In this protocol we demonstrate how to use a free living method c. elegans to elucidate the pathogenicity caused by hydrogen peroxide produced via Mitis Group streptococci. The main advantage of this technique is that we can study pathogenicity and the activation of stress responses in the worm by using a sustainable biological source of reactive oxygen species as opposed to chemical sources For one liter of media add 30g of Todd Hewitt powder, 2g of yeast extract and 20g of ager to a 2 liter Erlin Myer flask.
Add 970ml of deionized water to the contents of the flask and put in a stir bar. Use aluminum foil to cover the opening. Autoclave the media in the flask at a temperature of 121 degrees Celsius and pressure of 15 pounds per square inch for 30 minutes.
Thereafter set the flask on a stir plate to cool down with gentle stirring. Under a laminar hood, pour the media into appropriately sized, sterile Petri dishes. Allow the media to dry for 2 hours.
Thereafter store the plates at 4 degrees Celsius for up to 1 month. Before the preparation of Mitis Group streptococci, warm the THY agar plates in an incubator to 37 degrees Celsius. After that use a sterile loop to streak out desired strains of strepticocci on the places.
Then put the plates in a candle jar and incubate the jar at 37 degrees Celsius overnight for around 18 hours to provide a microerophilic environment. The next day, remove the plates from the candle jar and use a sterile loop to pick isolated colonies. Inoculate 15 ml sterile conical tubes containing 2 ml of THY broth.
Close the caps tight and incubate the tubes at 37 degrees Celsius under static conditions. Pre-warm THY plates in an incubator to 37 degrees Celsius add 50 microliters of solution containing 1000 units of catalase on to the THY plate. Use a sterile spreader to spread the catalase solution and allow the plates to dry on the laminar flow hood for 30 minutes.
To seed the plates with the streptococcus strains, use a pipette to add 80 microliters of overnight grown cultures to the plate and use a sterile spreader to spread the bacteria completely across the agar surface. Incubate the plates at 37 degrees Celsius in a candle jar overnight. As a control, on two NGM plates, add 80 microliters of overnight grown cultures of E.coli OP-50 for seeding.
Incubate the plates at 37 degrees Celsius overnight. The next day, remove the plates from the candle jar and allow the plates cool to room temperature. Using a sterile worm pick, transfer 30 L4 larvae from the NGM feeding plates to the streptococcus seeded THY plates.
Incubate the plates at 25 degrees Celsius. Under a dissecting microscope, count the number of live and dead L4 larvae on each plate at several time points. Use the sterile worm pick to gently prod the worms and determine if they are dead or alive.
Initially, score the worms as dead or live every 30 minutes. When worms rapidly start to die, score them at 15 minute intervals. The assay will take 5 to 6 hours to complete.
After that, repeat the experiment two more times. To prepare the agarose pad, stick labtape lengthwise along two glass slides. This will determine the thickness of the agarose pads.
Place a clean glass slide between the two taped slides. Dissolve agarose in deionized water to make 2 weight volume percent and heat the solution in a microwave. Use a pipette to place 100 microliters of molten agarose on the center of the clean slide.
Immediately place another clean glass slide on top of the molten agarose and gently press down to make a pad. Allow the agarose to solidify and subsequently remove the top slide. The agarose pad is ready for use.
Pre-warm THY plates to 37 degrees Celsius. Seed the plates with a streptococcus strains as done previously in the survival assays. Seed 3 NGM plates with E-coli OP-50 as control and incubate at 37 degrees Celsius overnight.
The next day, cool the plates to room temperature. Use M9W buffer to wash L4 larvae from NGM or NGM RNAi feeding plates into 15 ml conical tubes. Spin the tubes at 450 times g, for one minute in a centrifuge.
Decant the supernatant and add 10 ml of M9W to each tube. Repeat the centrifugal step three more times. Re-suspend the worms in 250 microliters of M9W and place three 5 microliter drops of the worm suspension on to a clean Petri dish lid.
Under a dissecting microscope, estimate the number of worms per microliter. Using a pipette, add approximately 100 L4 larvae to each THY streptococcus seeded and NGM E.coli seeded plates. Use three plates per strain of bacteria.
Incubate the plates for 2 to 3 hours at 25 degrees Celsius. Thereafter, remove the plates from the incubator. Wash them with M9W and collect the worms in 15ml conical tubes.
Wash the worms three times as done previously in the centrifugal step. Remove most of the M9W by aspiration. And add 500 microliters of M9W containing 2 millimolar sodium azide or tetramisole hydrochloride to the worm pellet for anesthesia.
Incubate the tube with worm pellets at room temperature for 15 minutes. Then, drop 15 microliters of the worm suspension on to a prepared agorose pad. Under a fluorescent microscope, visualize the localisation of skn-1 utilizing Fitzy and Dappy filters.
Image worms at 10 times and 20 times magnifications. Score the worms based on three levels of localisation;low localisation, where no nuclear localisation is observed, medium localisation, with SKN 1 BCGFP in the anterior or posterior of the worm, and high localisation, where nuclear localisation of skn-1 BCGFP is observed in all intestinal cells. In this experiment, members of the Mitis Group rapidly killed the worms, as opposed to S.mutans, S.salivarius and non pathogenic E.coli OP-50.
When catalase was supplemented to THY ager, the killing of the worms was abolished. Death of the worms was not observed on the delta spxB mutant strain compared to the wild-type and compliment strains. These data suggest that the hydrogen peroxide produced by the Mitis Group mediates killing of the worms.
A significant decrease in the survival of the skn-1 knockdown worms was observed compared to the vector control treated worms. A similar killing phenotype was observed with the skn-1 mutant strain and the N2 wild-type worms. This demonstrates that skn-1 influence the survival of the worms on the Mitis Group.
Localisation of skn-1 BCGFP was observed in worms exposed to the wild-type N-compliment strains and not in response to the Delta spxB mutant strain of the S cordonae. After components of the P38 map kinase pathway were knocked down, reduced localisation of skn-1 BCGFP and knockdown treated worms, relative to the vector control treated worms, was observed. By using the streptococci c.
elegans system, the effects of hydrogen peroxide on endoplasmic verticular stress, mitochondrial damage, mitophogy, and tophogy can be further studied. Further more, mechanisms by which hydrogen peroxide acts as a virilescence factor to elicit immune responses by disrupting core processes of the cell can be identified.
