The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Podsumowanie

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

Streszczenie

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

Wprowadzenie

C. elegans has been used as a powerful model organism for more than 50 years. In the 1960s, South African biologist Sydney Brenner pioneered the use of C. elegans to study neuronal development, paving the way for a long lineage of scientists to study various aspects of cell and animal biology in nematodes. This lineage includes Nobel Prize laureates Craig Mello and Andrew Fire for their RNAi work¹, Robert Horvitz and John Sulston for their work on organ development and apoptosis^2,3,4, and Martin Chalfie for his work on green fluorescent protein⁵. Although this model organism has been traditionally used to study molecular and developmental biology, over the past 15 years, researchers have begun to use C. elegans to investigate the biology of various human pathogens including Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella enterica, and Serratia marcescens^6,7,8,9,10. These studies revealed that many of the mechanisms involved in the human-pathogen interaction are conserved in nematodes, but also that there are some immunity mechanisms that are unique to this model organism^11,12. In nature, C. elegans encounters a variety of threats from ingested pathogens present in the soil and this has provided a strong selective pressure to evolve and maintain a sophisticated innate immune system in its intestinal lumen. Many of the genes and mechanisms involved in the protection of intestinal lumen are orchestrated by highly-conserved elements that also exist in higher mammals^11,13. C. elegans therefore represents a great model to study gastrointestinal pathogens like Salmonella enterica¹⁴, Shigella boydii¹⁵, or Vibrio cholera¹⁶.

Here we highlight the remarkable versatility of C. elegans as a model host to study infectious agents such as C. albicans. C. elegans as a model host allows for high throughput screening for virulence that is less expensive and time-consuming than a mouse model, which is commonly used to study candidiasis⁴².

In this study, we show that this model and the assosiated survival assay can be reliably used for studying host innate immune effectors important to counteract infections, pathogen determinants that drive virulence, and pharmacological compounds that can intervene in pathogenesis. Dissimilar to previously described assays, this method provides a means of studying exposure to a pathogen over the lifetime of the animal, from the larval stage to adulthood, rather than only adulthood to death^43,44. In summary, our C. elegans - C. albicans model is a versatile and powerful tool that can be used not only to study the genetic bases that drive infection and immunity but also to identify new compounds for therapeutic intervention.

Protokół

1. Preparation of Nematode Growth Medium (NGM)

1. For 1 L of media, combine 20 g agar, 2.5 g organic nitrogen source (e.g., bacto-peptone), and 3 g sodium chloride in a 2 L flask. Add 975 mL of sterile water.
   1. Add in a sterile stir bar. If using an automatic media pourer, autoclave tubing and media for 15 min; media should be autoclaved for longer if a higher volume is made.
2. Set media on stir plate, and allow to cool.
   1. Once media is warm to touch (approximately 60 °C) aseptically add 1 mL of 1 M MgSO₄● H₂O, 1 mL of 1M CaCl₂, 25 mL of KPO₄, and 1 mL of 0.5% cholesterol in ethanol.
   2. Pour media (by hand, or by automatic pump) under laminar flow into 35 mm x 10 mm sterile petri dishes. Allow to dry under hood for 1-2 days before use.
      NOTE: Plates should be stored at 4 °C to prevent contamination.

2. Making a Worm Pick

1. Cut a 1.5 cm piece of aluminum wire. Carefully insert approximately 0.5 cm of this length into the tip of a Pasteur pipette.
   1. 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.

3. Preparation of E. coli Culture

1. Using a sterile loop, inoculate single colony of E. coli strain OP50 in 200 mL of Luria Broth. Incubate overnight with shaking at 37 °C.
2. The liquid culture is ready for use the following day and may be stored at 4 °C when not in use.
   NOTE: The E. coli strain, OP50, is used as a food source for C. elegans. This culture may be used for up to several months when stored at 4 °C.

4. Essential C. elegans Maintenance

1. Seed prepared NGM agar plates with E. coli by spotting approximately 50 - 100 µL of prepared OP50 liquid culture onto the center of the plate.
2. Cover plates and allow them to dry at room temperature for 24 h. Once dry, place plates at 37 °C overnight for rapid growth or keep at room temperature, if slower growth is desired.
3. Add worms to the plate by either "chunking," (see protocol step 5 "Chunking and Picking Worms"), picking worms individually, or placing worm eggs directly onto the plate (see protocol step 6 "Egg Preparation").

5. Chunking and Picking Worms

NOTE: "Chunking," refers to a practice that is common in C. elegans maintenance. This involves cutting a section of the NGM agar plate that contains worms, and transferring this piece onto a new plate, thus also transferring a large number of worms in the process. Contamination may also be cut out of the plate in this manner. When picking worms, it is important to maintain the integrity of the agar because worms can be lost in holes in the agar. In addition, it is important to allow the pick to cool before touching the plate to prevent melting the agar or burning the worms.

1. In order to quickly transfer large amounts of worms onto new plates, cut a piece of NGM agar containing the selected worms using a spatula.Remove the cut piece and place it face down on the edge of the lawn of OP50 on a new plate.
2. Transfer worms individually as described below in protocol step 8.2, "Survival Assay," using a worm pick. Flame the wire in the worm pick until it is red. Remove it from the flame and allow it to cool for a few seconds.
3. Using the wire on the pick, gently scrape the lawn of the OP50 culture grown on the new plate, covering the wire on the pick.
4. The viscous culture covering the tip of the wire makes worms stick to the tip of the pick, thus gently pick up selected worms using a swiping motion.
5. Once selected worms are gathered, place them onto a new plate by gently swiping culture across the agar. Place the wire into the flame to remove remaining culture on the pick.

6. Egg Preparation

1. Place approximately 20 adult animals (approximately 2 - 3 days after hatching when grown at 20 °C) by either picking or chunking as described in protocol step 5, "Chunking and Picking Worms," onto a plate seeded with 50 µL of E. coli strain, OP50.
2. To collect the eggs, flood the plate with 10 mL of M9 buffer after 1-2 days. Using a glass serological pipette, swirl and gently agitate the contents on the agar plate to release eggs stuck to OP50 or adult animals. Collect all the liquid containing the eggs and adults.
3. Transfer M9 and OP50 solution into a 15 mL conical tube. Centrifuge 15 mL conical tube at 2,100 x g for 2 min.
4. Aspirate approximately 9 mL of supernatant without disturbing the OP50 pellet.
5. Resuspend the pellet in 2 mL of sterile water, 1 mL of bleach, and 1 mL of 0.25 M NaOH.
6. Mix gently by inversion until the majority (approximately 70 percent) of adult animals appear lysed; typically, eggs remain intact during this short bleach treatment because of their shell. Centrifuge the conical tube at 990 x g for 2 min.
7. Aspirate supernatant without disturbing the pellet. Re-suspend the pellet in 10 mL of M9 buffer.
8. Centrifuge the conical tube in 990 x g for 2 min. Aspirate supernatant without disturbing pellet. Re-suspend pellet with 200 µL of M9 buffer.
   NOTE: Egg preparation may require multiple trials. Eggs may be destroyed if they are exposed to bleach for too long. If eggs do not hatch, either decrease the concentration of bleach in solution or decrease the duration of exposure to bleach.

7. Infection Plate Set-up

1. Dispense 3-5 mL of YPD into a sterile test tube and inoculate it with a single colony of Candida albicans using a sterile loop.
   1. Place the tube on a rotatory drum for approximately 16-18 h at 30 °C.
2. Label one 1.5 mL microfuge tube to contain C. albicans, and another to contain OP50. Record the weight of each empty tube.
   1. Place 500 µL of the overnight C. albicans culture into tube labeled C. albicans and 1,500 µL of the overnight OP50 culture (from step 3.1) into the tube labeled OP50.
   2. Centrifuge for 10 min at 16,100 x g. Aspirate supernatant without disturbing pellet.
   3. Re-suspend each pellet with 500 µL of sterile water. Centrifuge for 5 min at 16,100 x g.
   4. Aspirate supernatant without disturbing the pellet. Record the final weight of microfuge tubes.
   5. Determine the weight of each pellet by subtracting the initial weight of the microfuge tubes from the final weight.
3. Using sterile water, re-suspend the C. albicans pellet to 10 mg/mL, and the OP50 pellet to 200 mg/mL. Make a master infection mix by combining 10 µL of a 50 mg/mL solution of Streptomycin, 2.5 µL of OP50 culture, 0.5 µL of C. albicans culture, and 7 µL of sterile water.
   1. For control plates, create a master mix by replacing the volume of C. albicans culture with sterile water. Seed 20 µL of infection mix or OP50 control solution onto the center of NGM plates using a micropipette.
      NOTE: Approximately 100 worms are needed to determine statistical significance during analysis. This assay is typically run in triplicate. Thus, a 3.2x infection mix as well as a 3.2x control solution is made. These mixes are made to be slightly over 3x the original to ensure that a full 20 µL is seeded onto each plate, allowing room for error. This master mix is created because the pharynx of the worm is too small during the initial larval stages (L1-L3) to consume C. albicans. For exposure to pharmacological agents such as fluconazole (Figure 3B), the agent is introduced to the infection mixture. The concentration of the agent is empirically determined. For example, in Figure 3B, 50 µM Fluconazole is represented, however 0, 12.5, 25, 50 and 100 µM Fluconazole were also tested using the same protocol.

8. Analysis of the Deformed Anal Region (Dar) Phenotype and Survival Assay

1. Visualize the Dar phenotype
   NOTE: Dar is best visualized at the L3 stage and beyond. Dar presents as a small protrusion in the post anal region of the worm (Figure 2A), which becomes more prominent over time.
   1. 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.
2. Survival assay
   1. Complete egg preparation as previously described in protocol step 6, "Egg Preparation". Count the eggs under the dissection scope, and dilute the egg solution with M9 until a concentration of approximately 5-6 healthy eggs per µL is achieved. Using a pipette, dispense 20 µL sample containing approximately 30 eggs onto prepared NGM plate, in the area between the bacterial culture and the side of plate (eggs and worm food, OP50, are in two distinct spots).
   2. Incubate plates at 20 °C for 48 h. Under a dissection microscope, count the number of live and dead adult worms on each plate, as well as the number of worms with Dar. Record the percent with Dar.
   3. Consider an animal dead if it does not respond to being tapped on the head by an aluminum wire or tapping on the plate.
   4. Every other day, transfer remaining adult worms by picking as described in protocol step 5, "Chunking and 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.
      NOTE: This assay may run for two weeks, beginning with egg preparation. In addition, the egg solution should not be dispensed onto the bacterial culture, as the solution may contain traces of bleach which can kill the OP50. The solution should also be dispensed approximately 0.5 cm away from the edge of the plate, as eggs can become stuck in between the sides of the plate and the agar. Additionally, due to the rapid proliferation of worms, transferring adults onto new plates is critical to separate them from their progeny.
3. Analyze Survival
   1. Analyze results using survival curve analysis software (refer to list of materials).
   2. Compare survival curves using the Grehan-Breslow-Wilcoxon method (assuming that the data from early survival times are more accurate than later times, weight the data accordingly) as well as Logrank statistical tools⁴⁵.
      NOTE: For example, in Figure 4B-C, the survival of worms treated with mutant C. albicans is compared to those treated with isogenic wild-type controls or complementarystrains.

Wyniki

A pathogenesis assay (Figure 1) using C. albicans and C. elegans has previously been described by our lab^17,18 and other labs^19,20. We demonstrate the amenability of using C. elegans to study C. albicans virulence showing that C. albicans cells are quickly ingested by the worms and accumulate in the...

Dyskusje

The methods for assaying C. elegans infection and survival over lifetime exposure to C. albicans that we have described can be modified to test another pathogen. Liquid cultures of another bacteria or fungus may be made and fed to C. elegans in a similar manner. Additionally, serial infections may be assayed by first exposing the larva to one pathogen as described, and then transferring the animals onto a new plate containing a separate pathogen after reaching adulthood.

Materiały

Name	Company	Catalog Number	Comments
Agar (granulated, bacterilogical grade)	Apex BioResearch Products	20-248
Aluminum Wire (95% Pt, 32 Gauge)	Genesee Scientific	59-1M32P
Axiovision Zeiss Inverted Microscope	Axiovision Zeiss
Bacto-Peptone	Fisher BioReagants	BP1420-500
C. elegans strain Bli-3	Caenorhabditis Genetics Center	Bli-3(e767) CB767
Calcium Chloride	Fisher Scientific	BP51-250
Cholesterol, Sigma Grade, minimum 99%	Sigma	C8667-25G
Disposable Culture Tubes (20 x 150 mm)	FIsherBrand	14-961-33
Dissection Microscope (NI-150 High Intensity Illuminator)	Nikon Instrument Inc.
E. coli	Caenorhabditis Genetics Center	OP50
GraphPad Prism (Survival Curve Analysis Software)	GraphPad Software
LB Broth (Miller's)	Apex BioResearch Products	11-120
Magnesium Sulfate	Fisher Scientific	10034-99-8
Medium Petri Dishes (35 X 10 mm)	Falcon	353001
Potassium Phosphate monobasic	Sigma	P0662-500G
Sodium Chloride	Fisher Scientific	BP358-1
Sodium Phosphate	Fisher Scientific	BP332-500
Wildtype C. albicans SC5314	ATCC	SC5314
Wildtype C. elegans	Caenorhabditis Genetics Center	N2