Friday, September 20, 2024
9/20/2024
Project Summary/Abstract The nematode Caenorhabditis elegans relies on small-molecule signals to control its development, metabolism, physiology, and behavior, and these signals play conserved roles in many parasitic nematode species. This MIRA application outlines our ongoing efforts to understand the structures, biosynthesis, and mechanisms of several important classes of small-molecule signals, including (1) the ascarosides – a broad family of pheromones secreted by C. elegans that the worm uses to induce the stress-resistant dauer larval stage and to coordinate various behaviors, (2) the N-acyl glutamine nacq#1 – a pheromone that males preferentially secrete to counter the effects of dauer-inducing ascarosides on hermaphrodites and promote reproductive development, and (3) the nemamides – a family of hybrid polyketide-nonribosomal peptides that serve as hormones in the worm and promote starvation survival through a poorly understood mechanism. We target the biosynthetic pathways to these signaling molecules in vivo by generating precise mutations in the worm genome using CRISPR-Cas9 and analyzing the effects of these mutations on the primary and secondary metabolome of the worm using comparative metabolomics. This approach enables us to map the biosynthetic pathways to these natural products, to identify additional signaling molecules produced by these pathways, and to determine how these pathways intersect with other metabolic pathways in the worm. We rigorously confirm the role of specific enzymes in the pathways by reconstituting the pathways using in vitro enzymatic assays, organic synthesis of biosynthetic intermediates, and structural studies. With the support of this award, we will investigate the biosynthesis and mechanism of the nemamides, as well as the biological roles of the two essential and enigmatic neurons where the nemamides are produced, the canal-associated neurons (CANs). We will use nemamide biosynthesis, which requires genes that are distributed throughout the worm genome, to understand how the biosynthesis of complex secondary metabolites is controlled in the context of an animal system. Furthermore, we will investigate how this biosynthetic pathway intersects with the biosynthetic pathways of other secondary metabolites, including the ascarosides and nacq#1. A central focus will be how the worm regulates the production and trafficking of these different small-molecule signals in response to different factors and environmental conditions in order to coordinate its development, metabolism, and physiology. This work will provide insights into how C. elegans and other nematode species use small-molecule signals to control important conserved downstream signaling pathways, such as the insulin pathway. Furthermore, given the conservation of these small-molecule signals in parasitic nematode species, this work will provide new chemical tools and strategies to interfere with the life cycles of those nematodes.
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