Research Article: cGMP and NHR Signaling Co-regulate Expression of Insulin-Like Peptides and Developmental Activation of Infective Larvae in Strongyloides stercoralis

Date Published: July 10, 2014

Publisher: Public Library of Science

Author(s): Jonathan D. Stoltzfus, Stephen M. Bart, James B. Lok, Stephen John Davies.


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.

Partial Text

Parasitic nematodes infect approximately one in four persons globally, with the vast burden of disease concentrated in tropical and developing regions [1]. The parasitic nematode Strongyloides stercoralis infects an estimated 30–100 million people worldwide [2]; in corticosteroid-treated or human T-cell lymphotropic virus 1 (HTLV-1) infected persons, infection with S. stercoralis can result in hyperinfection and potentially fatal disseminated strongyloidiasis [3]. Like many soil-transmitted helminths, the infectious form of S. stercoralis is a developmentally arrested third-stage larva (L3i), which is non-feeding, long-lived, and stress-resistant [4]. S. stercoralis L3i exhibit thermotaxis and chemotaxis in response to a range of host-like cues [5], including host body temperature [6], carbon dioxide [7], sodium chloride [8], and urocanic acid [9]. Upon entering a suitable host, L3i quickly activate and resume feeding and development [4]. However, the molecular mechanisms by which S. stercoralis L3i sense and transduce host cues and subsequently initiate resumption of development are poorly understood.

In this study, we sought to both describe the upstream components that regulate the second messenger cGMP in S. stercoralis, including chemosensory 7TM GPCRs and heterotrimeric G proteins, and determine whether cGMP pathway signaling regulates S. stercoralis L3i activation. Additionally, we sought to elucidate the epistatic relationships between cGMP signaling, IIS, and DAF-12 NHR signaling pathways during L3i activation. We hypothesized that the cGMP-regulated chemosensory pathway may be one of the first to transduce host cues when S. stercoralis L3i encounter a permissive host. This hypothesis was based on our previous observation that the transcripts of multiple cGMP pathway components are increased in S. stercoralis L3i, suggesting that this pathway may be “poised” to transduce host cues [14], and studies demonstrating that exogenous application of 8-bromo-cGMP activates L3i of hookworm species [51]–[53]. We therefore sought to describe the components of a chemosensory 7TM GPCR signaling pathway in S. stercoralis and determine whether cGMP signaling regulates L3i activation as well as IIS and other signaling pathways.




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