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The infection of Caenorhabditis elegans by the microsporidian parasite Nematocida parisii enables the worms to produce offspring that are highly resistant to the same pathogen. This is an example of inherited immunity, a poorly understood epigenetic phenomenon. The present protocol describes the study of inherited immunity in a genetically tractable worm model.
Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in their progeny and promote survival. While inherited immunity has been reported in many invertebrates, the mechanisms underlying this epigenetic phenomenon are largely unknown. The infection of Caenorhabditis elegans by the natural microsporidian pathogen Nematocida parisii results in the worms producing offspring that are robustly resistant to microsporidia. The present protocol describes the study of intergenerational immunity in the simple and genetically tractable N. parisii -C. elegans infection model. The current article describes methods for infecting C. elegans and generating immune-primed offspring. Methods are also given for assaying resistance to microsporidia infection by staining for microsporidia and visualizing infection by microscopy. In particular, inherited immunity prevents host cell invasion by microsporidia, and fluorescence in situ hybridization (FISH) can be used to quantify invasion events. The relative amount of microsporidia spores produced in the immune-primed offspring can be quantified by staining the spores with a chitin-binding dye. To date, these methods have shed light on the kinetics and pathogen specificity of inherited immunity, as well as the molecular mechanisms underlying it. These techniques, alongside the extensive tools available for C. elegans research, will enable important discoveries in the field of inherited immunity.
Inherited immunity is an epigenetic phenomenon whereby parental exposure to pathogens can enable the production of infection-resistant offspring. This type of immune memory has been shown in many invertebrates that lack adaptive immune systems and can protect against viral, bacterial, and fungal disease1. While inherited immunity has important implications for understanding both health and evolution, the molecular mechanisms underlying this protection are largely unknown. This is partly because many of the animals in which inherited immunity has been described are not established model organisms for research. In contrast, studies in the transparent nematode Caenorhabditis elegans benefit from an extensive genetic and biochemical toolkit2,3, a highly annotated genome4,5, and a short generation time. Indeed, research in C. elegans has enabled fundamental advances in the fields of epigenetics and innate immunity6,7, and it is now an established model for studying immune memory8,9.
Microsporidia are fungal pathogens that infect almost all animals and cause lethal infections in immunocompromised humans10. Infection begins when a microsporidia spore injects or "fires" its cellular contents (sporoplasm) into a host cell using a structure called a polar tube. Intracellular replication of the parasite results in the formation of meronts, which ultimately differentiate into mature spores that can exit the cell11,12. While these parasites are detrimental to both human health and food security, there is much still to learn about their infection biology12. Nematocida parisii is a natural microsporidian parasite that replicates exclusively in the intestinal cells of worms, resulting in reduced fecundity and, ultimately, death. The N. parisii -C. elegans infection model has been used to show: (1) the role of autophagy in pathogen clearance13, (2) how microsporidia can exit infected cells non-lytically14, (3) how pathogens can spread from cell-to-cell by forming syncytia15, (4) the proteins N. parisii use to interface with its host16, and (5) the regulation of the transcriptional intracellular pathogen response (IPR)17,18.
Protocols for the infection of C. elegans are described in the current work and can be used to reveal the unique microsporidia biology and dissect the host's response to infection. The microscopy of fixed worms stained with the chitin-binding dye Direct Yellow 96 (DY96) shows the infection spread of chitin-containing microsporidia spores throughout the intestine. DY96 staining also enables the visualization of chitin-containing worm embryos for the simultaneous assessment of worm gravidity (ability to produce embryos) as a readout of host fitness.
Recent work has revealed that C. elegans infected with N. parisii produce offspring that are robustly resistant to the same infection19. This inherited immunity lasts a single generation and is dose-dependent, as offspring from more heavily infected parents are more resistant to microsporidia. Interestingly, N. parisii-primed offspring are also more resistant to the bacterial gut pathogen Pseudomonas aeruginosa, though they are not protected against the natural pathogen Orsay virus19. The present work also shows that immune-primed offspring limit host cell invasion by microsporidia. The method also describes the collection of immune-primed offspring and how FISH can be used to detect N. parisii RNA in intestinal cells to assay host cell invasion and spore firing20.
Together, these protocols provide a solid foundation for studying microsporidia and inherited immunity in C. elegans. It is hoped that future work in this model system will enable important discoveries in the nascent field of inherited immunity. These techniques are also likely to be starting points for investigating microsporidia-induced inherited immunity in other host organisms.
The present study uses wild-type C. elegans Bristol strain N2 grown at 21 °C.
1. Preparation of media
2. Maintenance of C. elegans
3. Synchronization of C. elegans populations using sodium hypochlorite (bleaching)
NOTE: This step is very time-sensitive, so ensure the centrifuge is available before beginning. Alternative, less rapid bleaching protocols are available in the literature and may be used if preferred. To prevent 6% sodium hypochlorite from losing activity over time, store the reagent in the dark at 4 °C and keep it for up to 1 year.
4. Preparation of N. parisii spores
5. Infection of C. elegans with N. parisii to yield immune-primed offspring
6. Testing inherited immunity to N. parisii in C. elegans
7. DY96 staining of C. elegans to visualize embryos and microsporidia spores
NOTE: DY96 is a green fluorescent chitin-binding dye that stains worm embryos and microsporidia spore walls19,15,25. This allows simultaneous monitoring of the fitness and infection status of the worms.
8. Imaging and analysis of DY96-stained worms to assess worm fitness and infection status
9. FISH assay to assess invasion of C. elegans by microsporidia and spore firing
NOTE: The MicroB FISH probe recognizes a conserved region of microsporidian 18s rRNA and can be used to label intracellular sporoplasms (i.e., invaded host cells) and the genetic material within spores.
In the present study, parental populations of C. elegans (P0) were infected at the L1 stage with a low dose of N. parisii spores. These infection conditions are typically used to obtain high numbers of microsporidia-resistant F1 progeny through bleaching of the parents. Infected parental populations and uninfected controls were fixed at 72 hpi and stained with DY96 to visualize the worm embryos and microsporidia spores (Figure 1A). Infected animals are small, contain many m...
The present protocol describes the study of microsporidia and inherited immunity in a simple and genetically tractable N. parisii -C. elegans infection model.
Spore preparation is an intensive protocol that typically yields enough spores for 6 months of experiments, depending on productivity24. Importantly, infectivity must be determined for each new spore "lot" before using it for the experiments. Due to the variability in infectivity between ...
The authors have nothing to disclose.
We are grateful to Winnie Zhao and Yin Chen Wan for providing helpful comments on the manuscript. This work was supported by the Natural Sciences and Engineering Research Council of Canada (Grant #522691522691).
Name | Company | Catalog Number | Comments |
2.0 mm zirconia beads | Biospec Products Inc. | 11079124ZX | |
10 mL syringe | Fisher Scientific | 1482613 | |
5 μm filter | Millipore Sigma | SLSV025LS | |
Axio Imager 2 | Zeiss | - | Fluorescent microscope for imaging of DY96- and FISH- stained worms on microscope slides |
Axio Zoom V.16 Fluorescence Stereo Zoom Microscope | Zeiss | - | For live imaging of fluorescent transgenic animals to visualize the IPR |
Baked EdgeGARD Horizontal Flow Clean Bench | Baker | - | |
Bead disruptor, Genie SI-D238 Analog Disruptor Genie Cell Disruptor, 120 V | Global Industrial | T9FB893150 | |
Cell-VU slide, Millennium Sciences Disposable Sperm Count Cytometers | Fisher Scientific | DRM600 | |
Direct Yellow 96 | Sigma-Aldrich | S472409-1G | |
EverBrite Mounting Medium with DAPI | Biotium | 23001 | |
EverBrite Mounting Medium without DAPI | Biotium | 23002 | |
Fiji/ImageJ software | ImageJ | https://imagej.net/software/fiji/downloads | |
Mechanical rotor | Thermo Sceintific | 415110 / 1834090806873 | Used to spin tubes of bleached embryos for overnight hatching |
MicroB FISH probe | Biosearch Technologies Inc. | - | Synthesized with a Quasar 570 (Cy3) 5' modification and HPLC purified, CTCTCGGCACTCCTTCCTG |
N2 | Wild-type, Bristol strain | Default strain | Caenorhabditis Genetics Center (CGC) |
Sodium dodecyl sulfate (SDS) | Sigma-Aldrich | L3771-100G | |
Sodium hydroxide solution (5 N) | Fisher Chemical | FLSS256500 | |
Sodium hypochlorite solution (6%) | Fisher Chemical | SS290-1 | |
Stemi 508 Stereo Microscope | Zeiss | - | For daily maintenance of worms and counting of L1 worms for assay set ups |
Tween-20 | Sigma-Aldrich | P1379-100ML | |
Vectashield + A16 | Biolynx | VECTH1500 |
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