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Updated: Oct 13, 2026
Using Caenorhabditis elegans to Screen for Tissue-Specific Chaperone Interactions
Published on: June 7, 2020
Characterization of microsporidia-induced developmental arrest and a transmembrane leucine-rich repeat protein in
Robert J Luallen1, Malina A Bakowski1, Emily R Troemel1
1Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America.
Abstract:
Microsporidia comprise a highly diverged phylum of intracellular, eukaryotic pathogens, with some species able to cause life-threatening illnesses in immunocompromised patients. To better understand microsporidian infection in animals, we study infection of the genetic model organism Caenorhabditis elegans and a species of microsporidia, Nematocida parisii, which infects Caenorhabditis nematodes in the wild. We conducted a targeted RNAi screen for host C. elegans genes important for infection and growth of N. parisii, using nematode larval arrest as an assay for infection. Here, we present the results of this RNAi screen, and our analyses on one of the RNAi hits from the screen that was ultimately not corroborated by loss of function mutants. This hit was an RNAi clone against F56A8.3, a conserved gene that encodes a transmembrane protein containing leucine-rich repeats (LRRs), a domain found in numerous pathogen receptors from other systems. This RNAi clone caused C. elegans to be resistant to infection by N. parisii, leading to reduced larval arrest and lower pathogen load. Characterization of the endogenous F56A8.3 protein revealed that it is expressed in the intestine, localized to the membrane around lysosome-related organelles (LROs), and exists in two different protein isoforms in C. elegans. We used the CRISPR-Cas9 system to edit the F56A8.3 locus and created both a frameshift mutant resulting in a truncated protein and a complete knockout mutant. Neither of these mutants was able to recapitulate the infection phenotypes of the RNAi clone, indicating that the RNAi-mediated phenotypes are due to an off-target effect of the RNAi clone. Nevertheless, this study describes microsporidia-induced developmental arrest in C. elegans, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved F56A8.3 gene and its protein product.
Insights
Researchers screened for host genes affecting microsporidia (Nematocida parisii) infection in Caenorhabditis elegans. An RNAi screen identified a gene, F56A8.3, but subsequent genetic analysis revealed this effect was off-target, not a true host resistance mechanism.
Area of Science:
- Microbiology
- Genetics
- Parasitology
Background:
- Microsporidia are eukaryotic intracellular pathogens, some causing severe disease in immunocompromised individuals.
- Understanding host-pathogen interactions is crucial for combating microsporidian infections.
- Caenorhabditis elegans serves as a model organism to study host responses to pathogens like Nematocida parisii.
Purpose of the Study:
- To identify host Caenorhabditis elegans genes that influence Nematocida parisii infection and growth.
- To investigate the role of the conserved gene F56A8.3 in microsporidian pathogenesis.
- To characterize the protein product of F56A8.3 and its cellular localization.
Main Methods:
- Conducted a targeted RNA interference (RNAi) screen in C. elegans using larval arrest as an infection assay.
- Utilized CRISPR-Cas9 gene editing to create frameshift and knockout mutants for F56A8.3.
- Performed protein expression and localization studies for the F56A8.3 gene product.
Main Results:
- An RNAi screen identified F56A8.3 as a potential host factor conferring resistance to N. parisii infection.
- CRISPR-Cas9 generated F56A8.3 mutants did not replicate the resistance phenotype observed with RNAi.
- These findings indicate the RNAi-mediated resistance was likely due to off-target effects.
Conclusions:
- The study highlights the importance of genetic validation following RNAi screens.
- It characterizes the expression and localization of the F56A8.3 protein, a transmembrane protein with LRR domains.
- This work contributes to understanding host-pathogen interactions in microsporidian infections using C. elegans.
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