RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available

Characterization of Microsporidia-Induced Developmental Arrest and a Transmembrane Leucine-Rich Repeat Protein in <i>Caenorhabditis elegans</i>

<div><p>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 <i>Caenorhabditis elegans</i> and a species of microsporidia, <i>Nematocida parisii</i>, which infects <i>Caenorhabditis</i> nematodes in the wild. We conducted a targeted RNAi screen for host <i>C</i>. <i>elegans</i> genes important for infection and growth of <i>N</i>. <i>parisii</i>, 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 <i>F56A8</i>.<i>3</i>, 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 <i>C</i>. <i>elegans</i> to be resistant to infection by <i>N</i>. <i>parisii</i>, 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 <i>C</i>. <i>elegans</i>. We used the CRISPR-Cas9 system to edit the <i>F56A8</i>.<i>3</i> 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 <i>C</i>. <i>elegans</i>, presents results from an RNAi screen for host genes important for microsporidian infection, and characterizes aspects of the conserved <i>F56A8</i>.<i>3</i> gene and its protein product.</p></div

Mutation of <i>F56A8</i>.<i>3a</i> does not recapitulate the larval arrest phenotype of <i>F56A8</i>.<i>3</i> RNAi.

<p>(A) <i>Top</i>: Schematic representation of the <i>F56A8</i>.<i>3a</i> and <i>F56A8</i>.<i>3b</i> pre-mRNA transcripts, with exons represented as black and gray blocks, indicated coding and non-coding sequences, respectively, and solid lines representing introns. The sequence covered by <i>F56A8</i>.<i>3</i> RNAi clone is indicated at the top as a dotted line. Image adapted from WormBase and based on EST data (WBGene00010139). <i>Bottom</i>: Schematic representation of F56A8.3a and F56A8.3b protein, with dotted lines showing the relative locations on the coding exons from which the main protein domains are derived (LRR is the leucine-rich repeat domain, CC is the coiled coil domain, TM is the transmembrane domain, and CTD is the C-terminal domain). (B) Representation of CRISPR-Cas9 genome editing of the 5'-most exon of the <i>F56A8</i>.<i>3</i> gene, with the <i>F56A8</i>.<i>3</i> start codon in bold, the sgRNA targeting sequence highlighted, and the protospacer adjacent motif (PAM) underlined (left). WT is the wild-type sequence found in N2, and -5 is a 5 bp deletion found in the mutant ERT327 (<i>jy4</i>), with representative 80 bp and 75 bp PCR products from the F1 screen shown (right). (C) Larval arrest of <i>eri-1</i> and <i>F56A8</i>.<i>3</i> frameshift mutant ERT360 <i>F56A8</i>.<i>3(jy4)</i> (in an <i>eri-1</i> background) on control or <i>F56A8</i>.<i>3</i> RNAi after <i>N</i>. <i>parisii</i> infection, measured as the percent animals reaching the L4 at 2 dpi. Data are represented as mean values with SEM from two independent experiments (*p = 0.013 (left), *p = 0.022 (right), unpaired two-tailed t-test). (D) F56A8.3 protein in N2 and <i>F56A8</i>.<i>3</i> frameshift mutation ERT327 <i>F56A8</i>.<i>3(jy4)</i> on either control (L4440) or <i>F56A8</i>.<i>3</i> RNAi. The top picture represents a single blot probed with anti-F56A8.3 antibodies, while the bottom represents a single blot probed with anti-actin. Indicated molecular weight markers are in kilodaltons (kD).</p

<i>F56A8</i>.<i>3</i> RNAi clone reduces the level of <i>N</i>. <i>parisii</i> infection at several stages of infection.

<p>(A) Pathogen load at 8 hpi on control or <i>F56A8</i>.<i>3</i> RNAi measured as the number of FISH-stained sporoplasms seen in intact <i>C</i>. <i>elegans</i> intestines. Data are represented as mean values with SEM from three independent, blinded experiments (**p = 0.002, paired two-tailed t-test). (B) Pathogen load at 30 hpi on control or <i>F56A8</i>.<i>3</i> RNAi measured as the fold change in <i>N</i>. <i>parisii</i> rDNA transcript by qRT-PCR relative to L4440 infected at the lowest dose. Data are represented as mean values with SEM from three independent experiments (*p = 0.033, two-way analysis of variation, testing RNAi treatment effecting pathogen load at all doses). (C) Pathogen load at 40 hpi with <i>C</i>. <i>elegans</i> infected at the L2 stage on control or <i>F56A8</i>.<i>3</i> RNAi measured as the average number of spores produced per animal. Data are represented as mean values with SEM from three independent experiments (***p = 0.0005, paired two-tailed t-test).</p

<i>F56A8</i>.<i>3</i> RNAi clone acts in the intestine, and the F56A8.3 protein localizes to lysosome-related organelles in the intestine.

<p>(A) Representative image of transgenic <i>C</i>. <i>elegans</i> expressing intestinal mCherry under control of the putative <i>F56A8</i>.<i>3</i> promoter. Scale bar = 100 μm. (B) Larval arrest of tissue-specific RNAi strains MGH167 (left, intestinal-specific) and SPC272 (right, muscle-specific) after <i>N</i>. <i>parisii</i> infection, measured as the percent animals reaching the adult stage at 3 dpi. Data are represented as mean values with SEM from two independent experiments (**p = 0.002; n.s. p = 0.26, unpaired two-tailed t-test). (C) Representative image of endogenous F56A8.3 localization in dissected intestines from wild-type N2 <i>C</i>. <i>elegans</i> (left) or from N2 treated with <i>F56A8</i>.<i>3</i> RNAi (right). F56A8.3 was detected with anti-F56A8.3 followed by goat anti-rabbit IgG conjugated to Cy3. Scale bar = 10 μm. (D) Representative image of endogenous F56A8.3 colocalization relative to CDF-2::GFP in the WU1236 transgenic strain. F56A8.3 was detected as in C; CDF-2 GFP was detected with anti-GFP followed by anti-mouse IgG conjugated to FITC. Scale bar = 10 μm.</p