RIOK-2 protein is essential for egg hatching in a common parasitic nematode.

The atypical protein kinase RIOK-2 is a non-ribosomal factor essential for ribosome maturation in yeast and human cells; however, little is known about its physiological role in pathogens. Our earlier work examined the expression profile of a RIOK-2 gene (Ss-riok-2) in Strongyloides stercoralis - a prevalent nematode parasite of dogs and humans. Herein, we demonstrate that Ss-RIOK-2 encodes a catalytically active kinase, distributed primarily in the cytoplasm of intestinal and hypodermal cells in transgenic larvae. Its expression oscillates as the free-living L1s develop into infective L3s. Overexpression of a catalytically impaired Ss-RIOK-2-D228A mutant delayed the development of transgenic larvae, while ectopic expression of another dominant negative isoform with a mutation in the ATP-binding site (K123A) abrogated the process of egg hatching, which could be rescued by co-expressing a wild-type Ss-RIOK-2 but not by its Ss-RIOK-1 ortholog. Collectively, our findings show a critical and specific role of Ss-RIOK-2 during the development of a pathogenic roundworm, which can be exploited to develop anti-infectives.

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

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.

Functional genomic exploration reveals that Ss-RIOK-1 is essential for the development and survival of Strongyloides stercoralis larvae.

Protein kinase RIOK-1 is a non-ribosomal factor essential for rRNA cleavage and ribosome small subunit maturation. It is encoded in all eukaryotic organisms. The RIOK-1 encoding gene of Caenorhabditis elegans (Ce-riok-1) is expressed in the neuronal and reproductive systems in larvae and adults of this free-living nematode, and it supports larval growth and development of the adult gonad. In spite of its recognised roles in model organisms such as C. elegans, little is known about the function of this molecule in parasitic nematodes. In a previous study, we characterised the structure, transcriptional profiles and in vivo transcriptional expression patterns of the Ss-riok-1 of human and canine parasitic nematode Strongyloides stercoralis. Here, we extend previous work to undertake functional studies, using transgenesis to assess the roles of Ss-RIOK-1 in the development of S. stercoralis. The results revealed that recombinant Ss-RIOK-1 with D282A mutation at its catalytic site lost its kinase phosphorylation activity in vitro. Both wild-type and mutant Ss-RIOK-1s were expressed in the cytoplasm of neurons and some hypodermal cells in the wild-type strain (UPD) of S. stercoralis. Larvae expressing the dominant negative mutant Ss-RIOK-1 that lost the catalytic activity had a decreased mobility and a severe defect in development to the infective L3 stage. Our findings demonstrated that Ss-RIOK-1 is essential for the development and survival of free-living larvae of S. stercoralis, and that catalytic activity is essential for its function in the parasitic nematode.

Exploring features and function of Ss-riok-3, an enigmatic kinase gene from Strongyloides stercoralis.

BACKGROUND: Right open reading frame protein kinase 3 (RIOK-3) belongs to the atypical kinase family. Unlike the other two members, RIOK-1 and RIOK-2, which are conserved from Archaea to humans, RIOK-3 occurs only in multicellular organisms. Studies on HeLa cells indicate that human RIOK-3 is a component of the 40S small ribosome subunit and supports cancer cell growth and survival. However, almost nothing is known about the function of RIOK-3. We explored the functional role of RIOK-3 encoding gene from Strongyloides stercoralis, a parasitic nematode of humans and dogs. METHODS: To analyze the gene and promoter structure of Ss-riok-3, RACE-PCR and Genome-walker PCR were performed to isolate the full length cDNA, gDNA and promoter region of Ss-riok-3. RNA-seq was conducted to assess the transcript abundance of Ss-riok-3 in different stages of S. stercoralis. Transgenesis was employed to determine the anatomic expression patterns of Ss-riok-3. RESULTS: The RIOK-3 protein-encoding gene (designated Ss-riok-3) of S. stercoralis was characterized. The full-length complementary and genomic DNAs of the RIOK-3 encoding gene (riok-3) were isolated from this nematode. The cDNA of Ss-riok-3 is 1,757 bp in length, including a 23 bp 5'-UTR, a 36 bp 3'-UTR and a 1,698 bp coding region encoding a protein of 565 amino acids (aa) containing a RIO kinase domain. RNA sequencing (RNA-seq) analysis revealed that Ss-riok-3 is transcribed in all developmental stages of S. stercoralis assessed, with transcripts being particularly abundant in parasitic females. Gene structure analysis revealed that Ss-riok-3 contains no intron. The putative promoter contains conserved promoter elements, including four TATA, two GATA, one inverse GATA and one inverse CAAT boxes. The promoter of Ss-riok-3 drives GFP expression in the head neuron, intestine and body wall muscle of transgenic S. stercoralis larvae, and the TATA boxes present in the 3'-UTR of the gene immediately upstream of Ss-riok-3 initiate transcription. CONCLUSIONS: The characterization of the RIOK-3 encoding gene from S. stercoralis provides a sound foundation for investigating in detail its function in the development and reproduction of this important pathogen.

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.

Strongyloides stercoralis age-1: a potential regulator of infective larval development in a parasitic nematode.

Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The 'dauer hypothesis' predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 µM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08-0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases.

Strongyloides stercoralis excretory/secretory protein strongylastacin specifically recognized by IgE antibodies in infected human sera.

The infective, microscopic Strongyloides stercoralis larvae in contaminated soil can penetrate human skin with the help of excretory/secretory proteases. These proteases play a critical role in infection and transmigration of the parasite to the intestines. Strongylastacin is similar to astacin (from the digestive gland of the crayfish Astacus astacus), a multi-domain protein with a signal peptide, a pro-enzyme, a catalytic domain containing the zinc binding consensus astacin family signature sequence HEXXHXXGFXHEXXRXDR, and a second conserved zinc binding motif SIMHY at N- terminal region. An EGF-1 like domain and a CUB domain are located at the COOH- terminal. In this study, the excretory/secretory Strongylastacin gene from S. stercoralis infective larval stage was cloned and expressed as a 45 kDa in Escherichia coli. Immunoblot analysis showed the presence of natural IgG antibodies against strongylastacin in six infected and six non-endemic normal sera. These findings were confirmed in an ELISA of 32 S. stercoralis infected and 32 presumed normal human sera; all contained natural anti-strongylastacin IgG antibodies. By contrast, IgE antibodies specific to strongylastacin were present in sera from individuals infected with S. stercoralis but not in uninfected control sera. Moreover, recombinant strongylastacin did not cross-react with IgE antibodies either from patients infected with filaria or patients with tropical pulmonary eosinophilic (TPE) who had increased IgE antibodies. The present authors conclude that strongylastacin, an excretory/secretory antigen, elicits specific IgE antibodies in S. stercoralis infected humans. Non-specific IgG antibodies to strongylastacin are present in both infected and normal humans. Further investigation is needed to understand the role of the host protective response against strongylastacin.

Identification of an astacin-like metallo-proteinase transcript from the infective larvae of Strongyloides stercoralis.

Strongyloides stercoralis, an important nematode pathogen of humans, is transmitted by contact with soil contaminated with the microscopic larvae of the parasite. We determined the cDNA sequence and deduced amino acid structure of a metallo-proteinase that is abundantly transcribed expressed by infective stage larvae of S. stercoralis. This deduced structure of the enzyme revealed a multi-domain protein that included an NH2-terminal peptidase. This peptidase consisted of a signal peptide, a pro-enzyme region, and a mature peptidase domain that included the metal ion co-ordinating motifs, HETSHALGVIH and SIMHY ("Met-turn"), characteristic of the catalytic active site of members of the metzincin superfamily of zinc metallo-endopeptidases. It was phylogenetically and structurally similar to astacin from the digestive gland of the crayfish Astacus astacus, to the HCH-1 peptidase of Caenorhabditis elegans required for hatching and migration of a post-embryonic neuroblast, and to the morphogenetically important peptidases, bone morphogenetic protein-1 (BMP-1) and Drosophila tolloid. In addition, the Strongyloides enzyme, designated strongylastacin, includes a central epidermal growth factor (EGF) domain followed by a carboxyl CUB (complement sub component C1r/C1s/embryonic sea urchin protein Uegf/bone morphogenetic protein) domain. Inspection of the dbEST database revealed the presence of at least 9 transcript clusters that are related to greater or lesser extent to strongylastacin; based on these expressed sequence tags, strongylastacin was expressed only in the infective third stage larvae, whereas other transcript clusters were expressed both in filariform and rhabditiform stages or only in the rhabditiform stage. Based on the deduced sequence, structure, and expression profile, strongylastacin is the probable candidate for the zinc-dependent metalloprotease, Ss40, known to be deployed by larvae of S. stercoralis to penetrate human skin to initiate infection.

Cloning and expression of isocitrate lyase from human round worm Strongyloides stercoralis.

A full length cDNA (1463 bp) encoding isocitrate lyase (EC 4.1.3.1) of Strongyloides stercoralis is described. The nucleotide sequence of this insert identified a cDNA coding for the isocitrate lyase. The conceptually translated amino acid sequence of the open reading frame for S. stercoralis isocitrate lyase encodes a 450 amino acid residue protein with an apparent molecular weight of 50 kDa and a predicted pl of 6.39. The sequence is 69% A/T, reflecting a characteristic A/T codon bias of S. stercoralis. The amino acid sequence of S. stercoralis isocitrate lyase is compared with bifunctional glyoxylate cycle protein of Caenorhabditis elegans and isocitrate lyases from Chlamydomonas reinhardtii and Myxococcus xanthus. The full length cDNA of S. stercoralis was expressed in pRSET vector and bacteriophage T7 promoter based expression system. S. stercoralis lyase recombinant protein, purified via immobilized metal affinity chromatography, showed a molecular mass of 50 kDa on polyacrylamide gels. The role of isocitrate lyase in the glyoxylate cycle and energy metabolism of S. stercoralis is also discussed.

A cDNA encoding the highly immunodominant antigen of Strongyloides stercoralis: gamma-subunit of isocitrate dehydrogenase (NAD+).

A full length cDNA encoding the highly immunodominant 41 kDa antigen of Strongyloides stercoralis (P5), recognized by 83% of human patients [Siddiqui et al. (1997) Parasitol Res 83:655-658], is obtained. A clone containing a 1371 bp insert was selected following screening of the S. stercoralis cDNA library with antibodies specific to antigen P5. The nucleotide sequence of this insert identified a cDNA coding for the gamma-subunit of isocitrate dehydrogenase (NAD+), GenBank Accession Number AF176568. The conceptually translated amino acid sequence of the open reading frame for the gamma-subunit of S. stercoralis isocitrate dehydrogenase (NAD+) encodes a 388 amino acid residue protein with an apparent molecular weight of 43 kDa and a predicted pI of 7.15. The sequence is 71% A/T, reflecting the characteristic A/T codon bias of S. stercoralis. The amino acid sequence of the S. stercoralis gamma-subunit of isocitrate dehydrogenase (NAD+) is compared with those of Caenorhabditis elegans, rat and human NAD(+)-ICDH. The diagnostic potential of the S. stercoralis gamma-subunit of isocitrate dehydrogenase (NAD+) is also discussed.

A cDNA encoding a pepsinogen-like, aspartic protease from the human roundworm parasite Strongyloides stercoralis.

Using degenerate oligonucleotide primers based on conserved active site residues, we have isolated a cDNA encoding an aspartic protease from the nematode parasite Strongyloides stercoralis, an important, enteric pathogen of humans. cDNAs encoding the aspartic protease were isolated from the infective, third stage larvae of the parasite as well as from free-living, rhabditiform larvae. Based on comparisons of other aspartic proteases, the cDNA encoded a short signal peptide, an enzyme pro-segment of 35 amino acid residues, and mature enzyme of 337 residues. Homology alignments using the proenzyme sequence showed that the novel S. stercoralis zymogen was 36% identical to human pepsinogen A and 36% identical to pepsinogen C (progastricin) from humans and macaques. Phylogenetic analyses using the Phylip program and analysis of Glx/Asx and Leu/Ile ratios indicated that the proenzyme was closely related to pepsinogen A-like enzymes from the free-living nematode Caenorhabditis elegans and Haemonchous contortus, a nematode parasite of the gastro-intestinal tract of sheep. We have termed this novel enzyme strongyloidespepsin.

Ss40: the zinc endopeptidase secreted by infective larvae of Strongyloides stercoralis.

Infective larvae of a pathogenic nematode of humans, Strongyloides stercoralis, release a potent zinc endopeptidase activity which has a broad substrate specificity for constituents of extracellular dermal matrix, including elastin. Specific inhibitors of zinc endopeptidases prevent the penetration of mammalian skin by S. stercoralis larvae. We now report the molecular size and isoelectric point of the S. stercoralis zinc endopeptidase at 40 kDa, pI 5 by zymogram analysis. The activity was not influenced by incubation with beta-mercaptoethanol at 22 degrees C, but was inactivated by incubation at 100 degrees C for 2 min. The enzyme, which we term Ss40, is immunogenic and stimulates humoral IgG antibodies during infection of humans; the activity was immunoprecipitable from ES with pooled infection sera. In addition, a HPLC-enriched Ss40 preparation stimulated the release of histamine from peripheral blood leukocytes of S. stercoralis-infected persons, suggesting that Ss40 is allergenic in humans.