Is Strongyloides stercoralis hyperinfection induced by glucocorticoids a result of both suppressed host immunity and altered parasite genetics?

The gastrointestinal (GI) nematode Strongyloides stercoralis (S.s.) causes human strongyloidiasis, a potentially life-threatening disease that currently affects over 600 million people globally. The uniquely pernicious aspect of S.s. infection, as compared to all other GI nematodes, is its autoinfective larval stage (L3a) that maintains a low-grade chronic infection, allowing undetectable persistence for decades. Infected individuals who are administered glucocorticoid therapy can develop a rapid and often lethal hyperinfection syndrome within days. Hyperinfection patients often present with dramatic increases in first- and second-stage larvae and L3a in their GI tract, with L3a widely disseminating throughout host organs leading to sepsis. How glucocorticoid administration drives hyperinfection remains a critical unanswered question; specifically, it is unknown whether these steroids promote hyperinfection through eliminating essential host protective mechanisms and/or through dysregulating parasite development. This current deficiency in understanding is largely due to the previous absence of a genetically defined mouse model that would support all S.s. life-cycle stages and the lack of successful approaches for S.s. genetic manipulation. However, there are currently new possibilities through the recent demonstration that immunodeficient NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice support sub-clinical infections that can be transformed to lethal hyperinfection syndrome following glucocorticoid administration. This is coupled with advances in transcriptomics, transgenesis, and gene inactivation strategies that now allow rigorous scientific inquiry into S.s. biology. We propose that combining in vivo manipulation of host immunity and deep immunoprofiling strategies with the latest advances in S.s. transcriptomics, piggyBac transposon-mediated transgene insertion, and CRISPR/Cas-9-mediated gene inactivation will facilitate new insights into the mechanisms that could be targeted to block lethality in humans with S.s. hyperinfection.

Transcriptional profiles in Strongyloides stercoralis males reveal deviations from the Caenorhabditis sex determination model.

The human and canine parasitic nematode Strongyloides stercoralis utilizes an XX/XO sex determination system, with parasitic females reproducing by mitotic parthenogenesis and free-living males and females reproducing sexually. However, the genes controlling S. stercoralis sex determination and male development are unknown. We observed precocious development of rhabditiform males in permissive hosts treated with corticosteroids, suggesting that steroid hormones can regulate male development. To examine differences in transcript abundance between free-living adult males and other developmental stages, we utilized RNA-Seq. We found two clusters of S. stercoralis-specific genes encoding predicted transmembrane proteins that are only expressed in free-living males. We additionally identified homologs of several genes important for sex determination in Caenorhabditis species, including mab-3, tra-1, fem-2, and sex-1, which may have similar functions. However, we identified three paralogs of gld-1; Ss-qki-1 transcripts were highly abundant in adult males, while Ss-qki-2 and Ss-qki-3 transcripts were highly abundant in adult females. We also identified paralogs of pumilio domain-containing proteins with sex-specific transcripts. Intriguingly, her-1 appears to have been lost in several parasite lineages, and we were unable to identify homologs of tra-2 outside of Caenorhabditis species. Together, our data suggest that different mechanisms control male development in S. stercoralis and Caenorhabditis species.

Dafachronic acid and temperature regulate canonical dauer pathways during Nippostrongylus brasiliensis infectious larvae activation.

BACKGROUND: While immune responses to the murine hookworm Nippostrongylus brasiliensis have been investigated, signaling pathways regulating development of infectious larvae (iL3) are not well understood. We hypothesized that N. brasiliensis would use pathways similar to those controlling dauer development in the free-living nematode Caenorhabditis elegans, which is formally known as the "dauer hypothesis." METHODS: To investigate whether dafachronic acid activates the N. brasiliensis DAF-12 homolog, we utilized an in vitro reporter assay. We then utilized RNA-Seq and subsequent bioinformatic analyses to identify N. brasiliensis dauer pathway homologs and examine regulation of these genes during iL3 activation. RESULTS: In this study, we demonstrated that dafachronic acid activates the N. brasiliensis DAF-12 homolog. We then identified N. brasiliensis homologs for members in each of the four canonical dauer pathways and examined their regulation during iL3 activation by either temperature or dafachronic acid. Similar to C. elegans, we found that transcripts encoding antagonistic insulin-like peptides were significantly downregulated during iL3 activation, and that a transcript encoding a phylogenetic homolog of DAF-9 increased during iL3 activation, suggesting that both increased insulin-like and DAF-12 nuclear hormone receptor signaling accompanies iL3 activation. In contrast to C. elegans, we observed a significant decrease in transcripts encoding the dauer transforming growth factor beta ligand DAF-7 during iL3 activation, suggesting a different role for this pathway in parasitic nematode development. CONCLUSIONS: Our data suggest that canonical dauer pathways indeed regulate iL3 activation in the hookworm N. brasiliensis and that DAF-12 may be a therapeutic target in hookworm infections.

Conserved residues are critical for Haloferax volcanii archaeosortase catalytic activity: Implications for convergent evolution of the catalytic mechanisms of non-homologous sortases from archaea and bacteria.

Proper protein anchoring is key to the biogenesis of prokaryotic cell surfaces, dynamic, resilient structures that play crucial roles in various cell processes. A novel surface protein anchoring mechanism in Haloferax volcanii depends upon the peptidase archaeosortase A (ArtA) processing C-termini of substrates containing C-terminal tripartite structures and anchoring mature substrates to the cell membrane via intercalation of lipid-modified C-terminal amino acid residues. While this membrane protein lacks clear homology to soluble sortase transpeptidases of Gram-positive bacteria, which also process C-termini of substrates whose C-terminal tripartite structures resemble those of ArtA substrates, archaeosortases do contain conserved cysteine, arginine and arginine/histidine/asparagine residues, reminiscent of His-Cys-Arg residues of sortase catalytic sites. The study presented here shows that ArtAWT -GFP expressed in trans complements ΔartA growth and motility phenotypes, while alanine substitution mutants, Cys173 (C173A), Arg214 (R214A) or Arg253 (R253A), and the serine substitution mutant for Cys173 (C173S), fail to complement these phenotypes. Consistent with sortase active site replacement mutants, ArtAC173A -GFP, ArtAC173S -GFP and ArtAR214A -GFP cannot process substrates, while replacement of the third residue, ArtAR253A -GFP retains some processing activity. These findings support the view that similarities between certain aspects of the structures and functions of the sortases and archaeosortases are the result of convergent evolution.

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.

ArtA-Dependent Processing of a Tat Substrate Containing a Conserved Tripartite Structure That Is Not Localized at the C Terminus.

Most prokaryote-secreted proteins are transported to the cell surface using either the general secretion (Sec) or twin-arginine translocation (Tat) pathway. A majority of secreted proteins are anchored to the cell surface, while the remainder are released into the extracellular environment. The anchored surface proteins play a variety of important roles in cellular processes, ranging from facilitating interactions between cells to maintaining cell stability. The extensively studied S-layer glycoprotein (SLG) of Haloferax volcanii, previously thought to be anchored via C-terminal intercalation into the membrane, was recently shown to be lipidated and to have its C-terminal segment removed in processes dependent upon archaeosortase A (ArtA), a recently discovered enzyme. While SLG is a Sec substrate, in silico analyses presented here reveal that, of eight additional ArtA substrates predicted, two substrates also contain predicted Tat signal peptides, including Hvo_0405, which has a highly conserved tripartite structure that lies closer to the center of the protein than to its C terminus, unlike other predicted ArtA substrates identified to date. We demonstrate that, even given its atypical location, this tripartite structure, which likely resulted from the fusion of genes encoding an ArtA substrate and a cytoplasmic protein, is processed in an ArtA-dependent manner. Using an Hvo_0405 mutant lacking the conserved "twin" arginines of the predicted Tat signal peptide, we show that Hvo_0405 is indeed a Tat substrate and that ArtA substrates include both Sec and Tat substrates. Finally, we confirmed the Tat-dependent localization and signal peptidase I (SPase I) cleavage site of Hvo_0405 using mass spectrometry.IMPORTANCE The specific mechanisms that facilitate protein anchoring to the archaeal cell surface remain poorly understood. Here, we have shown that the proteins bound to the cell surface of the model archaeon H. volcanii, through a recently discovered novel ArtA-dependent anchoring mechanism, are more structurally diverse than was previously known. Specifically, our results demonstrate that both Tat and Sec substrates, which contain the conserved tripartite structure of predicted ArtA substrates, can be processed in an ArtA-dependent manner and that the tripartite structure need not lie near the C terminus for this processing to occur. These data improve our understanding of archaeal cell biology and are invaluable for in silico subcellular localization predictions of archaeal and bacterial proteins.

Perusal of parasitic nematode 'omics in the post-genomic era.

The advent of high-throughput, next-generation sequencing methods combined with advances in computational biology and bioinformatics have greatly accelerated discovery within biomedical research. This "post-genomics" era has ushered in powerful approaches allowing one to quantify RNA transcript and protein abundance for every gene in the genome - often for multiple conditions. Herein, we chronicle how the post-genomics era has advanced our overall understanding of parasitic nematodes through transcriptomics and proteomics and highlight some of the important advances made in each major nematode clade. We primarily focus on organisms relevant to human health, given that nematode infections significantly impact disability-adjusted life years (DALY) scores within the developing world, but we also discuss organisms of veterinary importance as well as those used as laboratory models. As such, we envision that this review will serve as a comprehensive resource for those seeking a better understanding of basic parasitic nematode biology as well as those interested in targets for vaccination and pharmacological intervention.

The genomic basis of parasitism in the Strongyloides clade of nematodes.

Soil-transmitted nematodes, including the Strongyloides genus, cause one of the most prevalent neglected tropical diseases. Here we compare the genomes of four Strongyloides species, including the human pathogen Strongyloides stercoralis, and their close relatives that are facultatively parasitic (Parastrongyloides trichosuri) and free-living (Rhabditophanes sp. KR3021). A significant paralogous expansion of key gene families--families encoding astacin-like and SCP/TAPS proteins--is associated with the evolution of parasitism in this clade. Exploiting the unique Strongyloides life cycle, we compare the transcriptomes of the parasitic and free-living stages and find that these same gene families are upregulated in the parasitic stages, underscoring their role in nematode parasitism.

Regulation of Life Cycle Checkpoints and Developmental Activation of Infective Larvae in Strongyloides stercoralis by Dafachronic Acid.

The complex life cycle of the parasitic nematode Strongyloides stercoralis leads to either developmental arrest of infectious third-stage larvae (iL3) or growth to reproductive adults. In the free-living nematode Caenorhabditis elegans, analogous determination between dauer arrest and reproductive growth is governed by dafachronic acids (DAs), a class of steroid hormones that are ligands for the nuclear hormone receptor DAF-12. Biosynthesis of DAs requires the cytochrome P450 (CYP) DAF-9. We tested the hypothesis that DAs also regulate S. stercoralis development via DAF-12 signaling at three points. First, we found that 1 µM Δ7-DA stimulated 100% of post-parasitic first-stage larvae (L1s) to develop to free-living adults instead of iL3 at 37°C, while 69.4±12.0% (SD) of post-parasitic L1s developed to iL3 in controls. Second, we found that 1 µM Δ7-DA prevented post-free-living iL3 arrest and stimulated 85.2±16.9% of larvae to develop to free-living rhabditiform third- and fourth-stages, compared to 0% in the control. This induction required 24-48 hours of Δ7-DA exposure. Third, we found that the CYP inhibitor ketoconazole prevented iL3 feeding in host-like conditions, with only 5.6±2.9% of iL3 feeding in 40 µM ketoconazole, compared to 98.8±0.4% in the positive control. This inhibition was partially rescued by Δ7-DA, with 71.2±16.4% of iL3 feeding in 400 nM Δ7-DA and 35 µM ketoconazole, providing the first evidence of endogenous DA production in S. stercoralis. We then characterized the 26 CYP-encoding genes in S. stercoralis and identified a homolog with sequence and developmental regulation similar to DAF-9. Overall, these data demonstrate that DAF-12 signaling regulates S. stercoralis development, showing that in the post-parasitic generation, loss of DAF-12 signaling favors iL3 arrest, while increased DAF-12 signaling favors reproductive development; that in the post-free-living generation, absence of DAF-12 signaling is crucial for iL3 arrest; and that endogenous DA production regulates iL3 activation.

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.

cGMP and NHR signaling co-regulate expression of insulin-like peptides and developmental activation of infective larvae in Strongyloides stercoralis.

The infectious form of the parasitic nematode Strongyloides stercoralis is a developmentally arrested third-stage larva (L3i), which is morphologically similar to the developmentally arrested dauer larva in the free-living nematode Caenorhabditis elegans. We hypothesize that the molecular pathways regulating C. elegans dauer development also control L3i arrest and activation in S. stercoralis. This study aimed to determine the factors that regulate L3i activation, with a focus on G protein-coupled receptor-mediated regulation of cyclic guanosine monophosphate (cGMP) pathway signaling, including its modulation of the insulin/IGF-1-like signaling (IIS) pathway. We found that application of the membrane-permeable cGMP analog 8-bromo-cGMP potently activated development of S. stercoralis L3i, as measured by resumption of feeding, with 85.1 ± 2.2% of L3i feeding in 200 µM 8-bromo-cGMP in comparison to 0.6 ± 0.3% in the buffer diluent. Utilizing RNAseq, we examined L3i stimulated with DMEM, 8-bromo-cGMP, or the DAF-12 nuclear hormone receptor (NHR) ligand Δ7-dafachronic acid (DA)--a signaling pathway downstream of IIS in C. elegans. L3i stimulated with 8-bromo-cGMP up-regulated transcripts of the putative agonistic insulin-like peptide (ILP) -encoding genes Ss-ilp-1 (20-fold) and Ss-ilp-6 (11-fold) in comparison to controls without stimulation. Surprisingly, we found that Δ7-DA similarly modulated transcript levels of ILP-encoding genes. Using the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002, we demonstrated that 400 nM Δ7-DA-mediated activation (93.3 ± 1.1% L3i feeding) can be blocked using this IIS inhibitor at 100 µM (7.6 ± 1.6% L3i feeding). To determine the tissues where promoters of ILP-encoding genes are active, we expressed promoter::egfp reporter constructs in transgenic S. stercoralis post-free-living larvae. Ss-ilp-1 and Ss-ilp-6 promoters are active in the hypodermis and neurons and the Ss-ilp-7 promoter is active in the intestine and a pair of head neurons. Together, these data provide evidence that cGMP and DAF-12 NHR signaling converge on IIS to regulate S. stercoralis L3i activation.

Strongyloides stercoralis daf-2 encodes a divergent ortholog of Caenorhabditis elegans DAF-2.

We hypothesise that developmental arrest in infectious larvae of parasitic nematodes is regulated by signalling pathways homologous to Caenorhabditis elegans DAF (dauer formation) pathways. Alignment of Strongyloides stercoralis (Ss) DAF-2 with DAF-2 of C. elegans and homologs of other species shows that most structural motifs in these insulin-like receptors are conserved. However, the catalytic domain of Ss-DAF-2 contains two substitutions (Q1242 and Q1256), that would result in constitutive dauer formation in C. elegans or diabetes in vertebrate animals. Ss-daf-2 also shows two alternately spliced isoforms, the constitutively expressed Ss-daf-2a, and Ss-daf-2b, which is only expressed in stages leading to parasitism.

RNAseq analysis of the parasitic nematode Strongyloides stercoralis reveals divergent regulation of canonical dauer pathways.

The infectious form of many parasitic nematodes, which afflict over one billion people globally, is a developmentally arrested third-stage larva (L3i). The parasitic nematode Strongyloides stercoralis differs from other nematode species that infect humans, in that its life cycle includes both parasitic and free-living forms, which can be leveraged to investigate the mechanisms of L3i arrest and activation. The free-living nematode Caenorhabditis elegans has a similar developmentally arrested larval form, the dauer, whose formation is controlled by four pathways: cyclic GMP (cGMP) signaling, insulin/IGF-1-like signaling (IIS), transforming growth factor ß (TGFß) signaling, and biosynthesis of dafachronic acid (DA) ligands that regulate a nuclear hormone receptor. We hypothesized that homologous pathways are present in S. stercoralis, have similar developmental regulation, and are involved in L3i arrest and activation. To test this, we undertook a deep-sequencing study of the polyadenylated transcriptome, generating over 2.3 billion paired-end reads from seven developmental stages. We constructed developmental expression profiles for S. stercoralis homologs of C. elegans dauer genes identified by BLAST searches of the S. stercoralis genome as well as de novo assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to C. elegans, S. stercoralis has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven S. stercoralis genes encoding homologs of the single C. elegans dauer regulatory TGFß ligand, three of which are only expressed in L3i. Putative DA biosynthetic genes did not appear to be coordinately regulated in L3i development. Our data suggest that while dauer pathway genes are present in S. stercoralis and may play a role in L3i development, there are significant differences between the two species. Understanding the mechanisms governing L3i development may lead to novel treatment and control strategies.

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.