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

ABSTRACT: 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 ?7-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 ?7-DA similarly modulated transcript levels of ILP-encoding genes. Using the phosphatidylinositol-4,5-bisphosphate 3-kinase inhibitor LY294002, we demonstrated that 400 nM ?7-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.

SUBMITTER: Stoltzfus JD

PROVIDER: S-EPMC4092141 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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

Stoltzfus Jonathan D JD Bart Stephen M SM Lok James B JB

PLoS pathogens 20140710 7

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-coupl ...[more]

PMID: 25010340

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Project description:BACKGROUND: Differences between noninfective first-stage (L1) and infective third-stage (L3i) larvae of parasitic nematode Strongyloides stercoralisat the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose. METHODS AND FINDINGS: Oligonucleotide hybridization probes for the array were designed to bind 3571 putative mRNA transcripts predicted by analysis of 11,335 expressed sequence tags (ESTs) obtained as part of the Nematode EST project. RNA obtained from S. stercoralis L3i and L1 was co-hybridized to each array after labeling the individual samples with different fluorescent tags. Bioinformatic predictions of gene function were developed using a novel cDNA Annotation System software. We identified 967 differentially expressed genes (457 L3i-biased; 510 L1-biased) having greater than two-fold expression differences and p < 0.01. Based on functional analysis, L1s have a larger number of genes putatively involved in transcription (p = 0.0158), and L3is have higher expression of stress-related genes (such as putative heat shock proteins dnj-12, daf-21, dnj-10). Genes with products that have been shown to be immunoreactive in S. sterocoralis-infected humans (SsIR and NIE) were additionally found to be L3i biased. Unique and abundant L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1, daf-12, and daf-21, which may be potential chemotherapeutic targets. The S. stercoralis ortholog of fatty acid and retinol binding protein-1, successfully used in a vaccine against Ancylostoma ceylanicum, was identified among the top 25 most L3i-biased genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in the L3i group and may be a valuable S. stercoralis target of interest. Conclusions A new DNA microarray tool for the examination of S. stercoralis biology has been developed and provides new and valuable insights regarding differences between infective and noninfective S. stercoralis larvae. Potential therapeutic and vaccine targets were identified for further study.

Project description: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 ?M ?7-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 ?M ?7-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 ?7-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 ?M ketoconazole, compared to 98.8±0.4% in the positive control. This inhibition was partially rescued by ?7-DA, with 71.2±16.4% of iL3 feeding in 400 nM ?7-DA and 35 ?M 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.

Project description:Background - Strongyloidiasis is a neglected tropical disease affecting an estimated 600 million people, particularly in resource-limited settings. The infection can persist lifelong due to Strongyloides stercoralis peculiar auto-infective cycle. The cumbersome diagnosis and the limited knowledge of the mechanisms underpinning this chronic infection are key issues in the disease management. To date, only a few proteomics studies have been conducted to elucidate the molecular mechanisms associated with Strongyloides parasitism or to highlight novel immunological markers and our knowledge of S. stercoralis proteome still limited. Methods - S. stercoralis infective larvae (iL3) we isolated from a faecal sample from an infected subject and analysed by high-throughput tandem mass spectrometry. To achieve a more comprehensive characterisation of iL3 proteome the experimental dataset was analysed through an automatic search strategy combined with manual annotation, which included gene ontology (GO) analysis, InterPro annotation, assessment of the homology with Homo sapiens and other pathogens of clinical importance and B-cell epitope prediction. Results – Our pipeline identified 430 S. stercoralis proteins, 187 (43%) of which corresponded to uncharacterized proteins. Oxidoreductases and peptidases were amongst the most represented protein categories as highlighted by GO analyses (molecular function terms); while membrane and mitochondrial proteins were the most represented cellular component GO categories. In our dataset we also identified a high proportion of proteins bearing CAP, SCP or thioredoxin domain or belonging to cysteine-rich secretory, transthyretin-like or peptidase protein families, confirming previous data on the most represented proteins associated with S. stercoralis parasitism, as inferred from genomic and transcriptomic data. Finally, we also highlighted 9 proteins bearing amino acid sequences with immunogenic properties that might represent novel candidates for serological tests. Conclusions – Here we provide a comprehensive description and annotation of S. stercoralis iL3 proteome and propose some proteins as potentially immunogenic, to be evaluated as novel serological diagnostic markers. In order to highlight novel targets to improve current serodiagnosis and treatment, it is in fact fundamental to expand the molecular understanding derived from ‘omics studies beyond the current state of the art.

Project description:BackgroundDifferences between noninfective first-stage (L1) and infective third-stage (L3i) larvae of parasitic nematode Strongyloides stercoralis at the molecular level are relatively uncharacterized. DNA microarrays were developed and utilized for this purpose.Methods and findingsOligonucleotide hybridization probes for the array were designed to bind 3,571 putative mRNA transcripts predicted by analysis of 11,335 expressed sequence tags (ESTs) obtained as part of the Nematode EST project. RNA obtained from S. stercoralis L3i and L1 was co-hybridized to each array after labeling the individual samples with different fluorescent tags. Bioinformatic predictions of gene function were developed using a novel cDNA Annotation System software. We identified 935 differentially expressed genes (469 L3i-biased; 466 L1-biased) having two-fold expression differences or greater and microarray signals with a p value<0.01. Based on a functional analysis, L1 larvae have a larger number of genes putatively involved in transcription (p = 0.004), and L3i larvae have biased expression of putative heat shock proteins (such as hsp-90). Genes with products known to be immunoreactive in S. stercoralis-infected humans (such as SsIR and NIE) had L3i biased expression. Abundantly expressed L3i contigs of interest included S. stercoralis orthologs of cytochrome oxidase ucr 2.1 and hsp-90, which may be potential chemotherapeutic targets. The S. stercoralis ortholog of fatty acid and retinol binding protein-1, successfully used in a vaccine against Ancylostoma ceylanicum, was identified among the 25 most highly expressed L3i genes. The sperm-containing glycoprotein domain, utilized in a vaccine against the nematode Cooperia punctata, was exclusively found in L3i biased genes and may be a valuable S. stercoralis target of interest.ConclusionsA new DNA microarray tool for the examination of S. stercoralis biology has been developed and provides new and valuable insights regarding differences between infective and noninfective S. stercoralis larvae. Potential therapeutic and vaccine targets were identified for further study.

Project description: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.

Dataset Information

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

Publications

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

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