Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

Summary

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.

Abstract

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.

Introduction

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 transpa....

Protocol

The present study uses wild-type C. elegans Bristol strain N2 grown at 21 °C.

1. Preparation of media

1. Prepare M9 media as per previous report^21,22.
2. Prepare nematode growth medium (NGM) as per previous report^21,22. Pour 12 mL of NGM per 6 cm plate or 30 mL per 10 cm plate.
3. Prepare Escherichia coli strain OP50-1 as per a previous report²³.
4. Prepare OP50-1-seeded NGM plates following the steps below.
   1. Resuspend 10....

Results

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.......

Discussion

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 .......

Materials

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