RNAseq Analysis of the Parasitic Nematode <i>Strongyloides stercoralis</i> Reveals Divergent Regulation of Canonical Dauer Pathways

<div><p>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 <i>Strongyloides stercoralis</i> 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 <i>Caenorhabditis elegans</i> 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 <i>S. stercoralis</i>, 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 <i>S. stercoralis</i> homologs of <i>C. elegans</i> dauer genes identified by BLAST searches of the <i>S. stercoralis</i> genome as well as <i>de novo</i> assembled transcripts. Intriguingly, genes encoding cGMP pathway components were coordinately up-regulated in L3i. In comparison to <i>C. elegans</i>, <i>S. stercoralis</i> has a paucity of genes encoding IIS ligands, several of which have abundance profiles suggesting involvement in L3i development. We also identified seven <i>S. stercoralis</i> genes encoding homologs of the single <i>C. elegans</i> 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 <i>S. stercoralis</i> 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.</p></div

Comparison of NHR pathway homologs and transcript abundances in <i>S. stercoralis</i> and <i>C. elegans</i>.

<p>Comparison of NHR pathway homologs and transcript abundances in <i>S. stercoralis</i> and <i>C. elegans</i>.</p

John Nichols to Sir James Edward Smith

Notes that although [Emanuel Mendez] da Costa's [(1717-1791), naturalist] manuscripts are chiefly concerned with mineralogy there are several on botany from [George] Edwards [(1694-1773)], [Georg Dionysius] Ehret [(1708-1770)], Knolles, [John] Martyn [(1699-1768)], [Philip] Miller, [Richard] Pulteney, and some others, and are available for Smith's use [for "A selection of the correspondence of Linnaeus" (1821)]

<i>S. stercoralis</i> RNAseq mean library sizes and number of reads aligning to the genome.

<p>A total of 21 libraries were derived from polyadenylated RNA and sequenced from seven developmental stages, each in biological triplicate. Paired-end 100 base-pair (bp) reads were generated from the following developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), <i>in vivo</i> activated third-stage larvae (L3+), parasitic females (P Female), predominantly (>95%) heterogonically developing post-parasitic first-stage larvae (PP L1), and post-parasitic approximately third-stage larvae heterogonically developing to free-living adults and enriched for females (PP L3). The mean number of reads generated per replicate refers to the mean number of 100 bp reads sequenced (black bars) per biological replicate from each developmental stage. The mean number of mapped reads per replicate refers to the mean number of 100 bp reads aligned to <i>S. stercoralis</i> genomic contigs using TopHat (white bars) per biological replicate from each developmental stage. Error bars represent +1 standard deviation.</p

Temporal regulation of <i>S. stercoralis</i> DAF-12 and genes putatively involved in dafachronic acid synthesis.

<p>Transcript abundances were determined for the coding region of: (A) <i>Ss-ncr-1</i>, a gene encoding a homolog of the intracellular cholesterol transporters <i>Ce</i>-NCR-1 and <i>Ce</i>-NCR-2; (B) <i>Ss-daf-36</i>, a gene encoding a homolog of the Rieske-like oxygenase <i>Ce</i>-DAF-36; (C) <i>Ss-scdh-16</i>, a gene encoding a short-chain dehydrogenase homolog most similar to <i>Ce</i>-DHS-16; (D) <i>Ss-cyp-9</i>, a gene encoding a cytochrome P450 homolog most similar to <i>Ce</i>-DAF-9; (E) <i>Ss-emb-8</i>, a gene encoding a homolog of the cytochrome P450 reductase <i>Ce</i>-EMB-8; (F) <i>Ss-strm-1</i>, a gene encoding a homolog of the methyltransferase <i>Ce</i>-STRM-1; and (G) <i>Ss-daf-12</i>, the homolog of the nuclear hormone receptor <i>Ce</i>-DAF-12. Transcript abundances were quantified in seven developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), <i>in vivo</i> activated third-stage larvae (L3+), parasitic females (P Female), post-parasitic first-stage larvae (PP L1), and post-parasitic third-stage larvae (PP L3). Transcript abundances were calculated as fragments per kilobase of coding exon per million mapped reads (FPKM) and log transformed. Error bars represent 95% confidence intervals. The y-axes were scaled from 0 to 3.5 to aid comparison between genes.</p

Comparison of IIS pathway homologs and transcript abundances in <i>S. stercoralis</i> and <i>C. elegans</i>.

<p>Comparison of IIS pathway homologs and transcript abundances in <i>S. stercoralis</i> and <i>C. elegans</i>.</p

<i>Caenorhabditis elegans</i> dauer pathways during reproductive development.

<p>Four developmental pathways regulate <i>C. elegans</i> dauer entry and exit: a cyclic guanosine monophosphate (GMP) signaling pathway, an insulin/insulin-like growth factor 1 (IGF-1) -like signaling pathway, a dauer transforming growth factor β (TGFβ) pathway, and a nuclear hormone receptor (DAF-12) regulated by a class of steroid ligands known as dafachronic acids (DAs). This simplified model depicts the four pathways under conditions favoring reproductive development and repression of dauer arrest. Proteins in light green are “active,” while proteins in dark red are “inactive.” Black circles represent phosphorylation sites and diamond-shaped boxes represent phosphatases. Green arrows represent either increases in metabolite concentration or increases in gene transcription. Solid black lines represent well-established pathways, while dashed lines represent putative pathways. Adapted from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001854#pntd.0001854-Fielenbach1" target="_blank">[12]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001854#pntd.0001854-McGrath1" target="_blank">[54]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001854#pntd.0001854-Narasimhan1" target="_blank">[111]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001854#pntd.0001854-Wollam1" target="_blank">[129]</a>.</p

Protein sequence diversity and temporal regulation of <i>S. stercoralis</i> insulin-like peptides.

<p>(A) A predicted protein sequence alignment of seven <i>S. stercoralis</i> insulin-like peptides (ILPs), <i>Ss</i>-ILP-1 through -7, was constructed using human insulin (<i>Hs</i>-INSULIN) and <i>Lymnaea stagnalis</i> molluscan insulin-related peptide I (<i>Ls</i>-MIP-1) as the references. Cysteine residues, which are predicted to form disulfide bonds, are in red letters. Predicted signal sequences are highlighted in yellow, predicted furin recognition motifs are highlighted in red, hydrophobic residues important for helix formation are highlighted in green, and a conserved glycine is highlighted in blue. Predicted C peptides are highlighted in gray with dibasic predicted cleavage sites underlined. The B peptide is N-terminal of the C peptide, while the A peptide is C-terminal of the C peptide. (B-H) Transcript abundances were determined for the coding region of seven <i>S. stercoralis</i> ILP-encoding genes (<i>Ss-ilp-1</i> through <i>-7</i>) in seven developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), <i>in vivo</i> activated third-stage larvae (L3+), parasitic females (P Female), post-parasitic first-stage larvae (PP L1), and post-parasitic third-stage larvae (PP L3). Transcript abundances were calculated as fragments per kilobase of coding exon per million mapped reads (FPKM) and log transformed. Error bars represent 95% confidence intervals. The y-axes were scaled from 0 to 3.5 to aid comparison between genes.</p

Phylogenetic analysis and temporal regulation of <i>S. stercoralis</i> TGFβ ligands.

<p>(A) Phylogenetic analysis of the transforming growth factor β (TGFβ) super-family ligands was performed; nematode TGFβ ligands resolved into three main families that share the same cysteine architecture. <i>Ss</i>-TIGL-1 groups with the <i>Ce</i>-TIG-2-like family; <i>Ss</i>-DBL-1 and <i>Ss</i>-DBL-2 group with the <i>D. melanogaster</i> decapentaplegic (DPP) and vertebrate bone morphogenetic protein (BMP) family; and <i>Ss</i>-TGH-1 through -7 group with the human TGFβ1 family that also includes <i>Ce</i>-DAF-7. A Clustal W alignment of the TGFβ ligands truncated at the first conserved cysteine was used to construct the neighbor-joining tree with 100 iterations of boot-strapping. Abbreviations: <i>Ancylostoma caninum</i> (Ac), <i>Ascaris suum</i> (As), <i>Brugia malayi</i> (Bm), <i>Caenorhabditis briggsae</i> (Cb), <i>Caenorhabditis elegans</i> (Ce), <i>Danio rerio</i> (Dr), <i>Drosophila melanogaster</i> (Dm), <i>Haemonchus contortus</i> (Hc), <i>Heligmosomoides polygyrus</i> (Hp), <i>Homo sapiens</i> (Hs), <i>Loa loa</i> (Ll), <i>Parastrongyloides trichosuri</i> (Pt), <i>Strongyloides ratti</i> (Sr), <i>Strongyloides stercoralis</i> (Ss), <i>Trichinella spiralis</i> (Ts), and <i>Xenopus laevis</i> (Xl). The scale bar represents substitutions per position. Accession numbers are listed in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001854#pntd.0001854.s017" target="_blank">Data S4</a>. (B–H) Transcript abundances were determined for the coding region of seven <i>S. stercoralis</i> genes, <i>Ss-tgh-1</i> through <i>-7</i>, encoding putative TGFβ ligands similar to <i>Ce</i>-DAF-7 in seven developmental stages: free-living females (FL Female), post-free-living first-stage larvae (PFL L1), infectious third-stage larvae (L3i), <i>in vivo</i> activated third-stage larvae (L3+), parasitic females (P Female), post-parasitic first-stage larvae (PP L1), and post-parasitic third-stage larvae (PP L3). Transcript abundances were calculated as fragments per kilobase of coding exon per million mapped reads (FPKM) and log transformed. Error bars represent 95% confidence intervals. The y-axes were scaled from 0 to 3.5 to aid comparison between genes.</p

Diagram of the <i>Strongyloides stercoralis</i> life cycle.

<p>Developmentally arrested infective third-stage larvae (L3i) can form by either a homogonic route (dark red) or a heterogonic route (light green). Female post-parasitic first-stage larvae (PP L1) passed in the feces of the infected host can develop homogonically through two larval molts directly to L3i or heterogonically through four larval molts to free-living females (FL Females). Post-parasitic L1 males invariably develop heterogonically through four molts to free-living males (FL Males). Post-free-living L1 (PFL L1), which are all female, molt twice and develop exclusively to L3i. Upon encountering and penetrating a susceptible host, activated third-stage larvae (L3+) resume feeding and development, migrate to the intestines, and molt twice into parasitic females (P Females). Post-parasitic L1 larvae can also precociously develop into auto-infective third-stage larvae (L3a) entirely within the host. Developmental stages marked with an asterisk (*) were interrogated by RNAseq. Adapted from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001854#pntd.0001854-Schad2" target="_blank">[7]</a>.</p