Toward understanding the functional role of Ss-RIOK-1, a RIO protein kinase-encoding gene of Strongyloides stercoralis

Abstract

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.

Figure 1. Alignment of the inferred amino acid sequences of Strongyloides stercoralis Ss-RIOK-1 with RIOK-1s from nine other species.

The nine selected species are Loa loa (XP_003135673.1, Ll-RIOK-1), Brugia malayi (EDP30009.1, Bm-RIOK-1), Caenorhabditis elegans (CCD67367.1, Ce-RIOK-1), Haemonchus contortus (ADW23592.1, Hc-RIOK-1), Trichostrogylus vitrinus (CAR64255.1, Tv-RIOK-1), Homo sapiens (NP_113668.2, Hs-RIOK-1), Mus musculus (NP_077204.2, Mm-RIOK-1), Danio rerio (NP_998160.1, Dr-RIOK-1), Arabidopsis thaliana (NP_180071.1, At-RIOK-1), Schistosoma mansoni (XP_002573653.1, Sm-RIOK-1). Alpha-helices A-I or beta-sheet structures are highlighted with light grey and marked under the alignment. The subdomains I-XI are marked above the alignment. Functional domains including ATP-binding motif (red), flexible loop (yellow), hinge (yellow), active site (red) and metal binding loop (yellow) are highlighted and labeled above the alignment. Asterisks indicate identical residues.

Figure 2. The Neighbor-joining tree of Strongyloides stercoralis Ss-RIOK-1 with 27 homologues from 23 selected species.

These species contain nine nematode species, two plant species, two insects, three fish and amphibian species and six mammalian species. The RIOK-1 from Saccharomyces cerevisiae (CAA99317.1) is used as the outgroup. GenBank accession numbers of the homologous sequences are listed beside the species name. Bootstrap values are displayed above or below the branches.

Figure 3. The gene structure of Ss-riok-1 with comparison to its homologues from Caenorhabditis elegans and Haemonchus contortus.

Black boxes indicate the exons, with the numbers above indicating the length of exon. Introns are indicated by slanted lines between the exons, with the numbers indicating the intron length. The 5′- and 3′- untranslated regions (UTR) of Ss-riok-1 and Ce-riok-1 are indicated with white boxes, with the numbers above the box indicating the length of the UTR.

Figure 4. Transcriptional profiles of Ss-riok-1.

Transcript abundances were determined for the C-terminal coding region of Ss-riok-1, in seven developmental stages: parasitic females (P Female), post-parasitic first-stage larvae (PP L1), post-parasitic third-stage larvae (PP L3), free-living females (FL Female), post free-living first-stage larvae (PFL L1), infectious third-stage larvae (iL3), and in vivo activated third-stage larvae (L3+). Transcript abundances were calculated as fragments per kilobase of coding exon per million mapped reads (FPKM). Bracket with 1 star represent the significant difference (p<0.05), brackets with 3 stars represent the significant difference (p<0.01) in transcript abundances between the two selected stages. Error bars represent 95% confidential intervals.

Figure 5. Autophosphorylation and phosphorylation activity of recombinant GST-Ss-RIOK-1.

A, Western-blot of GST-Ss-RIOK-1 (89 kDa) and GST (28 kDa) are immunoprecipitated with GST antibody. B, Kinase assay showing autophosphorylation and phosphorylation activities of recombinant GST-Ss-RIOK-1 (89 kDa). Substrate in phosphorylation assay is MBP (18 kDa). Amounts of each protein in the reaction are indicated in the labels. Purified GST constitutes the negative control.

Figure 6. The anatomical expression pattern of Ss-riok-1 in the early post-free-living first stage larvae.

(A, B) and in infective third-stage larvae (C, D). (A, B) strong GFP expression is found at the sphincter connecting pharynx and intestine (S). (C, D) GFP expressed in the pharynx muscle (PM) and body wall muscle (BM). Scale bars = 100 µm.

Figure 7. The spatial expression pattern of Ss-riok-1 in the post-free-living first-stage and second-stage larvae.

DIC (A, C) and fluorescence (B, D) images of transgenic S. stercoralis post-free-living L1-L2 stage larvae expressing Ss-riok-1p::gfp::Ss-era-1t. (A, B) GFP expression in the pharynx (P), head neurons (HN), body neurons (BN) and tail phasmidial neurons (ph) and longitudinal nerve tracts (L), GFP expression is also observed in the neurons (R) with positional homology to neurons of C. elegans in retrovesicular ganglion (RVG). (C, D) GFP expression in the commissures between body neurons and longitudinal nerve tracts (arrow). Scale bars = 100 µm.