RNAseq Analysis of the Parasitic Nematode Strongyloides stercoralis Reveals Divergent Regulation of Canonical Dauer Pathways

Abstract

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 β (TGFβ) 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 TGFβ 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.

Hc-daf-2 encodes an insulin-like receptor kinase in the barber’s pole worm, Haemonchus contortus, and restores partial dauer regulation

“…Hc-daf-2 transcripts are present throughout the entire life cycle, but are significantly upregulated in the iL3 stage. This upregulation might relate directly to developmental arrest, in preparation for further reproductive development, and is consistent with the transcription profiles for Ss-daf-2b , Ss-age-1 , Ss-pdk-1 and Ss-akt-1 in S. stercoralis (see Stoltzfus et al, 2012b; Massey et al, 2013). This information indicates that the insulin-like receptor encoded by Hc-daf-2 plays a crucial role in the transition from a developmentally arrested to an activated state in H. contortus .…”

Strongyloides stercoralis: current perspectives

“…102 Polyadenylated transcriptome analysis of S. stercoralis and comparison with dauer stage C. elegans revealed that in both S. stercoralis and C. elegans, the genes encoding Cyclic Guanosine Monophosphate (cGMP) pathway components were coordinately upregulated in L3i, suggesting that dauer pathway genes are present in S. stercoralis and may play a role in L3i development. 103 Microarray analysis of L3i and L1 of S. stercoralis helped to identify 935 differentially expressed genes between these two stages, 104 and similar results were reported using S. ratti transcriptome. 100 Overall, using Strongyloides EST representation and microarray data, there was rather little evidence for the conservation of transcriptional profiles between S. ratti and S. stercoralis or C. elegans.…”

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

“…The S. stercoralis (PV001 line), derived from a single female worm of the UPD strain, was maintained as described previously [24,[34][35][36][37]. Transcription profiles of Ss-riok-3 were assessed in seven developmental stages of the PV001 line of S. stercoralis, including free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infective third-stage larvae (iL3), in vivo activated third-stage larvae (L3+), parasitic females (P Female), post-parasitic first-stage larvae (PP L1) and post-parasitic third-stage larvae (PP L3).…”

“…The parasites from different stages were isolated as described previously [34,35]. Transcription was quantified using RNA-seq [35]. Raw RNA-seq data for the seven developmental stages are available at http://www.…”

“…ebi.ac.uk/arrayexpress/ under accession numbers E-MTAB-2192 (iL3) and E-MTAB-1164 (FL Female, PFL L1, L3+, P Female, PP L1, and PP L3). Ss-riok-3-specific transcript abundances were calculated as fragments per kilobase of coding exon per million fragments mapped (FPKM), and significant differences in FPKM values between developmental stages were determined using Cufflinks v.2.0.2 (http://cufflinks.cbcb.umd.edu/) [35,[38][39][40]; p-values <0.05 were considered statistically significant, and p-values <0.001 were considered statistically highly significant. FPKM and 95% confidence intervals were plotted in Prism version 5.01 (GraphPad Software, Inc., http://www.graphpad.com/).…”