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 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.

Transgenesis inStrongyloidesand related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing

“…These results are consistent with similar patterns of DAF-2 expression in S. stercoralis and C. elegans and so with similar functions of this insulin-like receptor kinase in regulation of third-stage larval development. Enhanced GFP (EGFP) expression under a putative promoter for Ss-age-1 , which encodes the ortholog of the insulin-regulated PI3 kinase AGE-1 in C. elegans , was observed in cells of the hypodermis, intestine and genital primordium, as well as in phasmidial and amphidial neurons of transgenic first-stage larvae of S. stercoralis (Stoltzfus et al 2014). A similar EGFP reporter construct incorporating the Ce-age-1 promoter had a comparable pattern of expression in transgenic first-stage larvae of C. elegans , including a striking concordance in the amphidial neurons.…”

“…To date, reporter constructs used in all but one study of anatomical gene expression patterns in Strongyloides and Parastrongyloides have constituted simple fusions of coding sequences of LacZ (Grant et al 2006 a ), gfp (Grant et al 2006 a ; Li et al 2006, 2011; Junio et al 2008; Massey et al 2013; Yuan et al 2014 a , b ) or egfp (Stoltzfus et al 2012 a , 2014) to putative promoters for the genes of interest. In contrast to these, a study employing transgenesis to investigate the function of Ss-daf-16 (previously named fktf-1 ) employed a construct designed to express an N-terminal fusion of GFP with wild-type Ss -DAF-16b (Castelletto et al 2009).…”

Development and application of three-dimensional skin equivalents for the investigation of percutaneous worm invasion

“…The infective larvae are attracted by physical factors e.g. by a thermal gradient (thermotaxis) and by host-emitted chemicals including the histidine derivative urocanic acid, cGMP, arginine, ceramide, and longchain fatty acids (Chaisson and Hallem, 2012;Haeberlein and Haas, 2008;Hawdon and Datu, 2003;Safer et al, 2007;Stoltzfus et al, 2014). Adhesion on the skin surface is promoted by wrinkles, skin appendages or sucker and adhesive molecules e.g.…”

“…It is comprehensible that the capability to invade into the non-host skin preparations is reduced, since the surface structure and also the chemical attractants may be different. Preliminary experiments with potential activators of larval infectivity like urocanic acid, dafachronic acid and serum filtrate <10 kDa indicated first increase in the invasion capability of the exposed infective larvae (Hawdon and Schad, 1990;Koga and Tada, 2000;Safer et al, 2007;Stoltzfus et al, 2014).…”

Perusal of parasitic nematode ‘omics in the post-genomic era

“…S. stercoralis transcripts encoding homologs of cGMP and TGFβ signaling proteins that regulate C. elegans dauer development are coordinately increased in infective larvae, and several insulin-like peptide-encoding transcripts are specifically regulated throughout the life cycle [72]. A putative pathway for in vivo production of dafachronic acid that regulates S. stercoralis development has been identified [72, 73], and dafachronic acid transcriptionally recapitulates in vivo activation of infective larvae [74]. The possibility of manipulating this crucial developmental regulatory pathway with administered small molecules underscores its potential as a chemotherapeutic target.…”