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

Collection of wild nematode-infecting microsporidia strains

<p>Collection of wild nematode-infecting microsporidia strains</p

Ultrastructural observations of <i>Enteropsectra longa</i>.

<p>Transmission electron micrographs of <i>E</i>. <i>longa</i> strain JUm408 after high-pressure freezing/freeze substitution. <b>A.</b> <i>E</i>. <i>longa</i> meront. A nucleus is visible in the cytoplasm full of ribosomes. <b>B.</b> Lower magnification with a multinucleated meront. Two meronts are indicated, one with a single nucleus in the plane of section (left) and one with several nuclei (right). Two host nuclei are visible on the right, with a dark nucleolus. Intestinal cells contain two nuclei. <b>C.</b> Early sporonts with an electron-dense coat indicated by arrowhead. <b>D.</b> Sporont undergoing a cell division (big arrow); small arrow indicates junction of host intestinal cells; the arrowhead indicates a host Golgi apparatus. <b>E.</b> Mitotic spindle (arrows designate microtubules) in a sporont; the spindle plaque is indicated by an arrowhead. <b>F.</b> Nascent polar tube (arrow) in a sporoblast. <b>G.</b> Wrinkled sporoblasts (*). Arrows indicate the host rough endoplastic reticulum folding around the microsporidia. <b>H.</b> Late stage sporoblast in the center, mature spore on the top left; arrows indicate polar tubes. <b>I.</b> Mature spore with the anterior part of the polar tube, including the anchoring disk. <b>J.</b> Cross-section of mature spores. The exospore and endospore layers are shown in the inset. Arrowheads indicate polar tubes. <b>K.</b> Two mature spores in the intestinal lumen that do not show an additional membrane around them. Low magnification inset shows the positions of the two spores in the lumen and arrowhead indicates host microvilli. <b>L.</b> Low magnification view of cross-section of host, with the intestinal lumen in the center. <i>E</i>. <i>longa</i> spores (arrowheads) concentrate around the apical membrane of intestinal cell, while meronts and early sporonts are on the basal side. Scale bar is 500 nm, unless indicated otherwise. A, anchoring disk; Chr: chromatin; Ex, exospore; En, endospore; Lu, lumen; M, meront; Mi: host mitochondrion; Mv, host microvilli; Nu, nucleus; HNu host nucleus; Pt, polar tube; RER, rough endoplastic reticulum; St: sporont.</p

Cell exit modes of <i>Nematocida ausubeli</i> and <i>Enteropsectra longa</i>.

<p><b>A.</b><i>Nematocida ausubeli</i>. The top panel is an electron microscopy image of a <i>Nematocida ausubeli</i> spore (large arrow) exiting from the intestinal cell into the lumen. Arrows indicate the apical membrane of the host intestinal cell. The hypothetical diagram below illustrates the exit of <i>N</i>. <i>ausubeli</i> spores from intestinal cells by exocytosis. As in <i>N</i>. <i>parisii</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006093#ppat.1006093.ref027" target="_blank">27</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006093#ppat.1006093.ref036" target="_blank">36</a>], spores appear surrounded by a membrane that fuses with the apical membrane of the host intestinal cell, resulting in the release of spores. We also observed apparently mature spores without an additional membrane and do not know whether they will later acquire a membrane or exit in another manner. Earlier stages were omitted here for simplicity. <b>B.</b> <i>Enteropsectra longa</i>. The top panel is an electron micrograph of <i>Enteropsectra longa</i> spores exiting from the intestinal cell into the lumen, with the host intestinal cell membrane folding out around the <i>E</i>. <i>longa</i> spores (arrow). The diagram below illustrates the exit of <i>E</i>. <i>longa</i> spore from the intestinal cell. The host intestinal cell membrane folds out around the spore until the whole spore exits the cell, after which the host membrane around the spore seems to disappear. Meronts and sporoblasts are not represented in either panel. Scale bars: 500 nm.</p

Responses of <i>C</i>. <i>elegans</i> strains with transcriptional reporters <i>C17H1</i>.<i>6p</i>::<i>GFP</i> and <i>F26F2</i>.<i>1p</i>::<i>GFP</i> to exposure by different microsporidia.

<p>Strains ERT54 carrying <i>C17H1</i>.<i>6p</i>::<i>GFP</i> (<b>A</b>) and ERT72 carrying <i>F26F2</i>.<i>1p</i>::<i>GFP</i> (<b>B</b>) were analyzed for GFP induction at different time points after infection with different microsporidia and the proportion of animals with GFP induction is shown. GFP was reproducibly induced in ERT54 and ERT72 upon infection with <i>N</i>. <i>parisii</i>, <i>N</i>. <i>major</i> and <i>N</i>. <i>homosporus</i>, while GFP signal was rarely observed in ERT54 and ERT72 inoculated with <i>N</i>. <i>ausubeli</i> or <i>E</i>. <i>longa</i> or the negative control. <i>N</i>. <i>ausubeli</i> did infect the <i>C</i>. <i>elegans</i> reporter strains, as monitored by DIC as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006093#ppat.1006093.t005" target="_blank">Table 5</a>. <b>C</b>. Transcript levels for three genes were measured after 4 hours of infection of N2 <i>C</i>. <i>elegans</i> by <i>N</i>. <i>parisii</i> (ERTm1) and <i>N</i>. <i>ausubeli</i> (ERTm2). The fold increase in transcript level was measured relative to uninfected N2 levels. Infection dose was normalized between <i>Nematocida</i> by successful invasion events counted as intracellular sporoplasms at 4 hpi. To independently compare the microsporidian doses in parallel to the transcript quantification, we also measure the levels of <i>Nematocida</i> SSU rRNA after 4 hours of infection of <i>C</i>. <i>elegans</i> in the same experiment: we found that the rRNA level measured after infection with ERTm2 was 1.25-fold higher than that with ERTm1.</p

Nematode-infecting microsporidia specificity.

<p>Nematode-infecting microsporidia specificity.</p

Molecular distances of microsporidia SSU rDNA.

<p>Molecular distances of microsporidia SSU rDNA.</p