Parasitic nematodes infect roughly 20% of the world's population and exact an enormous toll in human illness. Most of these parasites infect their hosts as developmentally arrested infective third- stage larvae (iL3) that resume development once they enter the host. Drawing upon findings in Caenorhabditis elegans, we have shown that the parasitic nematode Strongyloides stercoralis has significantly modified insulin-like (ILS) and steroid-nuclear hormone receptor (NHR) signaling to regulate iL3 development during the infective process. In this renewal application, we propose to expand our studies of regulation of iL3 development and morphogenesis by ILS and NHR signaling in S. stercoralis, deploying our new method for CRISPR/Cas9 mutagenesis to unambiguously assess gene function and RNAseq to determine transcriptomic changes resulting from knockout of key signaling elements.
Specific Aim 1 asks whether ILS regulates formation and development of L3i, and what genes in S. stercoralis are regulated by ILS downstream. To this end, we will knock out Ss-daf- 16, which encodes an ILS-regulated transcription factor and Ss-daf-2, which encodes the insulin-like receptor kinase in S. stercoralis. We will subject knockout worms to RNAseq to ascertain global transcriptional changes and specific ones involving cytochrome P450 genes that could act in biosynthesis of Ss-DAF-12 NHR ligands.
Aim 2 will assign functions to insulin-like peptides (ILPs) in S. stercoralis as agonists or antagonists of ILS by expressing them from transgenes designed to alter temporal patterns of ilp transcription in the worms.
Aim 2 will also determine whether Ss-DAF-12 NHR signaling feeds back to positively regulate ILS by enhancing expression of agonistic ILPs in S. stercoralis.
Aim 3 will employ vivo and in vitro studies to test the hypothesis that Ss-DAF-12 NHR signaling regulates lifespan in S. stercoralis, lengthening it in free-living adults and shortening it in parasitic females. All three aims will contribute to future development of much needed new anthelmintics targeting ILS and NHR signaling in parasitic nematodes. Elucidating functional links between these pathways will enable new combination therapies that target both pathways, acting additively or synergistically to block development and accelerate death and expulsion of adult worms.

Public Health Relevance

Parasitic roundworms adversely affect the health of 1.5 billion people worldwide. Although a small number of effective drugs are now available to treat these infections, there are early signs that some parasitic roundworms are becoming resistant to them. This application proposes to study aspects of parasite metabolism that could form the basis for new drugs to prevent or treat roundworm infection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
2R01AI050668-14
Application #
9670410
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2002-04-01
Project End
2023-10-31
Budget Start
2018-11-09
Budget End
2019-10-31
Support Year
14
Fiscal Year
2019
Total Cost
Indirect Cost
Name
University of Pennsylvania
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
042250712
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
Lok, J B; Shao, H; Massey, H C et al. (2017) Transgenesis in Strongyloides and related parasitic nematodes: historical perspectives, current functional genomic applications and progress towards gene disruption and editing. Parasitology 144:327-342
Shao, Hongguang; Li, Xinshe; Lok, James B (2017) Heritable genetic transformation of Strongyloides stercoralis by microinjection of plasmid DNA constructs into the male germline. Int J Parasitol 47:511-515
Lok, James B (2016) Signaling in Parasitic Nematodes: Physicochemical Communication Between Host and Parasite and Endogenous Molecular Transduction Pathways Governing Worm Development and Survival. Curr Clin Microbiol Rep 3:186-197
Hunt, Vicky L; Tsai, Isheng J; Coghlan, Avril et al. (2016) The genomic basis of parasitism in the Strongyloides clade of nematodes. Nat Genet 48:299-307
Mohandas, Namitha; Hu, Min; Stroehlein, Andreas J et al. (2016) Reconstruction of the insulin-like signalling pathway of Haemonchus contortus. Parasit Vectors 9:64
Albarqi, Mennatallah M Y; Stoltzfus, Jonathan D; Pilgrim, Adeiye A et al. (2016) Regulation of Life Cycle Checkpoints and Developmental Activation of Infective Larvae in Strongyloides stercoralis by Dafachronic Acid. PLoS Pathog 12:e1005358
Wang, Zhu; Stoltzfus, Jonathan; You, Young-Jai et al. (2015) The nuclear receptor DAF-12 regulates nutrient metabolism and reproductive growth in nematodes. PLoS Genet 11:e1005027
Li, Fa-Cai; Gasser, Robin B; Lok, James B et al. (2014) Exploring the role of two interacting phosphoinositide 3-kinases of Haemonchus contortus. Parasit Vectors 7:498
Yuan, Wang; Liu, Yingying; Lok, James B et al. (2014) Exploring features and function of Ss-riok-3, an enigmatic kinase gene from Strongyloides stercoralis. Parasit Vectors 7:561
Li, Facai; Lok, James B; Gasser, Robin B et al. (2014) Hc-daf-2 encodes an insulin-like receptor kinase in the barber's pole worm, Haemonchus contortus, and restores partial dauer regulation. Int J Parasitol 44:485-96

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