This study investigates the interaction between C. elegans and the pathogenic bacterium Aeromonas, focusing on the effects of bacterial virulence factors on muscle integrity. The methodology involves monitoring muscle disruption through fluorescence imaging of GFP-expressing worms.
Begin with a nutrient-rich nematode growth medium plate containing a lawn of the pathogenic bacterium Aeromonas.
Transfer L4-stage C. elegans worms expressing muscle-specific GFP onto the bacterial lawn and incubate.
The worms ingest bacteria along with nutrients. This allows the bacteria to reach and colonize the intestine.
Using a specialized secretion system, Aeromonas delivers virulence factors into the worm’s intestinal cells.
These virulence factors diffuse to the body wall, where they disrupt muscle, causing structural damage and necrosis.
Transfer the worms daily to a fresh plate with a bacterial lawn to maintain continuous pathogen exposure.
Place the worms onto an agarose gel supplemented with an anesthetizing agent to anesthetize them, then place a coverslip.
Using a GFP filter, capture fluorescence images at regular intervals.
The progressive loss of the GFP signal indicates muscle disruption in C. elegans, representing Aeromonas-induced muscle necrosis during host-pathogen interaction.
After culturing Aeromonas strains and adjusting the absorbance according to the text protocol, spot and spread the bacterial broth on NGM plates.
After this, transfer 50 L4 worms to each NGM plate. Every 24 hours transfer the worms to fresh NGM plates. Randomly select 10 worms and transfer them to a 2% agarose gel in M9 medium on a slide.
Finally position a coverslip on the gel and capture muscle images with a green fluorescent protein filter on a fluorescent microscope equipped with a camera.
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