Structural and developmental expression of Ss-riok-2, an RIO protein kinase encoding gene of Strongyloides stercoralis

Abstract

RIO kinases are essential atypical protein kinases in diverse prokaryotic and eukaryotic organisms, playing significant roles in yeast and humans. However, little is known about their functions in parasitic nematodes. In the present study, we have isolated and characterized the full-length cDNA, gDNA and a putative promoter of a RIOK-2 protein kinase (Ss-RIOK-2) encoding gene (Ss-riok-2) from Strongyloides stercoralis, a medically important parasitic nematode (Order Rhabditida). A three-dimensional structure (3D) model of Ss-RIOK-2 was generated using the Chaetomium thermophilum RIOK-2 protein kinase (Ct-RIOK-2) crystal structure 4GYG as a template. A docking study revealed some critical sites for ATP binding and metal binding. The putative promoter of Ss-riok-2 contains a number of conserved elements. RNAseq analysis revealed the highest levels of the Ss-riok-2 transcript in free-living females and parasitic females. To identify anatomical patterns of Ss-riok-2 expression in S. stercoralis, we observed expression patterns of a transgene construct encoding green fluorescent protein under the Ss-riok-2 promoter in post free-living S. stercoralis. Expression driven by this promoter predominated in intestinal cells. This study demonstrates significant advancement in molecular and cellular biological study of S. stercoralis and of parasitic nematodes generally, and provides a foundation for further functional genomic studies.

Figure 1

Multiple sequence alignment among predicted RIOK-2 proteins from eight organisms (Strongyloides stercoralis, Loa loa, Haemonchus contortus, Caenorhabditis elegans, Apis florea, Danio rerio, Canis familiaris, Homo sapiens, Chaetomium thermophilum). The N-terminal domain contains the conserved wHTH do main (red). The RIO kinase domain contains the ATP-binding motif (yellow), the flexible loop (yellow), the hinge region (yellow), and the catalytic (red) and metal-binding loops (yellow) as determined from the structure of the Archaeoglobus fulgidus RIOK-2 protein. The predicted subdomains I-IX are marked above the alignment. Asterisks indicate identical residues. Dashes indicate gaps in the sequence, included for alignment purposes.

Figure 2

The neighbour-joining tree of RIOK-2 amino acid sequences from a range of organisms. These species are Ascaris suum (ADY41687.1), Caenorhabditis elegans (NP_493544.2), Caenorhabditis remanei (XP_003097720.1), Haemonchus contortus (ADW23593.1), Loa loa (XP_003139548.1), Aedes aegypti (XP_001655107.1), Apis florea (XP_003692456.1), Canis familiaris (XP_536291.3, XP_005618121.1), Drosophila melanogaster (NP 651365.1), Dictyostelium discoideum (XP_640350.1), Dictyostelium fasciculatum (XP_004351182.1), Chaetomium thermophilum (XP_006693771.1), Monodelphis domestica (XP_001364030.1), Xenopus laevis (NP 001086801.1, NP 001088220.1). Accession numbers for the various sequences in the NCBI Protein Database are given next to each species name. Numbers represent bootstrap values (after 1000 iterations) and the scale bar represents branch length as a fraction of the total tree length. The bootstrap values of >95% were displayed in the tree.

Figure 3

Homology model of the RIOK-2 protein kinase from Strongyloides stercoralis. The modeled structure of the Ss-RIOK-2 protein kinase shows different structural elements, N-terminal helix winged domain (blue), ATP binding motif (red), β3 (green), αC (pink), Hing (yellow), active site (cyan), metal-binding loop (grey) and αI (Amaranth).

Figure 4

Surface structure of the Ss-RIOK-2 ATP binding pocket and interaction of ATP within modeled structure of Ss-RIOK-2-ATP. (A) Surface structure of the ATP binding pocket of Ss-RIOK-2-ATP. Blue is positive, red is negative, Mg²⁺ is shown as a purple sphere, ATP and catalytic residues are shown in stick representation. (B) Interaction of ATP with key residues present within the binding pocket of Ss-RIOK-2-ATP.

Figure 5

Diagrammatic representation of the genomic organizations of the riok-2 from Strongyloides stercoralis (Ss-riok-2) and orthologues from Chaetomium thermophilum (Ct-riok-2), Caenorhabditis elegans (Ce-riok-2) and Haemonchus contortus (Hc-riok-2). The organization of each gene was determined by aligning the cDNA and genomic DNA sequences, with intron-exon boundaries being defined using the GT-AG rule. Black boxes represent exons, whilst horizontal lines represent introns. Numbers below the boxes indicate the sizes of exons (in nucleotides), whereas numbers above the lines indicate the intron sizes.

Figure 6

Transcriptional profile of Ss-riok-2 in seven Strongyloides stercoralis life stages. Transcript abundances were compared in biological triplicate. Stages examined were: 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 are expressed as fragments per kilobase of coding exon per million mapped reads (FPKM). Error bars represent 95% confidence intervals.

Figure 7

Representative expression pattern of GFP under the putative Ss-riok-2 promoter in a transgenic second-stage larva of Strongyloides stercoralis. Differential interference contrast (DIC) and fluorescence (GFP) images showing the expression of construct S. stercoralis Ss-riok-2p::gfp (pRP2; Fig. S1) in a post-free-living second-stage larva. GFP reporter expression predominated in intestine. Scale bars = 10 μm.

Figure 8

Autophosphorylation of recombinant His-Ss-RIOK-2. (A) Western-blot of His-Ss-RIOK-2 immunoprecipitated with His antibody. (B) Kinase assay showing autophosphorylation activities of recombinant His-Ss-RIOK-2.