Toward Understanding the Functional Role of Ss-riok-1, a RIO Protein Kinase-Encoding Gene of Strongyloides stercoralis

Background Some studies of Saccharomyces cerevisiae and mammals have shown that RIO protein kinases (RIOKs) are involved in ribosome biogenesis, cell cycle progression and development. However, there is a paucity of information on their functions in parasitic nematodes. We aimed to investigate the function of RIOK-1 encoding gene from Strongyloides stercoralis, a nematode parasitizing humans and dogs. Methodology/Principal Findings The RIOK-1 protein-encoding gene Ss-riok-1 was characterized from S. stercoralis. The full-length cDNA, gDNA and putative promoter region of Ss-riok-1 were isolated and sequenced. The cDNA comprises 1,828 bp, including a 377 bp 5′-UTR, a 17 bp 3′-UTR and a 1,434 bp ORF encoding a protein of 477 amino acids containing a RIOK-1 signature motif. The genomic sequence of the Ss-riok-1 coding region is 1,636 bp in length and has three exons and two introns. The putative promoter region comprises 4,280 bp and contains conserved promoter elements, including four CAAT boxes, 12 GATA boxes, eight E-boxes (CANNTG) and 38 TATA boxes. The Ss-riok-1 gene is transcribed throughout all developmental stages with the highest transcript abundance in the infective third-stage larva (iL3). Recombinant Ss-RIOK-1 is an active kinase, capable of both phosphorylation and auto-phosphorylation. Patterns of transcriptional reporter expression in transgenic S. stercoralis larvae indicated that Ss-RIOK-1 is expressed in neurons of the head, body and tail as well as in pharynx and hypodermis. Conclusions/Significance The characterization of the molecular and the temporal and spatial expression patterns of the encoding gene provide first clues as to functions of RIOKs in the biological processes of parasitic nematodes.


Introduction
Strongyloides stercoralis is a parasitic nematode infecting human beings and dogs, and causes a fatal, disseminated hyperinfection in immuno-compromised patients [1,2]. The life cycle of S. stercoralis, like other members of Strongyloides and related genera, is more complicated than that of most obligatory parasitic nematodes. S. stercoralis can execute both parasitic and freeliving generations of development. Parasitic female adults (P Female) live in the host intestine and produce sexually differentiated eggs by mitotic parthenogenesis. Eggs of S. stercoralis hatch in the host intestine and in immune-competent hosts, newly hatched post parasitic first-stage larvae (PP L1) are passed in the feces. Once in the environment, female post-parasitic L1 can either develop directly (homogonically) to infective third stage larvae (iL3) and infect a host or develop heterogonically to free-living female adults (FL Female). Male PP L1s invariably develop via the heterogonic route to the free-living male adults (FL Male). Post-free-living L1 (PFL L1) produced by FL Female and FL Male are all female and develop to iL3. Female PP L1 of S. stercoralis may develop precociously to autoinfective L3 (aiL3) within the intestine, penetrate the intestinal wall, invade the somatic tissues and ultimately establish as a new generation of P Female in the primary host intestine. This process of autoinfection may proceed for sequential generations in an immuno-compromised host, with geometric expansion of parasite numbers and involvement of multiple body tissues, possibly leading to a fatal outcome for such immuno-compromised hosts [3].
In contrast to the relative wealth of information on the complex life cycle of this parasite, the understanding of molecular factors regulating its developmental biology is limited. Elucidating the functions of the essential genes that regulate the development and reproduction of S. stercoralis could facilitate the discovery of novel interventions for strongyloidiasis and other related parasitic nematode diseases.
Protein kinases are a large group of enzymes that are crucial in the regulation of a wide range of cellular processes, including cellcycle progression, transcription, DNA replication and metabolic functions [4]. Based on their structures, protein kinases can be classified into eukaryotic protein kinases (ePKs) and atypical protein kinases (aPKs) [5]. The ePKs contain a conserved catalytic domain that phosphorylates enzymes of signal transduction pathways that regulating many biological processes. The aPKs are active kinases containing kinase domains with limited sequence similarity to the conserved catalytic domain of ePKs. According to their characteristics in their kinase domains and functions in different biological processes, the aPKs have been divided into 13 families, one of which contains the RIO kinases. There are currently four members of the RIOK family, RIOK-1, RIOK-2, RIOK-3 and RIOK-B [6,7]. RIOK-1 and RIOK-2 are strongly conserved from archaea to human, whereas RIOK-3 is only found in metazoans, and RIOK-B is restricted to eubacteria [6]. RIOK-1 controls cell cycle progression and chromosome maintenance in yeast [8][9][10] and participates in aspects of ribosomal biogenesis including 20S rRNA cleavage and maturation of ribosomal small subunits in both yeast and human cells [8,11]. The genome of the free-living nematode Caenorhabditis elegans also encodes RIOK-1 [12]. A large-scale double-stranded RNA interference (RNAi) study of C. elegans showed that the silencing of Ce-riok-1 leads to embryonic lethality and arrest of larval development [13][14][15][16][17]. This finding suggests that RIOK-1 is essential for development and growth of nematodes. In spite of the functional importance of this molecule in C. elegans, there is no published information on the functions of RIOK-1 in any related parasitic nematodes, other than DNA sequence characterization and bioinformatic analyses of RIOK encoding genes of the ovine parasitic nematodes Trichostrongylus vitrinus [18] and Haemonchus contortus [19] These studies revealed that riok-1 of T. vitrinus is transcribed at the highest level in iL3 and proposed that riok-1 of H. contortus is a potential drug target. However, almost nothing is known about the function of this gene for any parasite.
Transgenesis, which is very useful for functional genomic studies in C. elegans [20], was successfully established in S. stercoralis [21,22], thus providing us with a technical platform to investigate the functions of genes in this parasite [21][22][23]. Because of the potential of this parasitic nematode for functional genomic studies, we aimed to isolate and characterize Ss-riok-1 and to explore the temporal and spatial expression patterns of this gene, with a view towards uncovering its function. Information on the function of Ssriok-1 will contribute to an evaluation of RIOKs as potential targets of drugs directed against S. stercoralis and related parasitic nematodes.

Ethics statement
The S. stercoralis (UPD strain) was maintained in prednisolonetreated Beagles in accordance with protocol (Permit Number: SYXK-0029) approved by the Committee on the Ethics of Animal Experiments of Hubei Province. The care and maintenance of animals were in strict accordance with the recommendations in the Guide for the Regulation for the Administration of Affairs Concerning Experimental Animals of P.R. China.

Parasite maintenance and culture
The UPD strain of S. stercoralis was maintained in prednisolone-treated dogs and cultured as described [24,25]. RNA and genomic DNA were extracted from iL3s concentrated from charcoal coprocultures using the Baermann funnel technique [26] after 7-10 days of incubation at 22uC. The iL3s were washed several times with a sterile buffered saline called BU buffer [24,27] to reduce bacterial contamination. Free-living adult S. stercoralis for micro-injection were isolated from charcoal coprocultures using the Baermann funnel, incubated for two days at 22uC and then placed on Nematode Growth Medium (NGM) agar plates seeded with Escherichia coli OP50 [24].

DNA and cDNA preparation
Total genomic DNA was extracted from ,10,000 iL3 larvae using a small-scale sodium proteinase K extraction [28] followed by mini-column (Promega) purification. Total RNA of S. stercoralis was extracted from ,30,000 iL3 by TRIzol reagent extraction (Life Technologies). RNA yields were estimated spectrophotometrically (NanoDrop Technologies, Thermo). Total 59-ends cDNA and 39-ends cDNA were synthesized by Smart RACE Kit (BD Bioscience) following the manufacturer's protocol; cDNAs were stored at 220uC.
Isolation of Ss-riok-1 cDNA and promoter region Degenerate primers 1F and 2R (Table S1) were designed based on the alignment of riok-1 homologues of H. contortus (GenBank

Author Summary
Parasitic nematodes cause serious global health problems and enormous economic losses. Control of these parasites is difficult due to their complicated life cycle and the lack of knowledge of their developmental biology at the molecular level. Protein kinases are key molecules regulating a range of biological processes of organisms. The atypical protein kinase RIOK-1 was reported to be indispensable in yeast, as well as in free-living nematode Caenorhabditis elegans, but little is known about its function in parasitic nematodes. In the present study, we investigate the RIOK-1 encoding gene (Ss-riok-1) and its predicted protein Ss-RIOK-1 from parasitic nematode Strongyloides stercoralis which causes canine and human diseases. We found that Ss-RIOK-1 has high sequence identities (50-65%) to its homologues from both vertebrates and invertebrates. It also has abilities of phosphorylation and auto-phosphorylation in vitro. Ss-riok-1 transcript is present in all stages of S. stercoralis with more abundance in the parasitic stages than in the free-living stages, along with the gene expression in neuron system of post free-living L1 and body muscle of iL3, indicating that it plays important role in the development and infection of S. stercoralis. The findings have important implications for understanding the function of RIOK-1 in the development of parasitic nematodes. accession no. HQ198854.1), C. elegans (NM_001026399) and an EST (BI324299.1) from Strongyloides ratti. A 210 bp fragment was amplified from the cDNA synthesized from total RNA extracted from S. stercoralis iL3s. This PCR product was cloned into the pMD-19T vector (Takara Biotechnology) and sequenced. Based on the isolated sequence, two gene-specific primers (designated 3F and 4R) were designed (Table S1). Using pairs of gene-specific primers and adaptor primers, two partially overlapping cDNA fragments were produced separately from total RNA from S. stercoralis iL3s by 59-and 39-RACE. After sequencing the two partial cDNAs, five gene-specific primers were designed to amplify the 59-and 39-terminal regions of the Ss-riok-1 cDNA (Table S1). The complete cDNA of Ss-riok-1 was assembled according to the sequence obtained through 59-and 39-RACE PCR. Then a pair of primers with restriction sites (Ss-riok1-BamHI and Ss-riok1-XhoI, Table S1) were designed to amplify the coding region of Ss-riok-1 using the following cycling conditions: initial 94uC, 5 min; then 94uC, 30 s, 60uC, 30 s, 72uC, 2 min for 30 cycles; final extension at 72uC for 10 min. The PCR product was then cloned into pMD19-T vector (Takara Biotechnology) and sequenced.
To isolate the promoter sequence, four genomic DNA libraries were constructed employing Genome-Walker Kit (BD Bioscience), following the manufacturer's instructions. Briefly, genomic DNA of S. stercoralis was digested with four restriction enzymes DraI, EcoRV, PvuII, StuI, respectively. Then, each of the four digested products was purified by phenol/chloroform extraction [29] and linked to an adapter provided in the kit, producing four libraries. Touch-down PCR was performed using one adapter primer with one gene-specific primer and the following protocol: 7 cycles for 94uC, 25 s, 72uC, 3 min; 32 cycles for 94uC 25 s, 67uC 3 min; and final extension at 67uC, 7 min. The PCR products from the four libraries were examined separately on agorose gels, and the products were gel-purified, cloned into pMD19-T vector (Takara Biotechnology) and sequenced. To isolate the entire promoter sequence, two primers (Ss-rio1-PstI and Ss-rio1-AgeI, Table S1) located in the 59-and 39-ends, respectively, were designed and used to amplify the merged sequence using the following protocol: initial 94uC, 5 min; then 94uC, 30 s, 60uC, 30 s, 72uC, 4 min 30 s for 30 cycles; final extension at 72uC for 10 min. The resultant PCR product containing the promoter of Ss-riok-1 in its entirety was cloned into pGMD19-T vector (Takara Biotechnology) and sequenced and then sub-cloned into pAJ01 [29].
For phylogenetic analysis, the amino acid sequences of 27 homologues were retrieved from GenBank databases and the alignment of protein sequences was carried out by Clustal X [35] and manually adjusted. The species selected were nine nematodes, including Ascaris suum ( The phylogenetic analysis was conducted using the neighborjoining (NJ), maximum parsimony (MP) and maximum likelihood (ML) methods based on Jones-Taylor-Thornton (JTT) model in the MEGA v.5.0 [36]. Confidence limits were assessed by bootstrapping using 1,000 pseudo-replicates for NJ, MP and ML, and other settings were obtained using the default values in MEGA v.5.0 [36]. A 50% cut-off value was implemented for the consensus tree.

Protein expression and purification
A full-length cDNA of Ss-riok-1 was amplified by PCR using primers Ss-riok1-BamHI and Ss-riok1-XhoI (Table S1). The PCR product was then cloned into pMD19-T and sequenced, and further subcloned into the vector pGEX-4T-1. The insert of the recombinant plasmid pGEX-4T-1-riok-1 was sequenced and its open-reading frame (ORF) encoding the fusion protein GST-Ss-RIOK-1 was confirmed [45]. This recombinant vector was then used to transform E. coli (Transetta; Transgene) cells for protein expression. The bacterial cells were diluted 1:100 into new LB/ Amp + medium, after 3 h of growth at 37uC. The bacteria were induced with IPTG (1:1000), grown at 28uC and 150 rpm/min overnight and then concentrated by centrifugation at 10000 rpm/ min for 2 min. The bacteria were re-suspended in 50 mM Tris-Cl with 0.1 M NaCl, passed through a 0.45 mm filter and loaded onto a 1 mL GST rap 4B affinity columns (GE Healthcare). The bound Ss-RIOK-1 was eluted with 50 mM Tris-HCl, 40 mM reduced glutathione, pH 8.0. The elution was concentrated using a Ultra-15 50 KD centrifugal filter devices (Millipore). The final concentration was 1 mg/mL. As a control, E. coli Transetta cells were transformed with null pGEX-4T-1 vector, incorporating a GST tag. The GST protein was purified using the same method as described above.

Kinase assays
All assays were performed in 20 mL reaction volumes containing 25 mM Tris pH 7.5, 50 mM NaCl and 2 mM MgCl 2 [46]. 10 mg purified GST-Ss-RIOK-1 were added into the autophosphorylation reaction; 2 mg GST-Ss-RIOK-1 and 9 mg myelin basic protein (MBP) were added to each phosphorylation reaction. In the control group, the GST-Ss-RIOK-1 was replaced with GST. All components were mixed prior to the addition of 1 mCi [c 32 P] ATP.

Transformation constructs and transformation of S. stercoralis
To make the plasmid for transgenesis, the promoter region of Ss-riok-1 was digested with restriction enzymes PstI and AgeI (Thermo) and gel-purified by Tiangen Gel purification kit (Tiangen Biotech). The purified product was then subcloned into the promoter-less vector pAJ01 [29] to create a plasmid pRP1 (Fig.  S1). The constructs was extracted by TIANpure Midi Plasmid Kit (Tiangen Biotech) and then was diluted to 30 ng/mL and stored at 220uC.
Adult FL Female S. stercoralis were transformed by gonadal micro-injection using an established approach [21]. Briefly, 30 ng/mL of plasmid Ss-riok-1p::gfp::Ss-era-1t (pRP1) were injected into the distal gonads of individual worms. Single females transformed with pRP1 were then paired with one or two FL adult males on an NGM+OP50 plate and incubated at 22uC for egg laying. F1 progeny were screened for fluorescence at 24, 48 and 72 h, respectively, after microinjection. S. stercoralis larvae were screened for expression of GFP fluorescent reporter transgenes using an Olympus SZX12 stereomicroscope with epifluorescence. Worms with GFP expression were examined in detail using an Olympus BX60 compound microscope equipped with Nomarski Differential Interference Contrast (DIC) optics and epifluorescence (Olympus America Inc.). Specimens were immobilized on a 2% agarose pad (Lonza), anesthetized using 20-50 mM levamisole (Sigma-Aldrich), and imaged using a digital camera (Spot RT Color, Model 2.2.1) and associated image analysis software (Diagnostic Instruments, Inc.) [37]. All images were processed using Photoshop CS 5.0. Image-processing algorithms, primarily brightness and contrast adjustments, were all applied in linear fashion across the entire image.

Characterization of the Ss-riok-1 cDNA
The full-length cDNA of Ss-riok-1 (GeneBank Accession No. KJ701282) is 1828 bp in length, including a 59-UTR of 377 bp, a 17 bp 39-UTR followed with the polyadenylation signal and a coding sequence of 1,434 bp encoding 477 amino acids. Neither a first nor a second spliced leader sequence (SL1 and SL2, respectively) was identified. In the protein sequence predicted from the gene, the RIOK-1 motif ''LVHADLSEYNTL'' [9] was identified (Fig. 1). The Ss-RIOK-1 shares high sequence identity (50-65%) to RIOK-1s from a diverse range of organisms, including vertebrates, amphibians, fish, plants and nematodes, with the highest identity (65%) to As-RIOK-1 from A. suum.
Alignment of the amino acid sequences of Ss-RIOK-1 with the homologues from selected species (Fig. 1) shows that the conserved regions include the ATP binding motif (sub-domains I and II), the flexible loop, the hinge region (subdomain V), the active site (subdomain VIb), the metal binding loop (DFG loop, subdomains VII and VIII) and other features of RIOK-1s, such as the C termini of ATP-biding motif G-x-[ILV]-S-T-G-K-E and the altered I-D-V- [SAQ] in the metal-biding motif of Ss-RIOK-1. The key residues ''Asp'' and ''Asn'' essential for protein kinase activity in the active sites of RIOK-1s, which are involved in catalytic function and are conserved in all ePKs [4,9]. The amino acid sequences in regions external to these functional subdomains were more divergent than the sequences within them (Fig. 1).

Relationship of Ss-RIOK-1 with orthologues from other species
Results of phylogenetic analyses (Fig. 2) showed that there is concordance in topology among the MP, ML and NJ trees. Ss-RIOK-1 groups with orthologues from clade V nematodes [47] with strong (99%) nodal support. The RIOK-1s from parasitic nematodes representing clade III [47] grouped together with strong (95%) support; all nematode RIOK-1s formed a cluster with absolute support to the exclusion of 17 RIOK-1s from 13 non-nematode species. Among the 17 RIOK-1s representing 13 non-nematode species, RIOK-1s from plants, mammals or insects each grouped together, respectively with high bootstrap support (99-100%). The RIOK-1s from other vertebrates, including fish and amphibians, grouped with the RIOK-1s from mammals with strong bootstrap support respectively (100%).
Genetic structure of Ss-riok-1 and comparison with orthologues from C. elegans and H. contortus The genomic DNA representing Ss-riok-1 (GeneBank Accession No. KJ701282) is 5889 bp in length. The 377 bp 59-UTR from cDNA is interrupted by two large introns of 711 bp and 3148 bp in length, respectively. The coding sequence of Ss-riok-1 encompasses 1,636 bp, containing three exons of 284-587 bp in size and two introns of 64 bp and 138 bp in size, respectively. The 17 bp 39-UTR of Ss-riok-1 follows the third exon of the coding sequence (Fig. 3). Comparison with Ce-riok-1 (MO1B12.5a, sequences were retrieved from WormBase) from C. elegans and Hc-riok-1 from H. contortus [19] showed that these two homologues contain more introns than Ss-riok-1. The coding sequence of Ce-riok-1 contains eight exons of 72-532 bp in size and seven introns of 58-857 bp in size, whereas Hc-riok-1 has 16 exons of 61-200 bp in size and 15 introns of 30-520 bp in size [19].

Analysis of the predicted Ss-riok-1 promoter
The isolated 59-upstream region of the start codon of Ss-riok-1 is 4,280 bp in size (GeneBank Accession No. KJ701282). Bioinformatic analysis of transcriptomic and genomic data from S. stercoralis revealed that the region between Ss-riok-1 and the upstream gene was 6,854 bp. The gene upstream of Ss-riok-1 encoded a putative falvin domain-containing protein and is transcribed in the opposite orientation of Ss-riok-1. The putative promoter region of this gene was 3,665 bp; the 4,280 bp DNA region upstream of the start codon of Ss-riok-1 overlapped by 1,091 bp with the flavin domain-containing protein-encoding gene. Comparison between the isolated 59-upstream region of Ss-riok-1 and the homologous region in Ce-riok-1 (4,242 bp upstream of the start code of Ce-riok-1 retrieved from WormBase) showed a sequence identity of 42.7% (Fig. S2). The putative promoter regions of both genes are A+T rich, with the A+T content of 81.1% for Ss-riok-1 and 68.1% for Ce-riok-1, respectively. Further analysis failed to detect CpG islands in either promoter region, but found a GC box (GGCGG) in the promoter region of Ce-riok-1 that is absent from that of Ss-riok-1. This analysis highlighted several promoter elements, including 38 TATA boxes, four CAAT (CCAAT) or inverse CAAT (ATTGG), 12 GATA (WGATAR), 19 inverse GATA (TTATC) and eight E-boxes (CANNTG) in the promoter region of Ss-riok-1. With the exception of the GC-boxes, CAAT boxes and inverse CAAT boxes, there are generally fewer such elements in the promoter region of Ce-riok-1 than in that of Ss-riok-1. There are 10 CAAT (CCAAT) or inverse CAAT (ATTGG), one GC-box, two GATA (WGATAR), seven inverse GATA (TTATC), seven E-boxes (CANNTG) and six TATA boxes in the promoter region of Ce-riok-1. The four nucleotides preceding the start codon (ATG) are AAGG for Ss-riok-1 and AAAC for Ce-riok-1. The AAAC sequence observed in Ce-riok-1 differs from the adenine tract AAAA more frequently seen in C. elegans genes [48]. The predicted promoter elements are scattered across the promoter regions of the two genes, with no apparent pattern to their distribution.

Transcriptional analysis of Ss-riok-1
Ss-riok-1-specific transcripts were detected in all developmental stages of S. stercoralis examined (Fig. 4). Abundance of these transcripts increases significantly during the transition from PFL   L1 to iL3, and remains at a high level in the host-derived L3+. L3+ develop to the parthenogenetic P female during their migration in the host and reach the intestine; a significant decrease with the transcripts abundance of Ss-riok-1 (p,0.05) was found during migration and development. The reduced abundance of Ss-riok-1 transcripts during development of PP L1s to FL females was also detected. The abundance of Ss-riok-1 transcripts in iL3 is significantly greater than in PP L3 (p,0.001). By contrast, the abundance of Ss-riok-1 transcripts are significantly higher in P female and PP L1 than in FL female and PFL L1, respectively (p, 0.001).
Protein kinase activity of recombinant Ss-RIOK-1 The activities of many protein kinases include phosphorylation and auto-phosphorylation. It is reported that RIOK-1 could also phosphorylate the common protein kinase substrate MBP as well as RIOK-1 itself [49]. To assess the kinase activity of Ss-RIOK-1, recombinant GST-Ss-RIOK-1 with a GST tag (designated GST-Ss-RIOK-1) was expressed in E. coli (Fig. 5A). Purified recombinant GST-Ss-RIOK-1 incubated with [c 32 P] ATP only or in the presence of MBP showed radioactive signals associated with the Ss-RIOK-1 and MBP, respectively, indicating that the GST-Ss-RIOK-1 is capable of both phosphorylation and auto-phosphorylation (Fig. 5B).

Localisation of Ss-RIOK-1 expression
To determine the anatomic expression pattern of Ss-riok-1, larval progeny of FL Female of S. stercoralis transformed with the construct pRP1 were screened for GFP expression. Some of the immature eggs had GFP expression, even when they were still in the vulva of the female adults (data not shown). After 24 h, newly hatched transgenic PFL L1s exhibited GFP expression throughout the body, with strongest expression at the boundary between pharynx and intestine ( Fig. 6A and B). After 72 h, strong GFP  expression under the Ss-riok-1 promoter was seen in the nervous system, including some head neurons, body neurons and tail neurons as well as in pharynx and hypodermis of transgenic PFL L1s and PFL L2s (Fig. 7A and B). Processes of head neurons go through the body of these larvae, connecting to neurons in the body and tail. The nervous system of S. stercoralis has not been mapped in its entirety, so that a neural map of the free-living nematode C. elegans [50,51] was employed as a model to tentatively identify of neurons expressing GFP under the Ss-riok-1 promoter. Using this comparative approach, we concluded that body neurons expressing the Ss-riok-1-based reporter are likely sensory neurons and ventral nerve cord motor neurons (Fig. 7C, D, E and F). Furthermore, as PFL L1s developed towards iL3s in the next 4-5 days in culture at 22uC, Ss-riok-1-specific reporter expression was localized to zones in the body wall muscle of the parasite (Fig. 6 C and D).

Discussion
The crucial role that RIOK-1 plays in the development of organisms was initially deduced from investigations in yeast as well as in C. elegans [8,[13][14][15][16][17]. In the present study, we laid the groundwork for functional studies of RIOK-1 in parasitic nematodes by isolating and characterizing the RIOK-1 encoding gene Ss-riok-1 from S. stercoralis, an important parasite causing disease in humans and dogs.
The present study revealed only one Ss-riok-1 transcript. By contrast, multiple riok-1 transcript variants, with shortened Cterminal and N-terminal ends, have been identified in C. elegans and humans, respectively. The presence of only one riok-1 transcript appears to be a common feature of parasitic nematodes as public database searches (results not shown) have failed to detect multiple riok-1 transcript variants in various species including A. suum, B. malayi, Dirofilaria immitis, H. contortus, L. loa, S. ratti, S. stercoralis, T. vitrinus and W. bancrofti. The functional significance of transcript variants encoding an incomplete RIOK-1 in C. elegans and human is yet unknown.
The main functional domains in RIOK-1 appear to be conserved among organisms studied to date, including Archaeoglobus fulgidus and humans. Previous studies in yeast and human cells revealed that RIOK-1s have several functional domains possessing different functions. RIOK-1s lack the substrate binding motif commonly found in ePKs, but have a flexible loop (between b3 and aC) which is absent from ePKs [7,52]. The conserved RIOK-1 signature sequence ''STGKEA'' in the ATP binding motif has higher similarity to the signature sequence ''STGKES'' in the ATP binding motif of RIOK-3 than to the analogous signature sequence ''GxGKES'' in RIOK-2. The Active site of RIOK-1 ''LVHxDLSEYN'' also has higher similarity to that of RIOK-3 ''LVHxDLSExN'' than to that of RIOK-2 ''IHxDoNEFN'', and the two residues Asp (D) and Asn (N) in this motif are present in the active sites of all ePKs [7]. The active sites in ePKs are usually involved in the transfer of phosphate groups from adenosine triphosphate (ATP) to substrate proteins, and such phosphorylation events are basic to signal transduction pathways regulating numerous cellular and metabolic processes [5,53]. Active site mutations that disrupt RIOK-1 kinase activity also interfere with recycling of two trans-activating factors (endonuclease hNobI and its binding partner hDim2) which are necessary for maturation of the human 40S ribosomal subunit [11]. Besides the active sites, the more divergent N-terminal and C-terminal regions of RIOK-1 also participate in some biological processes. The first 120 amino acids of the N-terminal region of human RIOK-1 interact with a complex consisting of protein arginine methyltransferase 5 (PRMT5) and methylosome protein 50 (MEP50), which are two components of the methylosome [54]. This RIOK-1-PRMT5 complex methylates the RNA binding protein nucleolin, which is involved in ribosomal maturation [55][56][57][58]. In addition to the active sites and the N-terminal region, the C-terminal region of yeast RIOK-1 is phosphorylated by the casein kinase 2 (CKII) to regulate the cell cycle in yeast [59]. The functions of the RIO domain and the N-and C-terminal regions of RIOK-1 in parasitic nematodes are unknown. In the present study, the predicted amino acid sequence alignment (Fig. 1) revealed that Ss-RIOK-1 shares common features with the RIOK-1 family. Ss-RIOK-1 has limited similarity in its Nterminal and C-terminal regions to yeast and human RIOK-1 homologues. In addition, Ss-RIOK-1 is capable of both phosphorylation and autophosphorylation, which is a property of the RIOK-1s from A. fulgidus, S. cerevisiae and humans [9,11,46]. Taken together, these findings suggest that Ss-RIOK-1 is an active protein kinase but its biological functions may differ from those of its homologues in yeast and humans.
Ss-riok-1 contained fewer introns than its homologues from C. elegans and H. contortus. This reduction in intron number has been a consistent trend in comparisons of genes in S. stercoralis and their orthologs in C. elegans and its parasitic counterparts in clade V [21,37,60,61]. The comparison of 59-UTRs in Ss-riok-1 and Ce-riok-1 revealed some shared promoter elements, though the sequence similarity was limited. The promoter elements included TATA box, CAAT, GATA box and E-boxes were all found in the regulatory region of Ss-riok-1 and Ce-riok-1. Along with the TATA box, the CAAT box is another common promoter element for protein-coding genes in eukaryotes [62]. The GATA box is recognized by GATA transcription factors and is necessary for regulation of eukaryotic development and reproduction [63][64][65][66][67]. E-boxes are recognized and bound by basic helix-loop-helix (bHLH) proteins which regulate a wide range of developmental process in eukaryotic organisms including neurogenesis and myogenesis [68][69][70]. 37 bHLH proteins have been identified in C. elegans, and some of them are associated with specification of neural lineages and differentiation of myogenic lineages [71,72]. E-boxes are also characterized as gene promoter elements in the parasitic nematode H. contortus [73] and, as demonstrated here, in S. stercoralis, suggesting that these elements are involved in regulating the development of parasitic nematodes.
Ss-riok-1 transcripts are present in all life stages of S. stercoralis, suggesting that this gene functions in the development of all stages of this parasite. The abundance of Ss-riok-1 transcripts varies during development, being higher in the iL3 and in parasitic and post-parasitic life stages, which are progressing towards the freeliving adults (FL Female and FL Male) than in the FL female and Ss-riok-1, a RIO Kinase Encoding Gene of Strongyloides stercoralis PFL L1. In order to explore the tissues that Ss-riok-1 may function in S. stercoralis, the anatomical expression pattern is analyzed by employing transgenesis in free-living stages of this parasite.
The results of transgenesis showed that the GFP expression under the Ss-riok-1 promoter occurs at embryos and PFL L1 suggesting that Ss-riok-1 begins early in embryogenesis in the postfree-living life stages of S. stercoralis. Ce-riok-1 expression in C. elegans occurs in a similar temporal pattern [74] and the embryos die when the expression of Ce-riok-1 is silenced by RNAi, highlighting the essential role of Ce-riok-1 in the embryogenesis of C. elegans [13,17]. This embryonic lethality phenotype, along with the similar embryonic and early larval pattern of Ss-riok-1 expression leaves open the possibility that this gene is also essential for embryogenesis in post-free-living stages of S. stercoralis.
As transgenic S. stercoralis developed from PFL L1s to PFL L2s in culture, strong GFP expression from the Ss-riok-1-based reporter persisted in the pharynx and nervous system (Fig. 7). With the exception of sensory neurons of the amphids and some related interneurons [75,76], the nervous system of S. stercoralis has not been mapped in detail. Despite this, studies on the nervous system of S. stercoralis published do show how C. elegans, with its morphological similarities to free-living stages of S. stercoralis can be employed as a model to identify neurons in this parasite [76]. The C. elegans hermaphrodite contains 302 neurons including 282 neurons in somatic nervous system and 20 neurons in pharyngeal nervous system [51,[77][78][79]. There were 113 motor neurons that control crawling and swimming behaviours as well as the motility of the alimentary and reproductive systems [50,51]. These motor neurons, along with some of the sensory neurons in the body, are connected by longitudinal nerve tracts and commissures [51]. The differentiation of C. elegans neurons begins at the proliferative phase of embryogenesis, and the nervous system mature at the late L1 and L2 stages. During the late L1 stage, some neurons, including five classes of ventral nerve cord (VNC) motor neurons, are generated from several cell lineages [78,79]. GFP expression from our Ss-riok-1-based transcriptional reporter occurs in late L1-L2, in the dorsal cord (DC) and VNC which connect the neurons from head, body and tail. In addition to DC and VNC, the Ss-riok-1 promoter is active in other longitudinal nerve tracts from head to tail and commissures sent by the body neurons and connected with DC in the PFL L1-L2 larvae of S. stercoralis. These findings suggest that Ss-riok-1 contributes to the development and function of the nervous system of S. stercoralis during the development in PFL L1-L2.
Studies on the maturation of neurons in the mammalian central nervous system (CNS) [80][81][82][83] have shown that polyribosomes contribute prominently to the synaptogenesis, leading to protein synthesis. During the development of the neuronal system of C. elegans, neurons project axons, which reach their synaptic partners to establish complex neuronal circuits [84]. This process might rely on one or more molecular signals in a neuron to recognize the receptor molecule on synaptic partners [85]. In some organisms, polyribosomes accumulate in growing synapses and contribute to postsynaptic membrane specialization [86]. Considering the important role of RIOK-1 plays in the maturation of 40S ribosomal subunits in other eukaryotes [10,11], Ss-RIOK-1, which our data suggest is localized in the nervous system of freeliving stage larvae, could also participate in the development of this system in S. stercoralis through its function in ribosomal maturation and resulting support of protein synthesis in developing neurons and synapses.
In contrast to PFL L1s and PFL L2s, activity of the Ss-riok-1 promoter is limited to the body wall of iL3. Ss-riok-1 transcripts are also most abundant at this stage. Both of these findings are consistent with the fact that the cuticles of S. stercoralis iL3 undergo significant remodeling and that L3i become radially constricted overall in the transition from actively developing postfree-living stage larvae [3]. The presence of eight E-boxes in the 59-UTR of Ss-riok-1 is consistent with a role in myogenesis that may accompany morphogenesis of the highly motile S. stercoralis iL3 [71,72]. Overall, the significant increase in abundance of Ssriok-1 transcripts in iL3 over PP L3 strongly suggested a pivotal role for Ss-RIOK-1 in the infective process and other aspects of parasitic life in iL3 of S. stercoralis. Localization of Ss-riok-1 expression in body wall muscle, along with the findings from other systems implicating RIOK-1s in myogenesis suggest that the parasite RIOK-1 kinase may support the increase in motility that is essential for host-finding and contact by iL3.
The conservation in RIO domains between Ss-RIOK-1 and homologues from selected species suggests that Ss-RIOK-1 may participate in the ribosomal process in S. stercoralis as did its homologues in yeast and human cells [8][9][10][11]. Whether the kinase activity of Ss-RIOK-1 supports its function in the maturation of ribosome as well as the development of S. stercoralis is unknown. Transformation of S. stercoralis with a transgene construct encoding a kinase dead mutant Ss-RIOK-1 that interact with other proteins or ribosomal particles but can't function as an active kinase may disrupt the function of the endogenous Ss-RIOK-1, which could be an effective way to analyze the function of Ss-RIOK-1 in the development and growth of S. stercoralis [23,24]. Understanding the function of RIOK-1 in regulating S. stercoralis' development may greatly help us to assess the potential of RIOK-1 as a drug target for the control of parasitic nematodes.
In conclusion, we have isolated and characterized the RIOK-1 encoding gene Ss-riok-1 from the zoonotic parasite S. stercoralis. Ss-RIOK-1 contains a RIO1 signature motif and has high similarity to a range of homologues from different species. Recombinant Ss-RIOK-1 has kinase activity. Ss-riok-1 transcripts are present throughout development in S. stercoralis with the highest abundance in iL3. The Ss-riok-1 promoter is active in head neurons, body neurons and tail neurons as well as in pharynx and hypodermis of S. stercoralis of PFL L1s and PFL L2s and in body wall muscle of iL3. These findings suggest that Ss-riok-1 plays an important role in regulating development of S. stercoralis, particularly in the formation of the nervous system in PFL L1 and L2s and in morphogenesis of the iL3 which is crucial to the infective process. Future work should focus on ascertaining whether Ss-RIOK-1 function is essential for the development or survival of S. stercoralis and by what mechanisms it exerts its function. Figure S1 Diagram of Ss-riok-1 transcriptional reporter construct pRP1 used to transform S. stercoralis. The 4280 bp promoter of Ss-riok-1 was inserted into pAJ01 between the PstI and AgeI restriction sites. Length of gfp with artificial introns and Ss-era-1 39 UTR are marked above them.  Table S1 The names and DNA sequences of primers used in the present study for isolating cDNA and promoter region of Ss-riok-1 and for constructing protein expression and transgenic plasmids. (DOC)