A consensus sequence of a cluster of eight A. caninum expressed sequence tag (EST) sequences homologous to 14-3-3 proteins was used to design oligonucleotide primers to isolate the complete Ac-ftt-2 cDNA from A. caninum. Using a hemi-nested strategy, the 5' end was isolated using the conserved nematode spliced leader sequence 29 as the forward primer, coupled with two nested gene-specific reverse primers in successive PCR reactions. The second reaction produced an amplicon of approximately 600 bp that contained the SL sequence and overlapped the 5' end of the A. caninum EST sequence. A similar strategy was used to isolate the 3' end from an A. caninum cDNA library. A reverse primer complementary to the flanking T7 sequence in the vector was used with two nested forward primers to generate an amplicon of approximately 700 bp that contained a poly d(A) tail, and overlapped the EST and the 5' end sequences. New primers were designed to amplify the entire coding sequence of A. caninum 14-3-3 cDNA, which was cloned into pET28 vector and confirmed by DNA sequencing of both strands. The full-length cDNA sequence was deposited in GenBank (accession number FJ842376).

The full-length 14-3-3 cDNA contained the conserved 22 nucleotide nematode SL at the 5' end 30, which was followed by 33 untranslated nucleotides and an ATG codon encoding the starting methionine at nucleotide 56. The cDNA is predicted to encode an open reading frame of 249 amino acids, ending with a TAA termination codon at nucleotide 803, and followed by a 327 bp 3'-untranslated region. A canonical AATAAT polyadenylation signal 31 was located 11 bp upstream of the poly (dA) tail (nucleotides 1116 to 1121). The deduced amino acid sequence has a predicted mass of 28174 Da and a calculated pI of 4.87. The hookworm 14-3-3 lacks a secretory signal peptide 32, and contains a nuclear export signal (LxxxLxL) 33 at amino acids 223 to 229, consistent with a role in nuclear transport 34.

A BLASTP search 35 of the non-redundant GenBank database using the deduced amino acid sequence confirmed that the hookworm protein was a member of the 14-3-3 protein family (Pfam

PF00244

IPR000308

The 14-3-3 proteins are a family of dimeric proteins that modulate protein-protein interactions 22. Phosphorylation of the interacting protein at sequence-specific sites mediates its interaction with the 14-3-3. Each subunit of the 14-3-3 dimer can bind to a phosphoserine or phosphothreonine ligand independently 34. The most highly conserved region, the peptide-binding pocket, contains the residues that contact the phosphorylated amino acids. These residues (Lys51, Arg58, Arg129 and Tyr130) are completely conserved in Ac-FTT-2 (Fig. 1A). The N-terminal portion of 14-3-3 proteins is required for dimerization, and contains two conserved phosphorylation sites (Ser59 and Ser65) that are substrates for several kinases in mammals, including Akt and protein kinase C (PKC) 22. Phosphorylation of Ser59 converted 14-3-3 dimers to monomers 36, suggesting a possible regulatory mechanism. However, a c-Jun N-terminal kinase (JNK) site at amino acid 186 22 is absent from both Ac-FTT-2 and Ce-FTT-2, but is conserved in PAR-5. This further supports the conclusion that Ac-FTT-2 represents the ortholog of Ce-FTT-2.

Given the high level of conservation in 14-3-3 molecules, we tested whether an antibody against the human β isoform of 14-3-3 could detect Ac-FTT-2. As shown in Figure 2A, the 14-3-3β antibody recognized a single band in lysates of both untreated and serum-stimulated (activated) A. caninum L3 by Western blot. Next, we expressed recombinant Ac-FTT containing a V5 epitope tag in HEK293 cells. Western blotting of cell lysates indicated that Ac-FTT-2 was expressed in mammalian cells (Fig. 2B). An anti-V5 body recognized a single band of the appropriate size in lysates of cells transfected with pcDNA3.1V5/Ac-ftt-2 plasmid, but not in lysates from cells transfected with empty vector. The 14-3-3β antibody recognized two bands in the transfected cells, and a single band in the mock transfected cells. The lower molecular weight band, present in both transfected and mock cells, is endogenous 14-3-3, while the higher molecular weight band is the expressed FTT-2/V5 fusion protein, which migrates more slowly in the gel because of the attached epitope tag. This indicates that recombinant Ac-FTT-2 can be expressed in mammalian cells, and recognized by an antibody against the epitope tag and an anti-human 14-3-3 antibody.

Dauer recovery in C. elegans is mediated by 14-3-3 through regulation of DAF-16 subcellular localization and transcriptional activities 26. This regulation is dependent on insulin-like signalling 23. Because the resumption of development associated with infection in hookworms is analogous to dauer recovery 1137, we hypothesized that DAF-16 is regulated by 14-3-3 in response to ILS as in dauer recovery in C. elegans. Previously, we demonstrated that Ac-DAF-16 binds to and drives transcription from a conserved hookworm DAF-16 DNA binding element in mammalian cells 38. As hookworms have yet to be transfected with foreign DNA, we again used mammalian cells to examine interactions between Ac-FTT-2 and Ac-DAF-16.

We first tested whether we could immunoprecipitate recombinant V5-tagged Ac-FTT-2 from HEK293 cell lysates. As shown in Figure 2C, anti-V5 antibody successfully pulled down recombinant A. caninum FTT-2, as detected by Western blot with HRP-conjugated anti-V5 antibody (lane 3) and with anti-14-3-3β (not shown). Ac-FTT was not detected in unbound supernatant from the immunoprecipitate (Fig. 2C, lane 2), nor in control (empty vector) transfected cell lysate supernatant or immunoprecipitate (lanes 4 and 5, respectively), indicating that the ant-V5 antibody specifically precipitated the recombinant Ac-FTT-2.

After confirming that we could express and immunoprecipitate recombinant Ac-FTT, we next tested if Ac-FTT-2 and Ac-DAF-16 interact in vivo. In C. elegans, FTT-2 interacts with and regulates Ce-DAF-16 by excluding it from the nucleus 26. HEK 293 cells were co-transfected with equal amounts of plasmid pcDNA3.1V5/Ac-ftt-2, encoding V5-tagged Ac-FTT-2, and plasmid pCMV4FLAG/Ac-daf-16 encoding FLAG-tagged A. caninum forkhead transcription factor DAF-16 38. After serum treatment for 24 hrs, reciprocal co-IP was performed on cell lysates using anti-V5 and anti-FLAG antibody resin.

As shown in Figure 3A, anti-FLAG agarose pulled down both a 64 kDa FLAG-tagged DAF-16 and the V5-tagged recombinant Ac-FTT-2 from co-transfected cells (lanes 3), but only recombinant DAF-16 from cells singly transfected with Ac-daf-16 (lane 1). As expected, anti-FLAG agarose did not pull down any protein from the Ac-ftt-2 singly transfected cells (lane 2). Conversely, anti-V5 agarose precipitated both proteins from co-transfected cells, but only recombinant 14-3-3 from cells singly transfected with the Ac-ftt-2 construct, and nothing from cells expressing recombinant Ac-DAF-16 only (Fig. 3B). This indicates that Ac-FTT-2 interacts with Ac-DAF-16 in mammalian cell culture, and suggests that a similar interaction occurs in worms.

Interaction of 14-3-3 with Ce-DAF-16 requires phosphorylation of specific serine or threonine residues by the protein kinase Akt (Cahill et al., 2001). To determine if the predicted Akt phosphorylation sites are important for the interaction of hookworm 14-3-3 and DAF-16, recombinant Ac-DAF-16 proteins with the phosphorylation sites mutated to alanine were tested for their ability to co-IP recombinant FTT-2 when co-expressed in HEK293 cells. Anti-FLAG M2 resin co-immunoprecipitated recombinant Ac-FTT-2 and wild type FLAG-tagged DAF-16 from cells expressing both proteins (Figure 4). However, mutation of serine107 to alanine (S107A) completely abolished the interaction with Ac-FTT-2, either singly or in combination with mutations at the other Akt phosphorylation sites (S107A:T312A, S107A:S381A, or triple). Mutation of threonine 312 (T312A) alone or in combination with mutations at the other sites also severely diminished the interaction with FTT-2 However, mutation of serine 381 (S381A) had no effect on the interaction with 14-3-3 (Figure 4). These data indicate that serine107 and threonine312 are essential for the interaction of Ac-DAF-16 with Ac-FTT-2.

Hookworm L3 activated in vitro by incubation with serum components under host-like conditions release multiple proteins, including the activation associated secretory proteins 1 and 2 (ASP-1 and ASP-2) 56 and a metalloprotease 4. Adult worms release molecules associated with feeding and survival in the host intestine, including anticoagulants 39, ASPs 40 and protease inhibitors 41. To determine if Ac-FTT-2 was secreted, we took advantage of the cross-reactivity with the anti-human 14-3-3β antibody to examine ESP from L3 and adult stages by Western blot. As seen in Figure 5A, 14-3-3β antibody failed to detect any bands in non-activated, activated L3, or adult ESP (lanes 1–3), but recognized recombinant Ac-FTT-2 expressed in HEK293 cells (lane 4). As a control, L3 ESP were probed with ASP-1 antiserum, which detects a band in activated but not non-activated ESP (Figure 5B, lanes 1–3) 5. Furthermore, antiserum against Ac-TMP-1, known to be released in adult ESP 42, detected Ac-TMP in the adult ESP (Figure 5B, lanes 4 and 5), indicating that the ESP contained secreted proteins. These data indicate that Ac-FTT-2 is not released in detectable amounts by either activated L3 or adult A. caninum.

The Baltimore strain of A. caninum (U.S. National Parasite Collection accession 100655.00) was maintained in beagles as described previously 67. The beagles were housed and treated according to a protocol approved by the George Washington University Institutional Care and Use Committee. Infective L3 were recovered from coproculture by a modified Baermann technique and stored at room temperature in buffer BU (50 mM Na₂HPO₄/22 mM KH₂PO₄/70 mM NaCL, pH 6.8) for up to one month 68, or snap-frozen in liquid N₂ and stored at -80 C until lysates were prepared.

Ancylostoma caninum L3 were activated under host-like conditions as described previously 6. Briefly, approximately 5000 decontaminated L3 were incubated at 37C, 5% CO₂ for 24 hr in 0.5 ml RPMI₁₆₄₀ tissue culture medium supplemented with 25 mM HEPES (pH 7.0) and antibiotics (RPMI-c) in individual wells of 24-well tissue culture plates. L3 were activated by the addition of 10% (v/v) of a less than 10 kDa ultrafiltrate of canine serum and 15 mM S-methyl-glutathione (GSM, Sigma Chemical, St. Louis, MO) in RPMI-c. Non-activated L3 were incubated with RPMI-c medium alone. The percentage of L3 feeding (or "activated") was determined as described elsewhere 2

Following incubation, medium containing L3 was transferred to microcentifuge tubes and centrifuged at 13,000 rpm for 5 min. The supernatant containing the excretory/secretory products (ESP) was collected and concentrated by ultrafiltration using Centricon YM-10 cartridge (Millipore). The retentate was washed with 0.5 mL PBS, and concentrated 10-fold for electrophoresis.

A consensus sequence derived from a cluster of eight expressed sequence tags encoding an A. caninum 14-3-3 (AC01065) 69 was used to design specific forward and reverse oligonucleotide primers for PCR. The Ac-ftt-2 cDNA ends were amplified in two separate PCR reactions. For the 5' end, the reverse primer R2 (5'-AAATTGAGAGCGAGGCCAAGGCG-3'), the forward nematode spliced leader primer SL (5'-GGTTTAATTACCCAAG TTTGAG-3') and first strand cDNA 70 were incubated in a PCR reaction for 35 cycles of 1 min at 94 C, 1 min at 57 C, and 2 min at 72, followed by a final extension for 4 min at 72 C.

The 3' end was isolated using a nested strategy from an A. caninum L3 directional cDNA library constructed in Lambda ZAP II 71. The first reaction employed the forward primer FTT-F1 (5'-GAACTCGTGTTGAGGGCCAAGC-3') together with a primer complementary to the T7 promoter site in the vector flanking the 3' end of the cDNA insert site (5'-TAATACGACTCACTATAG-3'). The primers and 1 μl of cDNA library were incubated in a PCR reaction of 1 min at 94 C, 1 min at 55 C, and 2 min at 72 C, and a final extension for 5 min at 72 C. The first round reaction was diluted 1:10 and used as template in a second reaction containing the nested forward primer FTT-F2 (5'-CGAGCTGGAGAGTTATTT CGTCG-3') and reverse primer T7-NEST (5'-ACTCACTATAGGGCGAATTG-3'), using identical cycling conditions. Amplicons of approximately 550 bp (5' end) and 900 bp (3'end) were gel purified, cloned using the pGEM-T Easy TA cloning kit (Promega, Madison, WI), and the DNA sequence of both strands determined. The DNA sequences of the 5' and 3' end clones were aligned and combined to give the 1165-bp full-length composite sequence of Ac-ftt-2, including a 32-bp poly-d(A) tail.

The composite sequence was used to design primers to amplify and clone the full-length coding sequence. Forward primer FTT-FX (5'-TTA

GGATCC

CTCGAG

The full length coding sequence of A. caninum 14-3-3 cDNA (Ac-ftt-2) in pET28a was used as a template to amplify the insert for subcloning into a mammalian expression vector. Forward primer FTT-FX and reverse primer FTT-RX2 (5'-ATAA

CTCGAG

Growing human embryonic kidney 293 (HEK293) cells were re-suspended in fresh, pre-warmed RPMI₁₆₄₀ containing 10% FBS (Biowhittaker, Walkersville, MD), 2 mM L-glutamine (Mediatech, Herndon, VA), 100 IU/mL penicillin (Mediatech, Herndon, VA), and 100 μg/ml streptomycin (Mediatech, Herndon, VA), and evenly distributed into individual wells of a 6-well tissue culture plate. Medium was added to a final volume of 2 mL in each well and the plate was incubated overnight at 37 C, 5% CO₂. The medium from each well was then replaced with fresh pre-warmed RPMI without disturbing attached cells and incubated for 2 h at 37 C. The cells were transfected with 4 μg of pcDNA3.1V5/Ac-ftt-2 plasmid DNA or empty pcDNA3.1V5 vector (mock) using Metafectene (Bionex, Munich) and incubated at 37°C for 48 h. Following incubation, media were removed and 2 mL of lysis buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 5 mM CHAPS, 1% Triton X-100, and 0.1% SDS) were added to each well to lyse the cells. Cell lysates were vortexed for 1 minute in microcentrifuge tubes, then frozen overnight at -80 C. Samples were thawed, vortexed again and centrifuged at room temperature for 5 min at 2300 × g. The supernatants were collected and stored at -20 C. Total protein concentrations were determined by bicinchoninic acid method (Micro BCA, Thermo Fisher) according to the manufacturer's protocol.

Co-transfections were performed as above with 2 μg each of pcDNA3.1V5/Ac-ftt-2 and pCMV4FLAG/Ac-daf-16 encoding the A. caninum forkhead transcription factor DAF-16 38, or 2 μg each of the expression constructs and the corresponding empty vector. Cells were incubated overnight, followed by 20% serum treatment for 24 h, and lysates prepared.

Western blotting with the specific anti-V5 antibody was used to determine if recombinant Ac-FTT-2 (rFTT-2) was expressed in HEK293 cells. Samples (16 μg of total protein) of mock and pcDNA3.1-V5-ftt-2 transfected cell lysates were separated in a 4–20% gradient pre-cast Novex Tris-glycine SDS polyacrylamide gel (Invitrogen). Separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane for 110 min at 22 V by standard methods 41. Following transfer, the membrane was blocked in 15 mL of 5% non-fat dry milk (NFDM) in PBS-T (0.05% Tween-20) overnight at 4 C. Membranes were incubated with mouse anti-human 14-3-3β/HRP antibody (Santa Cruz Bioechnology, Santa Cruz, CA) (1:20,000) or anti-V5/HRP mouse antibody (Invitrogen) (1:5,000) in 1% NFDM in PBS-T for 2 h at 22 C. After 3 × 10 min washes in PBS-T and 2 × 10 min washes in distilled water, the membrane was incubated in Enhanced Chemoluminescence (ECL) solution (Thermo Fisher), and chemoluminescent immunoreactive bands were detected by exposure to radiographic film.

To determine if we could immunoprecipitate hookworm recombinant Ac-FTT-2, 5 μL of anti-V5 antibody (Invitrogen) were incubated for 16 h at 4 C with lysates of mock or Ac-ftt-2 transfected HEK 293 cells in a total volume of 500 μL of PBS-T (0.1% Tween-20). Protein A-agarose (Invitrogen) was pre-cleared with PBS-T and incubated with the antibody-antigen complexes for 1.5 h at 22 C with rotation. Following centrifugation for 30 s at 2300 × g, the supernatant was removed and stored frozen as the unbound sample, and the agarose complexes washed by rotation 3 × 5 min with 1 mL PBS-T. The agarose-antibody-antigen complexes were separated by SDS-PAGE and visualized by Western blot with HRP conjugated α-human 14-3-3β antibody or α-V5/HRP as described above.

HEK293 cells co-transfected with Ac-daf-16 and Ac-ftt-2 were lysed with 200 μL of cold buffer containing 50 mM NaCl, 50 mM Tris-HCl pH7.5, 0.1% NP-40, 5 mM EDTA, 1× Phosphatase Inhibitor Cocktail II (Calbiochem), and 1× Halt Protease Inhibitor cocktail (Pierce). Lysates were centrifuged at 4 C for 10 min at 10,000 × g, and the protein concentration of the supernatants determined by Micro BCA. Forty μg of anti-FLAG M2 resin (Sigma, St. Louis, MO) was prepared according to manufacturer's directions, added to ~1 mg of cell lysates, and mixed by rotation overnight at 4 C. Tubes were centrifuged for 30 s at 8200 × g and washed 3 times in TBS (50 mM Tris HCl pH7.4, 150 mM NaCl). Co-immunoprecipitation (co-IP) complexes were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane for 7.5 min in an iBlot apparatus (Invitrogen). Following blocking, the membrane was probed with anti-V5/HRP (1:5000 dilution) at 22 C for 2 h. The membrane was washed 3 times in PBS-T and 2 times in distilled deionized water (ddH₂O), dried, and incubated with ECL solution as above. Following visualization, the membrane was stripped with Restore buffer (Pierce) and blocked overnight in 5% NFDM in PBS-T at 4 C. The stripped membrane was re-probed with a specific Ac-DAF-16 polyclonal antiserum (1:20,000 dilution) 38, and visualized as above. The reciprocal co-IP was performed as above, using anti-V5 resin (Sigma) to pull-down protein complexes, and anti-DAF-16/anti-rabbit IgG-HRP and anti-V5/HRP to probe the membranes.

To determine the importance of phosphorylation at the predicted Akt phosphorylation sites for the interaction between 14-3-3 and Ac-DAF-16, the serine or threonine residue at each site was changed to alanine by site-directed mutagenesis. Using a Quickchange Site-Directed mutagenesis kit (Stratagene, La Jolla, CA), mutant Ac-DAF-16 plasmids were constructed in which each site was mutated separately (S107A, T312A, S381A), in pairs (S107A/T312A; S107A/S381A; T312A/S381A), or at all three phosphorylation sites (3A). The mutated plasmid constructs were confirmed by DNA sequencing.

HEK293 cells were co-transfected with 2 μg of pcDNA3.1V5/Ac-ftt-2 and 2 μg of wild-type or mutant pCMV4FLAG/Ac-daf-16 as above. Mock cells received 4 μg of empty pcDNA3.1/V5-His vector. After 24 hrs incubation, the cells were fed with 20% serum and grown overnight. Following cell lysis, co-IPs were performed using anti-FLAG agarose, separated by electrophoresis, and visualized by Western blot with anti-V5/HRP antibody as above. After stripping, the gel was re-probed with DAF-16 antiserum to visualize Ac-DAF-16.

Ancylostoma caninum L3 were activated by incubating with canine serum fractions and S-methylglutathione as described above 6. Approximately 100,000 frozen non-activated and activated L3 A. caninum larvae were ground to fine powder with a sterile mortar and pestle pre-chilled with liquid N₂. The powder was added to 500 μL of cold PBS plus 1% EDTA, 1% Halt Protease Inhibitor Cocktail, and 1% Phosphatase Inhibitor Cocktail II and mixed by inversion. The lysates were centrifuged at 16,100 × g for 6 min at 4 C. Aliquots of the supernatants were removed to determine protein concentration by Micro BCA, and the remaining supernatants were stored at -20 C until needed.

To determine if Ac-FTT-2 was secreted during L3 activation, excretory/secretory products (ESP) from 6000 non-activated and activated L3 were collected as described previously 6. Adult A. caninum ESP, recombinant tissue inhibitor of metalloproteases (Ac-TMP-1), and anti-TMP-1 antisera were gifts of Dr. Bin Zhan. Adult ESP were collected following overnight incubation of adult worms in vitro. Lysates (20 μg) of HEK293 cells expressing recombinant Ac-FTT-2, 80 ng of recombinant ASP-1 5, and 130 ng of recombinant TMP-1 42 were included as positive controls. Samples were separated at 140 V, 40 mA for 1.5 h in a 4–20% gradient pre-cast Novex Tris-glycine SDS polyacrylamide gel (Invitrogen), and transferred to PVDF membrane for 7.5 min in an iBlot apparatus (Invitrogen). Blots were incubated with ASP-1 antiserum (1:4000), TMP-1 antiserum (1:5000), or 14-3-3β antibody (1:20,000), respectively, and visualized using ECL Plus as described above.

Dideoxy DNA sequencing using the ABI BigDye Terminator Cycle Sequencing Ready Reaction Kit v3.1 was performed by the Nevada Genomics Center 72, and the reactions run on an ABI3730 DNA Analyzer. Sequence analysis was performed using BioEdit Sequence Alignment Editor version 5.0.9 73, and multiple alignments performed using CLUSTAL W at Swiss EMBnet 74. Neighbor-joining trees were constructed using MEGA version 3.1 75. Homology searches were done using BLASTP 3576 at the National Center for Biotechnology Information 77 and WU-BLAST Parasite Genome Database Query at the European Bioinformatics Institute 78. Percentage identity and similarity was determined using the BLASTP algorithm with the BLOSUM62 matrix. Conserved motifs were identified by searching Scansite at MIT 79 using MotifScan software 80, and nuclear export signals identified by searching the NetNES 1.1 server 3381 at the Center for Biological Sequence Analysis, Technical University of Denmark.