Structure and developmental expression of Strongyloidesstercoralis fktf-1, a proposed ortholog of daf-16 inCaenorhabditis elegans☆

Holman C. Massey Jr.*, Manami Nishi, Kshitiz Chaudhary, Nazzy Pakpour, and James B.LokDepartment of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800Spruce Street, Philadelphia, PA 19104-6050, USA

AbstractA forkhead transcription factor gene, fktf-1, which we propose to be orthologous to theCaenorhabditis elegans dauer-regulatory gene daf-16 has been discovered in the parasiticnematode Strongyloides stercoralis. Genomic and cDNA sequences from both species predictalternately spliced a and b message isoforms. In contrast to C. elegans, where two a isoforms,daf-16a1 and daf-16a2, are found, a single fktf-1a isoform is found in S. stercoralis. Five of the 10introns found in the C. elegans gene are found in the proposed S. stercoralis ortholog. Functionalmotifs common to DAF-16 and several mammalian forkhead transcription factors are conserved inFKTF-1. These include the forkhead DNA binding domain, four Akt/protein kinase Bphosphorylation sites and a C-terminal domain that may associate with factors such as the steroidreceptor coactivator and other factors necessary for transcriptional regulation. An N-terminalserine-rich domain found in DAF-16A is greatly expanded in FKTF-1A. This domain is missing inDAF-16B, FKTF-1B and all mammalian orthologs. FKTF-1 shows the closest phylogeneticrelationship to DAF-16 among all known mammalian and nematode forkhead transcriptionfactors. Like its proposed Caenorhabditis ortholog, the fktf-1 message is expressed at all stages ofthe life cycle examined thus far. Discovery of fktf-1 indicates the presence of an insulin-likesignalling pathway in S. stercoralis similar to that known to regulate dauer development in C.elegans. This pathway is a likely candidate to control infective larval arrest and reactivation aswell as regulation of the switch between parasitic and free-living development in the parasite.

Keywordsfktf-1; Strongyloides stercoralis; daf-16; Forkhead transcription factor; Dauer larva; Infectivelarva

1. IntroductionParasitic nematodes of many species, including some of medical or veterinary importanceinvade their definitive hosts as infective third-stage larvae (L3i). The host may acquire theseL3i by various routes and from various sources including direct skin penetration fromcontaminated soil, ingestion with contaminated herbage, intermediate or paratenic hosts, or

☆Nucleotide sequence data reported in this paper are available in the EMBL, GenBank™ and DDJB databases under the followingaccession numbers: Strongyloides stercoralis fktf-1a genomic fragment AY281752, and cDNA sequence AY281749, S. stercoralisfktf-1b genomic sequence, AY281750, and cDNA sequence, AY28175.

© 2003 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.*Corresponding author: Tel.: +1-215-573-4756; fax: +1-215-573-7023. hmassey@vet.upenn.edu (H.C. Massey Jr.).

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Published in final edited form as:Int J Parasitol. 2003 November ; 33(13): 1537–1544.

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transfer during blood or tissue feeding by an arthropod vector (Noble et al., 1989; Bowman,1995; Despommier et al., 2000). Despite the varied nature of their hosts, habitats and modesof transmission, parasitic nematode L3i share some common biological attributes. Basicamong these is their programmed developmental arrest followed by reactivation uponencountering the host or host-like conditions. In vitro culture methods have allowed someorganism-scale studies of extrinsic factors governing the reactivation of L3i (Franke andWeinstein, 1984a, b; Farrar and Klei, 1985; Abraham et al., 1987; Wisnewski andWeinstein, 1993) and of the early events following reactivation (Abraham et al., 1987;Lustigman et al., 1995, 1996). Pioneering microsurgical techniques have been used recentlyto study the neuronal control of arrest and reactivation of Strongyloides stercoralis L3i(Ashton et al., 1998). Recent studies of growth regulating effects of muscarinic agentsprovide indirect evidence that insulin-like signalling participates in reactivation ofAncylostoma caninum L3i (Tissenbaum et al., 2000). An understanding of the molecularmechanisms underlying programmed arrest and reactivation of L3i has been slow todevelop. This is due in large part to the fact that the complex and often protracted lifehistories of parasitic nematodes have hampered the development of basic tools for genetic,cell and molecular biological investigation.

The free-living nematode Caenorhabditis elegans is, in many respects, an ideal subject forgenetic and molecular biological study, and consequently a relative wealth of basicinformation on the molecular, cellular, and developmental biology of that organism exists(Epstein and Shakes, 1995; Riddle and Albert, 1997). Caenorhabditis elegans has beenproposed by numerous authors as a general model for many aspects of basic molecular,cellular and developmental biology in the less tractable parasitic nematodes (Blaxter, 1998;Burglin et al., 1998; Aboobaker and Blaxter, 2000). One of the most frequent of suchanalogies is that drawn between the arrest and reactivation of parasitic L3i and the dauerdevelopmental pathway in C. elegans (Hotez et al., 1993). In response to adverseenvironmental conditions, larval C. elegans arrest their development at a specialised third-stage form termed the dauer larva. These dauer larvae re-enter the continuous, reproductivedevelopmental pathway (i.e. reactivate) if optimal growth conditions are restored.

In contrast to parasitic L3i, the developmental biology of C. elegans dauers is well studied atthe molecular genetic level. Currently, over 30 genes, designated daf- for their direct effecton dauer formation, and numerous genes not bearing daf designations have been shown tobe necessary for normal dauer development (Riddle and Albert, 1997). Epistasis analysis(Vowels and Thomas, 1992; Thomas et al., 1993) coupled with cloning of many daf andrelated genes in C. elegans has revealed that dauer development is controlled in part by asignal transduction pathway closely resembling mammalian insulin signalling (Gottlieb andRuvkun, 1994; Morris et al., 1996; Kimura et al., 1997; Ogg et al., 1997; Ogg and Ruvkun,1998; Paradis and Ruvkun, 1998; Paradis et al., 1999). Under optimal growth conditions,signalling through DAF-2, an insulin/insulin-like growth factor (IGF) receptor ortholog, andthe intervening kinase cascade negatively regulate the forkhead transcription factor DAF-16,allowing development through the continuous reproductive pathway. Under dauer inducingconditions, insulin-like signalling is downregulated and DAF-16 is released to promotedauer development. If the development of dauer larvae and parasitic L3i is controlled bysimilar molecular signalling mechanisms as has been proposed by Beall and Pearce (2002),then we would expect that this latter condition operates constitutively in the preinfectivelarvae of most parasitic nematodes for which dauer-like arrest of the L3i is the soledevelopmental alternative. On the other hand, in the case of the thread-worm, S. stercoralis,a subset of postparasitic larvae develop to free-living adults while others arrest as L3i. Insome related species, Strongyloides ratti, Strongyloides planiceps and Strongyloidesransomi, the proportions of postparasitic larvae entering the two alternative developmentalpathways are governed both by the genetic background of the worms (Viney et al., 1992)

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and by environmental factors such as pH, temperature and the concentration of organicnutrients (Arizono, 1976a, b; Moncol and Triantaphyllou, 1978; Minematsu et al., 1989).Therefore, if dauer and L3i development are similarly regulated in C. elegans andStrongyloides, then insulin-like signalling, particularly involving a pivotal regulatorymolecule like DAF-16, could be critical in switching between these developmentalalternatives. For this reason, we are conducting a PCR based search for insulin-like signalpathway intermediates in S. stercoralis. In the present paper we report the discovery of fktf-1in S. stercoralis, a proposed ortholog of daf-16 in C. elegans, and present information on itsdevelopmental expression patterns and its phylogenetic relationship to other forkheadtranscription factors.

2. Materials and methods2.1. Parasite culture

All studies were performed on the University of Pennsylvania dog strain of S. stercoralis(SsUPD), which is maintained as described (Schad et al., 1984). Free-living adults and alllarval stages were isolated by Baermannisation of faecal-charcoal cultures established withfreshly deposited faeces from an infected dog (Nolan et al., 1993) and maintained at 70%relative humidity under different time and temperature regimens. Postparasitic first-stagelarvae were obtained immediately after dispersing fresh faeces on activated charcoal. Mixedmale and female free-living adults were obtained from cultures maintained for 48 h at 22 °C.Post-free-living L1 were manually selected from the mixed population of old free-livingadults and newly hatched L1 in the Baermann proceeds from cultures incubated for 24 h at22 °C and then for 24 h at 25 °C. L3i were obtained from cultures incubated for 7 days at 25°C. Parasitic adult female worms were obtained by allowing them to emerge from slitintestines of experimentally infected jirds hanging in Dulbecco’s minimal essential medium(DMEM) with gentamicin for 1 h at 37 °C (Nolan et al., 1993).

2.2. DNA and RNA preparation from mass L3i culturesLarge scale DNA and RNA preparations were made from L3i of SsUPD grown in faecal-charcoal culture as described (Massey et al., 2001).

2.3. PCR screening and sequencing of S. stercoralis daf-16 sequencesPCR reactions were primed with degenerate guessmers FKHF1 (AAC AAG AMG WAATGC WTG GGG), FKHR2 (GGA ACR TTT TNW ACC ATC CAT TC), FKHR3 (CAGCWG AWS WAT TWG AAT CTC C), FKHF3 (TTC WAA TWS WTC WGC TGG ATGG), and FKHR4 (SCW TCT GGR TTW ATW ACC CAC C) (where M = A + C; W = A +T; R = A + G; N = A + T+ G + C; and S = G + C) designed using codon frequencies for S.stercoralis (Moore et al., 1996; Massey et al., 2001) and based upon sequences conservedbetween C. elegans daf-16a, daf-16b and the human forkhead transcription factors AFX andFKHR (Ogg et al., 1997) using conditions modified for the (A + T)-rich genome of thisspecies (Massey et al., 2001). Primer combinations FKHF1 plus FKHR2 and FKHF1 plusFKHR3 yielded 236 and 274 bp fragments, respectively, from the fktf-1a region, andFKHF1 plus FKHR4 and FKHF3 plus FKHR4 yielded 431 and 287 bp fragments,respectively, from the fktf-1b region. These fragments were sequenced directly from PCR asdescribed (Massey et al., 2001). Outward facing gene-specific primers (not shown) weredesigned and the cDNA sequence of fktf-1b was amplified by modified 5′ and 3′ rapidamplification of cDNA ends (RACE) procedures (Massey et al., 2001) and sequenced. Lowextension temperature inverse PCR (Massey et al., 2001) with outward facing gene-specificprimers SsFKHF162 (GCA ACA TCA TGT TCA ATT CAC AAT GG) and SsFKHR65(TGT ATC CTG TCA TTG GTG TAT CTG G) was used to capture 2.6 kb of sequencereading into the large intron between fktf-1a and fktf-1b from a circularised Bgl II digest of

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SsUPD genomic DNA, and primers SsFKHF4880 (CAT GAA ATT TCT ATA AAT AGTAAT GCT CC) and SsFKHR4822 (CAT TAA TGA CTC AAA ATC CAT TTC CG) wereused to capture 1.5 kb of 3′ flanking sequence from a circularised Sau3AI digest. Gene-specific primers SsFKHaFA (GCA TGA GAC TAA TTC ATT TAC TG) and SsFKHaRB(CTC ATG CCT ATT ATTAAGAAGTG) were used to capture most of the coding regionof fktf-1a from a circularised Msp I digest, and 600 bp reading into the large intron from acircularised Sau3AI digest. Two kilobases of the fktf-1a 5′ flanking region was capturedfrom a circularised Eco RI digest with primers SsFKHaMF (GTT CTG GTG CCT CTTCAG C) and SsFKHaMR (CTG TTT CAT AAG AAG GAA TAC CG).

To determine intron/exon boundaries, complete cDNA sequences were compiled from 5′and 3′ RACE products generated and sequenced directly from PCR using sequence-specificprimers and anchor primers directed to the SL1 5′ splice leader or the 3′ polyadenylated tailas described (Massey et al., 2001).

2.4. RT-PCR assays for stage-specific expression of fktf-1a and fktf-1b mRNAsFive hundred freshly collected worms at selected developmental stages were concentratedby centrifugation to a volume of 50 μl in 1.5 ml polypropylene microcentrifuge tubesimmediately after collection. Worms were never stored on ice, since this resulted in loss ofcertain labile mRNAs (unpublished results). An equal volume of 2 × RNA lysis buffer (100mM Tris–HCl, pH 8.0, 200 mM NaCl, 2% SDS, 200 mM EDTA) and 7 μl proteinase K (15mg/ml) were added and the tubes were frozen for at least 5 min at −20 °C followed byincubation at 55 °C for 15 min. One millilitre of TRIZOL reagent (Invitrogen) was addedand incubation was continued for 15 min at 55 °C. After cooling to ambient temperature for3 min, 200 μl of chloroform was added and RNA extraction was continued according to themanufacturer’s protocol. After final resuspension of the product in 50 μl diethylpyrocarbonate treated water, 1.0 μl aliquots were amplified using Super-Script One-StepRT-PCR with Platinum Taq (Invitrogen) with an annealing temperature of 47 °C andextension at 60 °C. Primer sets FK1aQF (ATA CAT GGC CAT TAA GAA GAG C) plusFK1aQR (TCC AAC CGG CAG AAC TAT) and FK1bQF (CTC CGA GCG CGT TGTTAC) plus FK1bQR (CCC CAT GGA TTA GGT TTT T) were used to amplify fktf-1a- andfktf-1b-specific message, respectively, and the RBPS21QF (ATG CAA AAC GAC AAAGGA GAA) plus RBPS21QR (AGC TAA ACG AAG AAG ACA ATC ATC) pair was usedto amplify a fragment from rbps-21, the cDNA for the ribosomal small subunit protein S21(Genebank BE579329) as an internal standard. In all cases, amplification sites crossedintrons to allow message to be distinguished from genomic sequences by size. Cyclenumbers (30–35) were empirically chosen to give sub-saturated levels of all products forrelative quantitation. Band densities on agarose gels stained with ethidium bromide werecompared using the Bio-Rad Gel Doc 2000 Gel Documentation System using Quantity Onequantitation software.

2.5. Sequence and phylogenetic analysisRaw sequence data were edited using SeqEd v1.0.3 (Applied Biosystems) and sequencehomologies detected using BLAST servers (Altschul et al., 1990) at the National Center forBiotechnology Information (http://www.ncbi.nlm.nih.gov/BLAST/), the Sanger Centre(http://www.sanger.ac.uk/Projects/C_elegans/blast_server.shtml), and the EuropeanBioinformatics Institute (http://www.ebi.ac.uk/blast2/parasites.html).

Alignment and analysis of protein sequences were performed using Clustal W algorithm inthe Lasergene software suite (DNASTAR) with manual adjustments. Evolutionary trees andBootstrap analysis were generated using the PAUP 3.1.1 program (Sinauer).

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3. Results3.1. Genomic organisation of fktf-1

The S. stercoralis fktf-1 gene is organised much like C. elegans daf-16, although introns arefewer and often smaller than in daf-16 (Fig. 1). Analysis of cDNA sequences from bothspecies shows alternately spliced isoforms differing in a region including their N-terminaldomains and an N-terminal portion of the forkhead domain. In C. elegans, the regionspecific to the two daf-16a isoforms consists of four exons separated by three introns.Isoforms daf-16a1 and daf-16a2 differ by two amino acid residues due to alternative spliceacceptor sites six bases apart at the end of the second intron. In S. stercoralis fktf-1, there isno intron corresponding to intron 2 of daf-16, allowing for only a single fktf-1a isoform. The(fktf-1/daf-16)a region is separated from the rest of the coding region by the large intron 4 indaf-16, or intron 3 in fktf-1, which is 6.2 kb in length in C. elegans and approximately 5 kbin S. stercoralis as judged by the size of PCR fragments spanning this region. A single(fktf-1/daf-16)b-specific exon in both species is located downstream of this large intron. Asmaller intron separates this region from a common region consisting of the remaining exonsof the gene. The messages for (FKTF-1/DAF-16)A or (FKTF-1/DAF-16)B contain theirrespective upstream regions spliced to this common region. A single intron corresponding toC. elegans intron 7 is found in this part of the S. stercoralis gene. Promoters upstream of thedaf-16a-specific exons and in the large intron upstream of the daf-16b-specific exon driveexpression of the respective isoforms of this gene in C. elegans. The region upstream of thefktf-1a-specific exons can drive expression of a green fluorescent protein reporter in C.elegans transformants in a pattern very similar to the daf-16α promoter (not shown),although very little sequence similarity exists between these promoters in the two species.There is likewise very little similarity between the two species in the presumptive (fktf-1/daf-16)b-specific promoter regions, although the large size of the intron in this region isconserved.

3.2. Three functional domains are conserved between the FKTF-1/DAF-16 isoforms andtheir mammalian orthologs

Alignment of the conceptual translations of S. stercoralis FKTF-1A and FKTF-1B with theC. elegans DAF-16 isoforms and the orthologous human forkhead transcription factorsAFX, FKHR and FKHRL1 shows conservation of the three domain structure common tothese transcription factors. This pattern consists of a characteristic N-terminal domain, acentral forkhead DNA binding domain, and a characteristic C-terminal domain. Thesequences of FKTF-1A, FKTF-1B and other members of this family of proteins are mosthighly conserved in the DNA binding forkhead domain, with, for example, 79.5% identitybetween DAF-16A1 and FKTF-1A in the forkhead domain, 51.4% in the N-terminal domain(minus the serine-rich domain described below), and 31.4% in the C-terminal domain. Adistinctive feature of the FKTF-1A sequence is a serine-rich region 181 residues long at theN-terminal end of the first domain. The frequency of serine residues in this region is 25.4%compared to 12.1% in the rest of the protein. A similar region consists of only 37 residues inDAF-16A1 and DAF-16A2 and is not found in FKTF-1B, DAF-16B, or any of the humanorthologs.

In S. stercoralis FKTF-1 and C. elegans DAF-16, three consensus Akt/protein kinase B(Akt/PKB) phosphorylation sites with the consensus sequence RxxRxR(S/T)Hyd (where R= arginine, (S/T) = serine or threonine, x = any residue, and Hyd = a hydrophobic residue)are found that are highly conserved within the DAF-16/FKHR/AFX family of transcriptionfactors, one in each of the conserved domains. A fourth site overlapping the conserved sitein the forkhead domain is also found in both S. stercoralis FKTF-1 and C. elegans DAF-16.

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The alignment of these sequences and other supplementary information can be found at thefollowing website: http://www.vet.upenn.edu/GraduateGroups/Parasitology/lok.htm.

3.3. Evolutionary relationships of fktf-1Analysis of an alignment of S. stercoralis FKTF-1A and FKTF-1B with their proposed C.elegans orthologs, the human forkhead proteins FKHR, FKHRL1 and AFX, and otherhuman and nematode forkhead transcription factors shows the S. stercoralis proteins to bemost closely related to their proposed C. elegans orthologs (Fig. 2). When sequences fromthe forkhead domain only are analysed, bootstrapping does not support a clade representedby FKTF-1A and DAF-16A1 separate from that including FKHR, FKHRL1 and AFX.When the sequences from the C-terminal domains of these proteins are added, however,DAF-16A1 and FKTF-1A clearly group together into a distinct clade as shown in Fig. 2.

3.4. Developmental expression patterns of the fktf-1 messageExpression of the fktf-1a and fktf-1b messages was found at all developmental stages of S.stercoralis examined. Signal ratios to control Ss rbps-21-specific mRNA, which encodes theribosomal small subunit protein S21, showed some variation but no consistentdevelopmental pattern at different stages of the S. stercoralis life cycle (Fig. 3).

4. DiscussionIt has been shown that the insulin/IGF receptor-like kinase DAF-2 in C. elegans mediates ashift between lipid storage metabolism in dauer larvae and carbohydrate catabolism inactively developing larvae and adults (Kimura et al., 1997). This activity is mediatedthrough a phosphoinositol-3-kinase pathway including orthologs of many of the knownintermediates of mammalian insulin and IGF pathways involved in the regulation of geneactivity in response to insulin and glucocorticoid signalling (Morris et al., 1996; Kimura etal., 1997; Paradis and Ruvkun, 1998; Paradis et al., 1999; Nasrin et al., 2000). The family offorkhead transcription factors that includes DAF-16 in C. elegans and the human factorsFKHR, FKHRL1 and AFX are the terminal effectors in these pathways. DAF-16A1 canfunctionally replace FKHR in mammalian reporter systems where transcriptional regulationwas mediated through binding to insulin response sequences in the promoters of the insulin-like growth factor binding protein 1 and phosphoenolpyruvate carboxykinase genes (Nasrinet al., 2000). Likewise, transgenic FKHRL1 can partially replace DAF-16 function in C.elegans (Lee et al., 2001). The presence of FKTF-1, a close ortholog of DAF-16, in S.stercoralis argues for conservation of this signalling pathway in the parasite.

The fktf-1 gene in S. stercoralis closely resembles daf-16 in C. elegans in its overallorganisation (Fig. 1), with an upstream region specific to the fktf-1a isoform separated fromthe region specific to the fktf-1b isoform by a large intron (Ogg et al., 1997). Transcriptionof the daf-16a isoforms is driven by an upstream promoter (α) in C. elegans (Ogg et al.,1997; Lee et al., 2001), and presumably also in the parasite. The large intron separatingdaf-16a-specific and daf-16b-specific portions of the daf-16 gene is conserved in the fktf-1gene, suggesting functional conservation in this region, which contains the promoter (β) forthe daf-16b message in C. elegans (Lee et al., 2001). The two promoters showed acomplementary pattern of expression in tissues, with the green fluorescent protein (GFP)reporter construct daf-16α::gfp driving GFP expression in most somatic tissues excludingthe pharynx and somatic gonad, and the daf-16β driven construct showing expression in thepharynx, somatic gonad and a subset of neurons (Ogg et al., 1997; Lee et al., 2001). Thesimilar architecture of the C. elegans daf-16 gene and the S. stercoralis fktf-1 gene regionsuggests two promoters may function in a similar fashion in both species.

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Strongyloides stercoralis fktr-1 has fewer and generally smaller introns than C. elegansdaf-16, a feature that has been noted in other genes sequenced in this parasite (Massey et al.,2001). In C. elegans, the number of introns can have a strong positive effect on theefficiency of expression of transgenes (Okkema et al., 1993), suggesting that certain featuresof message processing in the two species may differ or vary in their degree of influence.Early transformation experiments in this laboratory with S. stercoralis showed high,potentially toxic levels of GFP production during embryogenesis using gfp constructscontaining multiple artificial introns designed for efficient expression in C. elegans (Lok andMassey, 2002). The consideration that introns may have greater efficacy in stimulatingtranscriptional processing in S. stercoralis is being investigated in the context of designingappropriate transformation reporters for the S. stercoralis system.

The DAF-16A1 and DAF-16B proteins were found to be functionally interchangeable intransgenic arrays driven by either of the two promoters in C. elegans (Lee et al., 2001),suggesting that the differential effects of these two factors on growth and longevity weremore the product of differential expression from the two promoters than intrinsic differencesbetween the proteins. However, FKTF-1A and DAF-16A share an N-terminal serine-richsequence, greatly expanded in FKTF-1A, that is not found in FKTF-1B, DAF-16B or theirmammalian orthologs. The function of this region is not known, but it might containregulatory features such as phosphorylation sites or signals for turnover allowing forregulatory pathways unique to these isoforms. It is possible that consequent differences inthe regulation of these proteins would not have been evident in transgenic studies wherethey were over expressed in extrachromosomal arrays (Lee et al., 2001).

FKTF-1A and FKTF-1B show strongest identity with DAF-16A1 and DAF-16B in thecentral forkhead DNA binding domain (79.5 and 81.1%, respectively), suggesting possibleconservation of sequence binding specificity. This domain binds specific DNA sites, whichinclude the insulin response element of the insulin-like growth factor binding protein 1promoter and the insulin response sequence of the phosphoenolpyruvate carboxykinase genein mammalian cells (Nasrin et al., 2000), and the DAF-16 family member binding elementof sod-3 in C. elegans (Furuyama et al., 2000). DAF-16A1 was shown to promote insulinresponsive gene transcription in a manner similar to its human orthologs when transfectedinto HepG2 cells, with a sensitivity to insulin inhibition being most similar to FKHR (Nasrinet al., 2000). All these proteins bound artificial oligonucleotides with the same eight basecore consensus sequence in vitro (Furuyama et al., 2000).

All of these transcription factors share three conserved regulatory Akt/PKB phosphorylationsites, one in each of the conserved domains. In addition to these sites, FKTF-1 and DAF-16share an additional overlapping Akt/PKB phosphorylation site in the forkhead domain (Ogget al., 1997). These sites in the C. elegans and human proteins form binding sites for 14-3-3proteins when phosphorylated that mediate transport out of the nucleus and consequentdownregulation of transcription directed by these factors (Cahill et al., 2001). Mutation ofthe phosphorylated serine or threonine residues in these sites to alanine preventsphosphorylation, resulting in constitutive nuclear retention and upregulation of transcription(Cahill et al., 2001). Ratios of fktf-1 message to a control mRNA indicate that thistranscription factor is expressed at similar levels in all life stages. These findings areconsistent with the developmental patterns of C. elegans daf-16 expression where daf-16-linked GFP expression occurs at all stages (Ogg et al., 1997; Lee et al., 2001). Since noconsistent pattern of stage-specific regulation of message level is apparent, regulation of theactivity of these proteins appears to occur primarily by phosphorylation and transport to andfrom the nucleus rather than at the level of transcription.

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In mammalian systems, the C-terminal domain is responsible for interactions with thesteroid receptor coactivator and other transcription factors such as p300/CREB bindingproteins, indicating a role in integrating glucocorticoid and insulin-like transcriptionalresponses. Despite limited sequence similarity in this domain, C. elegans DAF-16 is able tomediate recruitment of these factors to promoters and regulate transcription of mammalianinsulin responsive genes in vitro and in vivo (Nasrin et al., 2000). The FKTF-1 sequence is34.1% identical to DAF-16 in this region, suggesting conservation of function in theparasite. The C-terminal domains of the human proteins in this family and DAF-16 havebeen shown to have similar functional properties despite very limited sequence identity(Nasrin et al., 2000; Lee et al., 2001), suggesting that such properties as conformation andhydropathy may play more important roles in the function of this domain than sequenceidentity. In C. elegans, daf-12 encodes a member of the steroid nuclear receptor family oftranscription factors, and daf-12 interacts with the insulin-like branch of the dauer pathwayto regulate dauer development (Riddle and Albert, 1997; Snow and Larsen, 2000). Aproposed S. stercoralis ortholog of daf-12 has been identified (Siddiqui et al., 2000). It ispossible that cross-talk between insulin-like and glucocorticoid-like regulatory pathways hasa regulatory role in dauer arrest in C. elegans and the arrest of L3i in S. stercoralis and otherparasitic nematodes, and that the C-terminal domains of forkhead transcription factors likeDAF-16 and FKTF-1 may function in this cross-talk.

The discovery of FKTF-1, a proposed DAF-16 ortholog in the parasitic nematode S.stercoralis supports the hypothesis that a dauer-like pathway is functional in this parasite andmay function in determining any or all of the following: (1) the choice between parasitic andfree-living life cycles, (2) developmental arrest and/or extended life span of infective L3i,and (3) reactivation of development after infection of the host (Hotez et al., 1993; Blaxter,1998; Burglin et al., 1998; Aboobaker and Blaxter, 2000). The C. elegans system has provenan invaluable tool in investigating many aspects of developmental biology that go farbeyond the biology of this species. No such facile system has been found among parasiticnematodes, but the discovery of key intermediates in the dauer pathway in S. stercoralis mayallow complementation of relevant mutations in C. elegans with parasite transgenes, as hasbeen done in a study of the function of the cathepsin L protease CPL-1 in parasiticnematodes (Britton and Murray, 2002). The ability of the C. elegans dauer-regulatory genesto function in systems as divergent as mammalian cells, and vice versa, argues for thesuccess of this strategy. The early success in this laboratory of experiments in which S.stercoralis has transiently expressed reporter transgenes (Lok and Massey, 2002) encouragesus that it may soon be possible to test the function of these genes in the parasite as well.

AcknowledgmentsThe authors are grateful to G.A. Schad for critical discussions and for starting material for S. stercoralis cultures.We also thank C.C. Ball, M. Brenes, M. Castelleto, W. Forbes and A. Freeman for technical assistance. This workwas supported by grants from the National Institutes of Health (AI-43616 and AI-50668 to J.B. Lok, AI-22662 toG.A. Schad, and RR02512 to M. Haskins), the Ellison Medical Foundation (ID-IA0037-02) and the University ofPennsylvania Research Foundation to J.B. Lok.

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Fig. 1.Genomic organisation of fktf-1 compared with its Caenorhabditis elegans ortholog daf-16(Paradis and Ruvkun, 1998). The genes are arranged similarly in the two nematode species.Narrow bars indicate untranscribed sequences or introns; wide bars indicate exons; white,noncoding; black, N- and C-terminal domains; grey, forkhead domain. Regions specific to aisoforms of the transcription factors are beneath bars marked a. Those specific to b isoformsare beneath bars marked b, and those common to both isoforms are beneath bars marked c.Introns 1, 2, 3, 4 and 5 of Strongyloides stercoralis fktf-1 correspond in position exactly tointrons 1, 3, 4, 5 and 7 of C. elegans daf-16, respectively. Arrows indicate direction oftranscription and splice acceptor sites for the SL1 splice leader. Promoters for a and bisoforms are marked α and β respectively for C. elegans, and in parentheses at theirexpected locations for S. stercoralis.

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Fig. 2.An unrooted phylogenetic tree shows the relationships of Strongyloides stercoralisFKTF-1A and FKTF-1B to other Caenorhabditis elegans and human forkhead transcriptionfactors. Only residues located in the forkhead domains of these proteins were used in theanalysis because residues in outlying domains of many of these proteins were too divergentto be aligned. Branches of the tree including FKTF-1A, FKTF-1B, DAF-16A1, DAF-16B,and the FKHR-like group of human forkhead transcription factors are highlighted inboldface. The tree was generated by PAUP 3.1.1 analysis of a Clustal W alignment(DNASTAR software suite) with manual editing. Bootstrapping scores (100 iterations) areshown for robust clades (>50% cutoff). Homo sapiens, Hs; Caenorhabditis elegans, Ce;Strongyloides stercoralis, Ss. GenBank™ accession numbers: Hs FKHR, S40521; Hs AFX,P98177; Hs FKHRL1, CAC26821; Ce DAF-16A1, AAB84390; Ss FKTF-1A, AY281749;Ce DAF-16B, AAB84392; Ss FKTF-1B, AY281750; Ce FKH-9, AAA81139; Ce FKH-10,Z81038; Ce LET-381, NP_491826; Hs HNF-3, AAD51979; Ce FKH-6, AAA80692; HsHBF-3, X74144; Ce FKH-2, AAL02521; Ce PES-1, CAA82224.

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Fig. 3.Expression of fktf-1a and fktf-1b messages in larval and adult stages of Strongyloidesstercoralis compared with internal standard rbps-21 (see text). Bands in agarose gels of RT-PCR products from postparasitic L1 larvae (ppL1), free-living mixed male and female adults(flAd), post-free-living L1 (pflL1), infective L3 (L3i), and parasitic adult females (pAd).

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