線虫の宿主微生物間相互作用を研究する多彩なIn Vivoモデル

The overall goal of this assay is to observe the effect of fungal infection in the model organism C.Elegans. This method can help answer key mechanistic questions in the field of infectious disease, such as pathogen virulence factors, host immune effectors, as well as drug discovery. The main advantage of this technique is that it is an experimentally tractable system to study host-pathogen interactions from both points of view.

It is a fast, facile and a cost-effective system. Though this particular method can provide insight into fungal pathogens, it can also be applied to other infectious agents, such as bacteria or even multiple organisms to study microbial interactions in a live host. To make a worm pick, cut a 1.5 centimeter piece of aluminum wire then carefully insert approximately 0.5 centimeters of the wire into the tip of a pasteur pipette.

Using a Bunsen burner or alcohol lamp melt the glass pipette tip by slowly rotating it in the flame until the wire is securely adhered to the pipette without compromising the original shape of the pipette tip. Finally, using forceps, carefully bend the end of the wire to create a small loop. For quickly transferring large amounts of worms onto new plates, use a spatula to cut a piece of NGM agar containing the selected worms.

Then remove the cut piece and place it facedown on the edge of the patch of OP50 on a new plate. To transfer worms individually, flame the wire in the worm pick until it is red then remove it from the flame and allow it to cool for a few seconds. Using the wire on the pick, gently scrape the patch of the OP50 culture grown on the new plate, covering the wire on the pick.

The viscous culture covering the tip of the wire makes worms stick to the tip of the pit. Thus use a swiping motion to gently pick up selected worms. Once selected worms are gathered place them onto a new plate by gently swiping the culture across the agar.

Then place the wire into the flame to remove remaining culture on the pick. Place approximately 20 adult animals by either picking or chunking them onto a plate seated with 50 microliters of E.coli strain OP50. To collect the eggs after one to two days flood the plate with 10 milliliters of M9 buffer.

Then using a glass serological pipette swirl and gently agitate the contents on the agar plate to release adult animals or eggs stuck to OP50. Collect all the liquid containing the eggs and adults. Transfer the M9 and OP50 solution into a 15 milliliter conical tube.

Centrifuge the tube at 2, 100 x g for two minutes. Then aspirate approximately nine milliliters of supernatant without disturbing the pellet. Next, use two milliliters of sterile water, one milliliter of bleach and one milliliter of 0.25 molar sodium hydroxide to resuspend the pellet.

Mix gently by inversion until approximately 70%of adult animals appear lysed. Typically eggs remain intact during this short bleach treatment because of their shell. It is critical that the amount of exposure to bleach is limited in this step.

Prolonged exposure to bleach will destroy the eggs. If egg preparation fails, either limit the duration of exposure to bleach or dilute the solution. Centrifuge the conical tube at 990 x g for two minutes.

Aspirate the supernatant without disturbing the pellet. Then resuspend the pellet in 10 milliliters of M9 buffer. After spinning again and removing the supernatant without disturbing the pellet, use 200 microliters of M9 buffer to resuspend the pellet.

Dispense three to five milliliters of YPD into a sterile test tube, and use a sterile loop to inoculate it with a single colony of Candida albicans. Place the tube on a rotary drum at 30 degrees Celsius for approximately 16 to 18 hours. Label one 1.5 milliliter microfuge tube to contain C.albicans and another to contain OP50, then record the weight of each empty tube.

Transfer 500 microliters of the overnight C.albicans culture into the tube labeled C.Albicans. And 1500 microliters of the overnight OP50 culture into the tube labeled OP50. Centrifuge the tubes for 10 minutes at 16, 100 x g, then aspirate the supernatant without disturbing the pellets.

Use 500 microliters of sterile water to resuspend each pellet. Then centrifuge the tubes at 16, 100 x g for five minutes. Aspirate the supernatant without disturbing the pellets.

Then record the final weight of the microfuge tubes. Determine the weight of each pellet by subtracting the initial weight of the microfuge tubes from the final weight. Using sterile water, resuspend the C.albicans pellet to 10 milligrams per milliliter and the OP50 pellet to 20 milligrams per milliliter.

Then make a master infection mix by combining 10 microliters of a 50 milligram per milliliter solution of Streptomycin. 2.5 microliters of OP50 culture. 0.5 microliters of C.Albicans culture, and 7 microliters of sterile water.

For control plates, create a master mix by replacing the volume of C.albicans culture with sterile water. Then use a micropipette to seed 20 microliters of infection mix or OP50 control solution onto the center of NGM plates. After preparing eggs as demonstrated earlier in this video, count the eggs under the dissection scope.

And use M9 to dilute the egg solution until a concentration of approximately five to six healthy eggs per microliter is achieved. Using a pipette, dispense a 20 microliters sample containing approximately 30 eggs onto a prepared NGM plate in the area between the bacterial culture and the side of the plate. Incubate the plates at 20 degrees Celsius for 48 hours.

Then, under a dissection microscope, count the number of live and dead adult worms on each plate. considering the animal dead if it does not respond to being tapped on the head by an aluminum wire or tapping the plate. Also count the number of worms with Dar and confirm if an animal has Dar by quickly tapping the plate on the stage of the dissection microscope.

Animals without Dar will move backwards immediately, while animals with Dar will either need repeated rounds of tapping to reverse their direction or they will not do so at all. Every other day transfer any remaining adult worms by picking worms onto a new infection or control plate. At this point, if needed, perform a survival assay by tracking the number of live, dead and missing worms each day.

It's important to transfer the worms every other day at minimum. This ensures that the adult worms that are being scored in this assay are not confused at their progeny. C.elegans were exposed to C.albicans over a period of six days and observed for signs of infection, progression of disease and death.

The Dar phenotype is most visible by day four of the survival assay as noted by a protruding anal region that is not visible in the uninfected animal. Worms infected by C.albicans are also known to exhibit swelling in the vulva region. In both cases the worm is unable to clear the infection after reaching this stage.

In order to visualize colonization of C.Albicans in the intestinal lumen, worms were fed wild type C.albicans tagged with RFP which cause areas of colonization to fluoresce red. C.albicans colonize the intestinal lumen by the third day of the assay. As shown here, infected worms showed more severe intestinal distension than observed in the uninfected control.

In this experiment, the ability of the worms to survive infection caused by the a virulent C.Albicans efg1/efg1 cph1/cph1 double mutant was tested. Worms infected with the double mutant live statistically significantly longer than controls. Suggesting that these two genes are required for C.albicans virulence against C.Elegans.

Once mastered, each part of this technique can be done in one to two hours each day of the lifespan of the worm. While attempting this procedure, it's important to remember that one might be using infectious agents. So appropriate biosafety precautions should be practiced.

Minor modifications can be made to this basic procedure, like introducing additional microbial agents in order to study microbial interactions in the context of a live host. After its development, this technique paved the way for researchers in the field of infectious disease to explore microbial virulence, mechanisms of host innate immune responses, as well as discovery of novel antimicrobial agents. After watching this video, you should have a good understanding of how to maintain and manipulate C.Elegans and use the organism to study host microbe interactions at the molecular level.

Don't forget that working with microbial pathogens and chemical reagents, like sodium azide, can be hazardous and appropriate safety precautions like PPE and waste disposal methods should be employed.