3s in a collaboration with the Parasitic Nematode Sequencing Project. The Ac-dbl-1 cDNA was amplified as a PCR product of 992 bp and subsequent sequencing confirmed the presence of the 5 0 end based on sequence homology and a methionine

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Fig. 1. Alignment of C-terminal deduced amino acid sequences from Ac-DBL-1 and three other members of the BMP/GDF/MIS subfamily of TGF-b proteins. Amino acids identical to those in Ac-DBL-1 are shaded. Gaps, represented by dashs, are introduced to aid in alignment. Numbers at the end of each line represent amino acid position in each respective sequence. Accession numbers for above sequences: Brugia malayi TGH-1 (Bm-TGH-1), AF012878; Caenorhabditis elegans DBL-1 (Ce-DBL-1), NM_072308; Homo sapiens BMP-2 (Hs-BMP-2), NM_001200.

downstream of the splice leader. The 3 0 end of the cDNA was isolated via 3 0 RACE with a gene specific primer designed from the genomic sequence and adult cDNA as template. A 1.5 kb product was cloned and sequencing confirmed the presence of the 3 0 end of the transcript. The 1.4 kb cDNA, generated from a splice of the overlapping RT-PCR products, codes for 354 amino acids and contains a number of characteristics of members of the TGF-b superfamily. The N-terminus contains a putative hydrophobic signal sequence shared by many of the secreted members of the superfamily. A putative basic proteolytic cleavage site (separating the pro-domain from the mature C-terminal peptide) is found at position 231 as RRDR. Downstream of the cleavage site, the C-terminal ligand domain contains all of the invariant amino acids, including seven conserved cysteine residues involved in the intra- and inter-disulfide bonds necessary to form the cysteine knot structure common to this superfamily of growth factors. Two other invariant amino acids include proline-274 and glycine-284, which are essential for the proper tertiary structure of the mature homodimer. For Ac-daf-7, the 1.5 kb EST clone obtained from the Parasitic Nematode Sequencing Project encodes a predicted 355 amino acids with closest homology to C. elegans daf-7 including a similarly positioned start

methonine and stop codon. This homolog does not appear to have a hydrophobic signal sequence at its N-terminus and SL1 primed PCRs failed to amplify a product, unlike its Ac-dbl-1 counterpart. However, other characteristics of the TGF-b superfamily are present. A predicted tetra-basic cleavage site is found starting at amino acid 237 as RRKR and there are nine conserved cysteine residues in the C-terminal domain of Ac-daf-7. The invariant proline and glycine residues can be found at amino acids 272 and 282, respectively. While the pro-domain of most TGF-b proteins is divergent, the active domain is more conserved with significant homology between the A. caninum sequences and those of C. elegans and B. malayi. Within the active domain, Ac-dbl-1 is 60% identical at the amino acid level to C. elegans dbl-1 and 54% identical to Bm-tgh-1 (Fig. 1). In addition, Ac-dbl-1 is 50% identical to mammalian BMP2 at the amino acid level suggesting that this protein belongs to the BMP/GDF/DPP subfamily of TGF-b. Acdaf-7 showed 46% amino acid identity to both C. elegans daf-7 and Bm-tgh-2 with less similarity to the mammalian TGF-b1 homolog at 28% (Fig. 2). Within their conserved C-terminal domains, the two A. caninum cDNAs, Ac-dbl-1 and Ac-daf-7, are 30% identical at the deduced amino acid level.

Fig. 2. Alignment of C-terminal deduced amino acid sequences from Ac-DAF-7 and three other members of the TGF-b/activin/nodal subfamily of TGF-b proteins. Amino acids identical to those in Ac-DAF-7 are shaded. Gaps, represented by dashs, are introduced to aid in alignment. Numbers at the end of each line represent amino acid position in each respective sequence. Accession numbers for above sequences: Brugia malayi TGH-2 (Bm-TGH-2), AF104016; Caenorhabditis elegans DAF-7 (Ce-DAF-7), NM_064864; Homo sapiens TGF-b1 (Hs-TGF-b1), NM_000660.

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the TGF-b/activin/nodal subfamily and is most similar to C. elegans daf-7 (Fig. 3). 3.2. Cloning and sequence analysis of Ac-dbl-1 and Ac-daf-7 genes

Fig. 3. Phylogenetic representation of Ac-DBL-1 and Ac-DAF-7 among members of the TGF-b superfamily within the conserved C-terminal region. Percentages at branch points are based on 1000 bootstrap runs. Accession numbers for sequences used in analysis: Brugia malayi TGH-1 (Bm-TGH-1), AF012878; B. malayi TGH-2 (Bm-TGH-2), AF104016; Caenorhabditis elegans DAF-7 (Ce-DAF-7), NM_064864; C. elegans DBL-1 (Ce-DBL-1), NM_072308; C. elegans TIG-2 (Ce-TIG-2), NM_071870; C. elegans UNC-129 (Ce-UNC-129), AF029887; Homo sapiens Activin (Hs-ACTIVIN), X82540; H. sapiens BMP-2 (Hs-BMP-2), NM_001200; H. sapiens GDF-8 (Hs-GDF-8, myostatin), AF104922; H. sapiens Inhibin (Hs-INHIBIN), X72498; H. sapiens TGF-b1 (Hs-TGF-b1), NM_000660; H. sapiens TGF-b2 (Hs-TGF-b2), NM_003238, Parastrongyloides trichosuri DAF-7 (Pt-DAF-7), BI743385; Strongyloides ratti (SrDAF-7); AY672707; Strongyloides stercoralis (Sst-TGH-1), AY662390.

Phylogenetic analysis further demonstrated that these two sequences belong to separate groups of the TGF-b superfamily. Ac-dbl-1 clearly groups with other sevencysteine members of the BMP/GDF/DPP subfamily with its closest relative being C. elegans dbl-1 (Fig. 3). Ac-daf-7 groups with other nine-cysteine containing proteins in

The 288 bp genomic PCR product for Ac-dbl-1 was used to screen a Bam HI lGem11 A. caninum genomic library resulting in the isolation of a single clone containing a 10 kb insert. Sequencing the Ac-dbl-1 portion of the insert showed the clone contained the entire gene organised into nine exons spanning 4.36 kb of DNA (Fig. 4(a)). The exons and introns range in size from 55 to 179 bp, and 56 to 1803 bp, respectively, with exon/intron boundaries conforming to the conventional splicing pattern (Mount, 1982). Exons I through V contain the pro-protein and exons V through IX contain the active portion of the protein. The entire coding sequence for Ac-daf-7 was used to screen a Bam HI and a Sau 3A lGem11 A. caninum genomic library. One clone from each library was isolated, each with approximately 10 kb inserts. Sequencing the Acdaf-7 portion of each insert produced the entire gene with partial sequence for the sixth intron (Fig. 4(b)). The gene is organised into 10 exons spanning at least 8.5 kb of DNA. The exons and introns range in size from 74 to 166 bp, and 64 to 2556 bp, respectively, and exon/intron boundaries also conformed to normal splicing (Mount, 1982). Exons I through VII encode the pro-protein while exons VII through X encode the mature product. 3.3. Transcription and protein expression profiles of Ac-dbl-1 and Ac-daf-7 Since the full-length cDNA for Ac-dbl-1 was obtained using adult worm cDNA as a template, and the Ac-daf-7 cDNA was obtained from an ssL3 EST clone, some of the life stage associations for these transcripts were known. To determine the overall transcription profile of expression,

Fig. 4. Genomic organisation of Ac-dbl-1 and Ac-daf-7. Shaded boxes represent exons with the numbers above designating length in nucleotides. Lines represent introns with the numbers below designating length in nucleotides.

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Fig. 6. Expression pattern of Ac-DBL-1 and Ac-DAF-7 native proteins. Antigen extracts from Ancylostoma caninum infective L3s, serum stimulated L3s, and mixed male/female (M/F) adult worms were loaded based on comparable staining profiles observed with Coomassie Blue staining of the gels, blotted, and probed with rabbit antisera directed against (a) recombinant Ac-DBL-1 or (b) recombinant Ac-DAF-7 (upper panels) and the corresponding pre-immunisation serum (lower panels). The normal dog serum (NDS) used for serum stimulation of larvae was also loaded as antigen to serve as negative control. Fig. 5. Stage specific expression of Ac-dbl-1 and Ac-daf-7 transcripts. First strand cDNA from various Ancylostoma caninum stages were used as template in real-time RT-PCR. (a) Ethidium bromide stained gels of realtime RT-PCR products after 45 cycles of amplification. Numbers to the right of the figure represent PCR product length for each transcript. The life-cycle stages are indicated on top of the gels. (b) Expression data from real-time RT-PCR. The life-cycle stage tested is indicated on the x-axis. The y-axis (on a scale of 10K3) represents ratios of Ac-dbl-1 and Ac-daf-7 expression relative to the Ac-60S reference gene. Data are presented as the meanGSEM from replicates within one representative experiment.

real-time RT-PCR was performed on cDNA from eggs, L1/L2, iL3, ssL3, and adult males and females relative to the previously validated 60S acidic ribosomal protein reference gene. Similar results were obtained in analyses of Ac-daf-7 expression levels relative to cyclic AMP-dependent protein kinase A (data not shown). Amplified products were not observed in the negative controls of water, E. coli, and dog liver cDNA (data not shown). As seen in Fig. 5(a), both Acdbl-1 and Ac-daf-7 transcripts were detected in all stages tested, but had maximal expression in specific stages. Acdbl-1 expression peaked in the adult male while Ac-daf-7 peaked at the iL3 and ssL3 stages (Fig. 5(b)). Furthermore, expression of Ac-dbl-1 was consistently higher than that of Ac-daf-7 relative to the endogenous Ac-60S reference gene (Fig. 5(b)). Ac-daf-7 expression detected in the embryos was not considered to be contamination from other developmental stages because the eggs were collected from fresh feces and immediately frozen. These expression patterns were reproducible between different real-time RT-PCR experiments and several distinct RNA/cDNA preparations. Western analyses using polyclonal antibodies against recombinant Ac-dbl-1 and Ac-daf-7 were used to determine their protein expression profiles in iL3, ssL3, and mixed adults, i.e. stages from which quantitative amounts of protein could be generated. A 20 kDa protein was

recognised by both anti-Ac-dbl-1 and anti-Ac-daf-7 in all stages tested (Fig. 6). The faint signal intensity for Ac-dbl-1 reactivity with adult stage antigens could be due to the greater percentage of larger female worms relative to male worms in the antigen preparation as well as the lesser sensitivity of the Ac-dbl-1 antibody which required longer autoradiographic exposure times than blots incubated with the anti-daf-7 antibody. The 20 kD proteins were not detected with the pre-immunisation sera or preabsorbed sera controls (data not shown). In immunolocalisation studies, anti-Ac-daf-7 antibodies showed specific binding to the anterior region of the larva (Fig. 7(a) and (b)). Neither the rabbit anti-Ac-dbl-1 nor antiH. glycines cellulase antibodies (de Boer et al., 1999) showed binding (data not shown) while anti-A. caninum sera from an iL3 infected mouse localised to antigens in many regions and cells throughout the larval sections (Fig. 7(c)). 4. Discussion TGF-b signalling is known to be involved in regulating the arrested development of the free-living nematode, C. elegans (Ren et al., 1996), and is hypothesised to be one of the pathways regulating recovery from environmental as well as host-associated arrest of A. caninum (Rajan, 1998; Arasu, 2001). This report describes the cloning and characterisation of TGF-b homologs from A. caninum to better decipher the role of parasite-encoded ligands in the arrest/reactivation process. Two TGF-b-like ligands were identified in A. caninum: Ac-dbl-1, which shares homology to C. elegans dbl-1, the ligand associated with growth regulation and development of the male tail, and Ac-daf-7, which shares homology to C. elegans daf-7, the ligand regulating arrest and reactivation.

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Fig. 7. Immunofluorescence localisation analyses. Panels to the left are bright-field images while panels to the right are fluorescent images of the larval sections (a) and entire larva (b) detected with anti-Ac-DAF-7 antibodies. Arrows point to localisation of Ac-DAF-7 to the anterior region of the larva. Solid triangles point to the non-specific labeling on the cut region of an iL3. The positive control mouse anti-Ancylostoma caninum iL3 antiserum recognised proteins in many regions and cells of the iL3 larva (c). Scale bar: 20 mm.

The nucleotide and deduced amino acid sequence of these two genes are most similar to TGF-b homologs from C. elegans, especially within the C-terminal mature peptide. Interestingly, the intron/exon structures of the genes differ between the two nematodes that belong to the same phylogenetic clade V (Blaxter et al., 1998). For both parasite-encoded genes, intron size, exon number, and overall length of the genes are greater as compared to their C. elegans counterparts but the significance of this finding is unclear. From a comparison of other A. caninum proteincoding genes with associated mRNAs in the GenBank database, the Ac-dbl-1 and Ac-daf-7 genes are the longest; however, the average intron size for all available sequences is 499 bp (G665 bp) versus an average of 466 bp (G852 bp) for C. elegans (Deutsch and Long, 1999) suggesting that A. caninum genes may not have significantly larger introns than C. elegans. These analyses of gene structure further demonstrate differences between phylogenetic clades as Sst-tgh-1, a daf-7 homolog from the

Clade IV nematode Strongyloides stercoralis (Genbank Accession number AY662390) contains only one, relatively short, intron (Massey et al., 2005). In addition, it is worth noting that introns in C. elegans have been shown to contain elements that regulate temporal/spatial expression patterns (Nam et al., 2002). The phylogram (Fig. 3) further shows that the daf-7 sequences of A. caninum and C. elegans from clade V are more similar to that of Brugia sp. from clade III as compared with Strongyloides sp. and Parastrongyloides sp. from Clade IV. Both Ac-dbl-1 and Ac-daf-7 cDNA sequences displayed important hallmarks of the TGF-b superfamily including at least seven conserved cysteine moieties, a putative tetra-basic proteolytic cleavage site, and two other invariant amino acids. Ac-dbl-1 also has an N-terminal hydrophobic region and the highly conserved SL1 spliced leader consensus sequence found on a large number of nematode transcripts (Blaxter and Liu, 1996), but neither was noted with Ac-daf-7.

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The transcriptional profile of Ac-dbl-1 matched the pattern for C. elegans dbl-1 with maximal expression in the adult male (Fig. 5(b)) suggesting that it may also be involved in patterning of the male tail (Suzuki et al., 1999). In contrast, the transcriptional profiles of Ac-daf-7 differed from that of daf-7. In C. elegans, daf-7 transcript is most abundant in the L1 when the decision on dauer or non-dauer development is made (Ren et al., 1996). Green fluorescent protein (GFP) reporter expression under the control of the daf-7 promoter (daf-7p::gfp) is detected from 4 to 5 h after hatching through development to the adult stage; however, if the worms are starved and exposed to high levels of pheromone, daf-7p::gfp expression is significantly reduced in larvae entering the dauer phase (Ren et al., 1996). Expression of daf-7p::gfp is, however, re-activated in dauer larvae that sense food and resume development through the L4 to adult stages (Ren et al., 1996). In A. caninum, Ac-daf-7 expression was detected in the embryo, peaked in the iL3, a developmentally arrested stage similar to the dauer of C. elegans, and remained at a high level of expression in the feeding, reactivated ssL3. Protein analysis by Western blotting demonstrated Ac-daf-7 in the iL3 and ssL3, suggesting that Ac-daf-7 transcripts are probably being actively translated and not merely transcribed in preparation for translation upon reactivation (Fig. 6); a similar finding has been reported with the A. caninum ASP protein that is secreted by serum stimulated L3s with apparently similar transcript levels in iL3s versus ssL3s (Hawdon et al., 1996). The possibility, however, that anti-Ac-daf-7 antibodies are cross-reacting with as yet unidentified A. caninum TGF-b homologs cannot be excluded. In C. elegans, expression of daf-7 is limited to the ASI sensory neuron pair in the worm’s anterior end with dendritic processes extending towards the chemosensory amphidial pores located on the exterior (Ren et al., 1996). These exposed chemosensory neurons (as well as several others) represent the regulatory site for the initiation and recovery cues of arrest/reactivation. Ac-daf-7 expression also appears to be associated with the anterior region of the iL3 in the area corresponding to the amphidial pores (Fig. 7(a) and (b)); this detection pattern was not observed with anti-Ac-dbl-1, the anti-H. glycines cellulase negative control (data not shown) or with the anti-A. caninum iL3 serum positive control (Fig. 7(c)). The similarity in expression pattern suggests that Ac-daf-7 activity may be related to that in C. elegans and influenced by environmental or host factors. A recent report of daf-7 homologs from Strongyloides ratti and Parastrongyloides trichosuri from the rat and opossum, respectively, showed that daf-7 transcript levels peaked at the iL3 stage but were down regulated in iL3 subjected to in vitro penetration of host skin or transient passage in a host but not by physiological temperatures (Crook et al., 2005). The authors hypothesised that unlike C. elegans, daf-7 homologs in these parasites may function to maintain developmental arrest until infection of a host

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(Crook et al., 2005). Similarly high levels of the daf-7 homolog (Bm-tgh-2) were reported in the developmentally arrested microfilarial or L1 stage of the B. malayi filarial nematode (Gomez-Escobar et al., 1998; 2000). In an extension of this difference from the C. elegans free living nematode, the high daf-7-like transcript levels in iL3 of A. caninum are maintained through the in vitro reactivated L3 in which stimulation by exposure to dog serum appears to be associated with the early transition to parasitism of the host (Hawdon et al., 1996). It is therefore possible that the obligate phase of developmental arrest in parasitic nematodes has evolved to constitutively repress the reactivation pathways as seen in C. elegans until triggered by host factors and the transition to parasitism with resumption of development. Ligands of the TGF-b as well as the insulin-like signalling pathways (Tissenbaum et al., 2000) might well be expressed and/or secreted upon exposure to host factors and activate the genes associated with resumption of development or repress the genes associated with developmental arrest. Expression of Ac-daf-7 during arrest might better prepare the worm for reactivation upon infection as an ‘armed and ready’ state. Acdaf-7 protein may be sequestered within the expressing cell or could be under the control of other negative regulators of TGF-b signalling such as by binding to latent proteins. If this were the case, reactivating cues from the host would cause the release of these regulatory controls allowing the ligand to become functional and stimulate reactivation. Several approaches can be taken to further elucidate the function of the parasite encoded TGF-b homologs. Studies of B. malayi have suggested an immunomodulatory role for Bm-tgh-2 based on evidence that the protein is secreted from the adult parasite and that recombinant Bm-tgh-2 can bind to and stimulate signalling from mammalian TGF-b receptors (Gomez-Escobar et al., 2000). To determine if the A. caninum TGF-b homologs might be released, preliminary analyses by Western blot were conducted but failed to identify Ac-dbl-1 or Ac-daf-7 in excretory-secretory (ES) products of ssL3 or adults (data not shown). Exposure to host factors other than serum may be critical for expression/ detection of these molecules. RNA interference (RNAi) has also been successfully used in some parasitic nematodes (Hussein et al., 2002; Urwin et al., 2002; Aboobaker and Blaxter, 2003; Lustigman et al., 2004) but indirect methods may be needed as both C. elegans dbl-1 and daf-7 lack an RNAi phenotype due possibly to their neuronal location (Gonczy et al., 2000; Winston et al., 2002; Kamath et al., 2003); as such the A. caninum genes may be studied by rescuing the respective C. elegans dbl-1 and daf-7 mutants or by transgenic analyses in closely-related parasitic nematodes.

Acknowledgements The authors would like to thank Shweta Trivedi for assistance with real-time PCR and Eric L. Davis and Hanane

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Diab El Arab for help with the immunofluorescent localisation assays. We would also like to thank Jim McCarter and Brandi Chiapelli of Washington University for supplying the A. caninum EST clone pb34h07.y1 and Peifeng Ren, Eric Davis and James Lok for helpful comments on the manuscript. TF was supported by an Integrative Graduate Education and Research Training NSF fellowship. This work was supported by grants from North Carolina Biotechnology Center and the National Institutes of Health (R01A142908).

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