title: Characterization of microsporidia-induced developmental arrest and a transmembrane leucine-rich repeat protein in Caenorhabditis elegans.
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.
authors: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America.
authors: Robert J
authors: RJ
authors: Luallen
authors: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America.
authors: Malina A
authors: MA
authors: Bakowski
authors: Division of Biological Sciences, Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California, United States of America.
authors: Emily R
authors: ER
authors: Troemel
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics / Q000254:growth & development* / Q000469:parasitology; D000917:Antibody Formation; D017173:Caenorhabditis elegans / Q000235:genetics / Q000254:growth & development* / Q000469:parasitology; D002462:Cell Membrane / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D015151:Immunoblotting; D007814:Larva / Q000235:genetics / Q000254:growth & development* / Q000469:parasitology; D000090263:Leucine-Rich Repeat Proteins; D016814:Microsporidia / Q000472:pathogenicity*; D016881:Microsporidiosis / Q000235:genetics / Q000469:parasitology; D011506:Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000276:immunology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D011817:Rabbits
chemical_list: D000090263:Leucine-Rich Repeat Proteins; D011506:Proteins; D034741:RNA, Small Interfering