Translational Fusion of a G‐protein Coupled‐Receptor from the Hookworm Ancylostoma ceylanicum Expressed in Caenorhabditis elegans

Abstract

Hookworms are infectious parasites that affect more than 500 million people worldwide, causing extensive morbidity and economic burden in developing countries. Mass drug administration is a current control method, but resistance is of concern. Many hookworm species, including those in the genus Ancylostoma, are specialists and require specific host signals to continue development upon infection. Little is known about the signaling mechanisms, although evolutionary conservation between parasitic nematodes and the model organism C. elegans suggests that G‐protein coupled receptors (GPCRs) expressed in amphid neurons are chemosensory detection candidates. The amphid neurons of C. elegans are exposed to the environment, even during the dauer stage, an alternate phase of larval development that is similar to the parasitic infective juvenile stage. Previous experiments involving A. ceylanicum transcriptional GFP fusions in C. elegans identified putative GPCR genes expressed in neurons during dauer stage. To confirm that hookworm proteins are neuronally localized under dauer conditions, a translational GFP fusion of a putative hookworm GPCR was generated. The entire coding sequence, plus introns, was cloned using homologous recombination‐based, yeast assembly methods to create a C‐terminal GFP fusion. The putative A. ceylanicum promoter consisted of the upstream intergenic region, which is approximately 5000 base pairs, and the transcriptional terminator from C. elegans unc‐54 was used. To generate the expression plasmid, amplicons containing homology to the adjacent segment in the assembly were amplified using polymerase chain reaction (PCR) and transformed into yeast. The plasmid was verified by junction‐confirming PCR analysis, restriction enzyme mapping, and sequencing. After microinjection, GFP expression in amphid neurons of stably transformed C. elegans during the dauer stage would support the hypothesis that this putative GPCR plays a role in reception of host signaling molecules and may provide a possible therapeutic target.Support or Funding InformationThis work was supported by the Salisbury University Faculty Mini‐Grant Program.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.