Name: the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions
Uploaded: 2017-10-18T14:00:00.000+00:00
Duration: 11 min 58 s
Description: Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

This work was performed at and supported by Worcester Polytechnic Institute.

1. Preparation of Nematode Growth Medium (NGM)
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Set media on stir plate, and allow to cool.
	<ol>
		<li>Once media is warm to touch (approximately 60 &#176;C) aseptically add 1 mL of 1 M MgSO4&#9679; H2O, 1 mL of 1M CaCl2, 25 mL of KPO4, and 1 mL of 0.5% cholesterol in ethanol.</li>
		<li>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 &#176;C to prevent contamination.</li>
	</ol>
	</li>
</ol>
2. Making a Worm Pick
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Preparation of E. coli Culture
<ol>
	<li>Using a sterile loop, inoculate single colony of E. coli strain OP50 in 200 mL of Luria Broth. Incubate overnight with shaking at 37 &#176;C.</li>
	<li>The liquid culture is ready for use the following day and may be stored at 4 &#176;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 &#176;C.</li>
</ol>
4. Essential C. elegans Maintenance
<ol>
	<li>Seed prepared NGM agar plates with E. coli by spotting approximately 50 - 100 &#181;L of prepared OP50 liquid culture onto the center of the plate.</li>
	<li>Cover plates and allow them to dry at room temperature for 24 h. Once dry, place plates at 37 &#176;C overnight for rapid growth or keep at room temperature, if slower growth is desired.</li>
	<li>Add worms to the plate by either &#34;chunking,&#34; (see protocol step 5 &#34;Chunking and Picking Worms&#34;), picking worms individually, or placing worm eggs directly onto the plate (see protocol step 6 &#34;Egg Preparation&#34;).</li>
</ol>
5. Chunking and Picking Worms
NOTE: &#34;Chunking,&#34; 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.
<ol>
	<li>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.</li>
	<li>Transfer worms individually as described below in protocol step 8.2, &#34;Survival Assay,&#34; 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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
6. Egg Preparation
<ol>
	<li>Place approximately 20 adult animals (approximately 2 - 3 days after hatching when grown at 20 &#176;C) by either picking or chunking as described in protocol step 5, &#34;Chunking and Picking Worms,&#34; onto a plate seeded with 50 &#181;L of E. coli strain, OP50.</li>
	<li>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.</li>
	<li>Transfer M9 and OP50 solution into a 15 mL conical tube. Centrifuge 15 mL conical tube at 2,100 x g for 2 min.</li>
	<li>Aspirate approximately 9 mL of supernatant without disturbing the OP50 pellet.</li>
	<li>Resuspend the pellet in 2 mL of sterile water, 1 mL of bleach, and 1 mL of 0.25 M NaOH.</li>
	<li>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.</li>
	<li>Aspirate supernatant without disturbing the pellet. Re-suspend the pellet in 10 mL of M9 buffer.</li>
	<li>Centrifuge the conical tube in 990 x g for 2 min. Aspirate supernatant without disturbing pellet. Re-suspend pellet with 200 &#181;L 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.</li>
</ol>
7. Infection Plate Set-up
<ol>
	<li>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.
	<ol>
		<li>Place the tube on a rotatory drum for approximately 16-18 h at 30 &#176;C.</li>
	</ol>
	</li>
	<li>Label one 1.5 mL microfuge tube to contain C. albicans, and another to contain OP50. Record the weight of each empty tube.
	<ol>
		<li>Place 500 &#181;L of the overnight C. albicans culture into tube labeled C. albicans and 1,500 &#181;L of the overnight OP50 culture (from step 3.1) into the tube labeled OP50.</li>
		<li>Centrifuge for 10 min at 16,100 x g. Aspirate supernatant without disturbing pellet.</li>
		<li>Re-suspend each pellet with 500 &#181;L of sterile water. Centrifuge for 5 min at 16,100 x g.</li>
		<li>Aspirate supernatant without disturbing the pellet. Record the final weight of microfuge tubes.</li>
		<li>Determine the weight of each pellet by subtracting the initial weight of the microfuge tubes from the final weight.</li>
	</ol>
	</li>
	<li>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 &#181;L of a 50 mg/mL solution of Streptomycin, 2.5 &#181;L of OP50 culture, 0.5 &#181;L of C. albicans culture, and 7 &#181;L of sterile water.
	<ol>
		<li>For control plates, create a master mix by replacing the volume of C. albicans culture with sterile water. Seed 20 &#181;L 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 &#181;L 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 &#181;M Fluconazole is represented, however 0, 12.5, 25, 50 and 100 &#181;M Fluconazole were also tested using the same protocol.</li>
	</ol>
	</li>
</ol>
8. Analysis of the Deformed Anal Region (Dar) Phenotype and Survival Assay
<ol>
	<li>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.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Survival assay
	<ol>
		<li>Complete egg preparation as previously described in protocol step 6, &#34;Egg Preparation&#34;. Count the eggs under the dissection scope, and dilute the egg solution with M9 until a concentration of approximately 5-6 healthy eggs per &#181;L is achieved. Using a pipette, dispense 20 &#181;L 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).</li>
		<li>Incubate plates at 20 &#176;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.</li>
		<li>Consider an animal dead if it does not respond to being tapped on the head by an aluminum wire or tapping on the plate.</li>
		<li>Every other day, transfer remaining adult worms by picking as described in protocol step 5, &#34;Chunking and Picking Worms,&#34; 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.</li>
	</ol>
	</li>
	<li>Analyze Survival
	<ol>
		<li>Analyze results using survival curve analysis software (refer to list of materials).</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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.
While conducting this assay, it is important to pay careful attention to timing. First, when completing egg preparation in order to harvest eggs for the assay, it is critical to limit the amount of time the eggs are exposed to bleach. The amount of time needed to dissolve adults in the bleach solution to release the eggs varies. Immediately after administering the bleach solution, the worm solution must be watched closely under a dissection microscope. Once approximately 70% of the worms have disintegrated, then the solution should be centrifuged. If egg preparation does not yield viable eggs, decrease either the concentration of bleach or the duration of exposure to bleach. In completing this assay, it is also crucial that attention is given to transferring adult worms onto new plates as needed to avoid confusing study subjects with their progeny. Limiting the number of eggs plated at the start of the assay to 25-30 eggs should prevent this as well. Finally, transferring adult worms onto new plates frequently also decreases the chances of worms crawling up the sides of the petri dish and dying.
We have utilized Caenorhabditis elegans as a versatile model host for studying pathogenic or commensal relationships between microbes or a microbe and its host. Our system can be used as a tool to study microbial or host-microbe interaction at the cellular and molecular level using forward and reverse genetic approaches but also for exploring new molecules for therapeutic intervention. We have demonstrated the versatility of this system by conducting a genetic screen to identify genes that contribute to virulence40, those linked to microbial fitness41, those that mediate host response to microbes17,18, as well using the model for high throughput applications for drug discovery24. This is a good model for use by undergraduate students because it does not need expensive infrastructure to maintain colonies, considering that animals can be cryo-preserved for future applications. Finally, this provides a perfect &#34;go-between&#34; for in vitro studies and murine models.

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 apoptosis2,3,4, 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 marcescens6,7,8,9,10. 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 organism11,12. 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 mammals11,13. 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 death43,44. 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.

<table><tbody><tr><td>Agar (granulated, bacterilogical grade)</td><td>Apex BioResearch Products</td><td>20-248</td><td></td></tr><tr><td>Aluminum Wire (95% Pt, 32 Gauge)</td><td>Genesee Scientific</td><td>59-1M32P</td><td></td></tr><tr><td>Axiovision Zeiss Inverted Microscope</td><td>Axiovision Zeiss</td><td></td><td></td></tr><tr><td>Bacto-Peptone</td><td>Fisher BioReagants</td><td>BP1420-500</td><td></td></tr><tr><td>C. elegans strain Bli-3</td><td>Caenorhabditis Genetics Center</td><td>Bli-3(e767) CB767</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>BP51-250</td><td></td></tr><tr><td>Cholesterol, Sigma Grade, minimum 99%</td><td>Sigma</td><td>C8667-25G</td><td></td></tr><tr><td>Disposable Culture Tubes (20 x 150 mm)</td><td>FIsherBrand</td><td>14-961-33</td><td></td></tr><tr><td>Dissection Microscope (NI-150 High Intensity Illuminator)</td><td>Nikon Instrument Inc.</td><td></td><td></td></tr><tr><td>E. coli</td><td>Caenorhabditis Genetics Center</td><td>OP50</td><td></td></tr><tr><td>GraphPad Prism (Survival Curve Analysis Software)</td><td>GraphPad Software</td><td></td><td></td></tr><tr><td>LB Broth (Miller&#039;s)</td><td>Apex BioResearch Products</td><td>11-120</td><td></td></tr><tr><td>Magnesium Sulfate</td><td>Fisher Scientific</td><td>10034-99-8</td><td></td></tr><tr><td>Medium Petri Dishes (35 X 10 mm)</td><td>Falcon</td><td>353001</td><td></td></tr><tr><td>Potassium Phosphate monobasic</td><td>Sigma</td><td>P0662-500G</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Sodium Phosphate</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Wildtype C. albicans SC5314</td><td>ATCC</td><td>SC5314</td><td></td></tr><tr><td>Wildtype C. elegans</td><td>Caenorhabditis Genetics Center</td><td>N2</td><td></td></tr></tbody></table>

the nematode caenorhabditis elegans - a versatile in vivo model to study host-microbe interactions

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.

A pathogenesis assay (Figure 1) using C. albicans and C. elegans has previously been described by our lab17,18 and other labs19,20. 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 intestinal lumen causing slower locomotion, deformed anal region (Dar) (Figure 2A), swelling of the vulva (Figure 2B), distention of the intestine (Figure 3A), and lethality (Figure 3, Figure 4). We also measure the life span of infected worms as a quantitative measure of fitness. For example, C. elegans infected with C. albicans live 10-12 days compared to 20-22 for uninfected controls. In C. elegans infected with the C. albicans double knock out mutant, efg1/efg1 cph1/cph1, which are two key virulence factors required for infection in mouse, rats and human epithelial models21,23, nematodes survived significantly longer than controls (Figure 3B). These experiments suggest that some of the lessons we learn in this simpler model about C. albicans virulence may remain valid in higher mammals and vice versa.
We also show that the nematode model system can be pharmacologically modulated (Figure 3B). In the presence of fluconazole, the most commonly prescribed antifungal drug, worms challenged with C. albicans survived significantly longer than controls. This proof of principle experiment suggests that the nematode model can be used for small molecule screening. Indeed, our C. elegans model was instrumental in identifying filastatin, a small molecule inhibiting various aspects of fungal virulence24.
Disease phenotypes and microscopic analysis of C. elegans infection 
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 4 of the survival assay, as noted by a protruding anal region that is not visible in the uninfected animal (Figure 2A). Worms infected by C. albicans are also known to exhibit swelling in the vulva region (Figure 2B). 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 (Figure 3A). In order to produce these images, worms were anesthetized on a 2% agarose pad containing 0.01 M sodium azide. Worms were exposed to either wild-type C. albicans or RFP-tagged C. albicans, and transferred into 5 &#181;M M9 buffer on the agarose pad. The agarose pad was then covered with a coverslip. Worms were viewed at 200X and 400X magnification using an inverted microscope with fluorescent microscopy capabilities. Images were created under differential interference contrast (Nomarski) and epifluorescence optics. The fluorescent images were enhanced using software (Figure 3A). Time series micrographs of infected worms determined that C. albicans colonized the intestinal lumen by the third day of the assay17. Infected worms showed more severe intestinal distension than observed in the uninfected control.
Genetic and pharmacological tools to study infection 
Next, we tested the model using genetic and pharmacological modulation. We also tested previously documented virulence factors that regulate hyphal transition of C. albicans25 and have been shown to be important in in vivo infections of mice and nematodes20,26. We tested the ability of the worms to survive infection caused by C. albicans efg1/efg1 cph1/cph1 double mutant. Efg1 is a highly-conserved transcription factor and an essential component of the cyclic AMP/protein kinase A (PKA) metabolic pathway. In C. albicans this pathway regulates hyphal morphogenesis27, white-opaque switching28, and an arsenal of key virulence factors. These virulence factors include Hwp1 and Hwp2, two yeast-specific cell wall proteins involved in adhesion and biofilm formation, Eap1, a cell-wall adhesin involved in binding to human epithelial cells29, and Sap5, a hydrolytic enzyme involved in epithelial tissue invasion30. Cph1 is a transcription factor that regulates many metabolic processes including mating, filamentation, and biofilm formation and has been shown to play a critical role in damaging epithelial cells31 and human reconstructed epithelium21. Disruption of either of these genes has a significant impact on virulence and simultaneous disruption of both in cph1/cph1efg1/efg1 results in dramatic virulence reduction in various animal models including mice25, Drosophila32, zebrafish33,34 and moth34. The cph1/cph1efg1/efg1 double mutant is considered by the C. albicans community as the avirulent strain by definition and the gold standard for validation of novel model systems. The efg1&#916; and cph1&#916; single mutants showed decreased Dar (~10% and ~50%, respectively) compared with the cognate wild type, while the efg1&#916; cph1&#916; double mutant failed to elicit the Dar response17. These results recapitulate the pattern of virulence in mice, where the cph1&#8710; mutant is slightly attenuated, the efg1&#8710; mutant is significantly attenuated, and the double mutant is completely avirulent25. Worms infected with cph1/cph1 efg1/efg1C. albicans double mutant lived statistically significantly longer than controls (p &#60;0.01 for both Log Rank and Breslow statistical test, n=60) suggesting that these two genes are required for C. albicans virulence against C. elegans (Figure 3B).
In order to explore the possibility of using our C. elegans model for potential drug screening, we tested the effect of the addition of fluconazole on the ultimate outcome of the infection. We used a variety of different concentrations of fluconazole and found that 50 &#181;M gave us the most significant results. Worms infected with C. albicans and 50 &#181;M fluconazole (Figure 3B) lived statistically significantly longer than controls (p&#60;0.01 at both Log Rank and Breslow statistical test, n=60). This concentration was empirically determined (see the note at the end of protocol step 7, &#34;infection plate set up&#34;). These proof of principle experiments showed that our nematode model can be used for small molecule antifungal screening. The model was in fact instrumental in the discovery of filastatin, a small molecule inhibiting various aspects of fungal virulence that is currently undergoing further preclinical studies24. Accordingly, our assay is suitable for exploring the virulence strategies of C. albicans and pharmacological agents.
Study of innate host response 
Next, we wanted to study the reciprocal host defenses against pathogens, since aspects of this innate immunity are conserved in mammals11,35. It is well known that C. elegans produces ROS upon both bacterial and fungal infections18,36,37 as part of its defense mechanism. ROS have a biocidal effect on invading organisms and play a major role in innate immunity. zcf15/zcf15 hyper susceptibility to ROS in vitro led us to hypothesize that its reduced virulence in C. elegans was due to a reduced ability to withstand the host&#39;s generated ROS. To test our hypothesis, we determined the ability of zcf15/zcf15 to kill either wild-type worms or worms with an impaired ability to produce ROS. Representative C. albicans mutant zcf15 that is sensitive to reactive oxygen species (Figure 4A) showed reduced virulence in wild-type C. elegans. C. elegans responds to ingestion of pathogen by producing extracellular ROS in the intestinal lumen via Ce-Duox1, an NADPH oxidase coded by the gene bli-3. During ROS production, the intestinal cells also produce intracellular antioxidants via DAF-16 to protect its own tissues from the ROS damaging effects36,38. Ce-Duox1 is a protein with an N-terminal peroxidase domain, a C-terminal superoxide-generating NADPH-oxidase domain and two central calmodulin-binding sites39. Upon microbial infection, this protein uses cytosolic NADPH to generate extracellular toxic ROS in the intestinal lumen to counteract the infection. Throughout a series of biochemical assays in a 2009 study, Jain et al. showed that ROS are abundantly produced upon yeast infection and that bli-3 loss of function via bli-3(e767) dramatically reduces the ability of nematodes to produce ROS. As shown in Figure 4B, worms challenged with zcf15/zcf15 survived significantly longer than wild type or complemented strains (p &#60;0.01 by the log-rank test) indicating that ZCF15 is required for virulence. However, when we challenged ROS-deficient C. elegansbli-3(e767), we obtained kinetics of killings that were comparable between zcf15/zcf15, wild type and complemented strain (Figure 4C). This evidence indicates that zcf15/zcf15 fail to kill nematodes unless the host&#39;s ability to produce ROS is compromised. Taken together, these results suggest that ZCF15 is required for full virulence and that this gene is likely involved in the pathogen&#39;s ability to resist host generated ROS. To the best of our knowledge, this is the first time that ZCF15 has been shown to be involved in pathogenicity and ROS resistance.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56487/56487fig1.jpg" /> 
Figure 1: A schematic representation of C. elegans infection. C. elegans are exposed to a mix of E. coli strain OP50 and a pathogen and observed over a period of 4 days after reaching adulthood for signs of infection and progression of disease.
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56487/56487fig2.jpg" /> 
Figure 2: Disease phenotypes. (A) Deformed anal region (Dar) is visible (indicated with an arrow) as a swelling in the post anal region of infected worms (right panel) four days post exposure. The worms on the left panel are uninfected and serve as a control. (B) A subset (~15%) of infected worms show a swelling of the vulva (right panel, indicated with an arrow) compared to uninfected control worms (left panel) that represents disseminated infection resulting in matricidal death. All images were taken as described in the representative results18.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56487/56487fig3.jpg" /> 
Figure 3: The C. albicans - C. elegans model is amenable to microscopic manipulation, and can be pharmacologically and genetically modulated. (A) C. albicans tagged with RFP accumulation in the nematode intestinal lumen day 3 post infection. Yeast cells are quickly ingested by the worms and accumulate in the intestinal lumen completely intact, indicating that they are able to survive the mechanical crushing of the pharynx. Images were taken as described in the representative results18. (B) Kaplan-Meier survival curves of nematodes challenged with wild-type C. albicans, cph1/cph1efg1/efg1 double mutant or wild-type C. albicans + 50 &#181;M of fluconazole compared to uninfected control worms.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56487/56487fig4.jpg" /> 
Figure 4. ZCF15 is required to withstanding C. elegans generated ROS. (A) ZCF15 is responsible for wild type resistance to paraquat, which is known to generate reactive oxygen species. Wild type, knockout or complemented strains were grown in liquid culture overnight and resuspended to OD=1. Cultures were each diluted 1:5 and plated onto YPD or YPD containing 1 mM paraquat. C. elegans produce ROS via bli-3 in order to combat pathogens that invade the intestinal lumen38. (B) Kaplan-Meier survival curves show that ZCF15 deletion limits the killing of wild-type worms. (C) The Kaplan-Meier survival curve shows similar rates of survival between zcf15/zcf15, wild type, and complemented strain when ROS-deficient C. elegansbli-3(e767) were infected.

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

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

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

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

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9.3K Views.  Worcester Polytechnic Institute. Hier presenteren we de nematode Caenorhabditis elegans als een veelzijdig hostmodel aan de studie van microbiële interactie.

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

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

Immunology and Infection

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human diseases have been reported to be associated with four species of bacteria: Aeromonas dhakensis, Aeromonas hydrophila, Aeromonas veronii, and Aeromonas caviae. The model organism Caenorhabditis elegans is a bacterivore that provides an excellent infection model by which to study the bacterial pathogenesis of Aeromonas. Here, we introduce three different experiments to study Aeromonas infection using a C. elegans model, including survival, liquid toxicity, and muscle necrosis assays. The results of the three methods determining the virulence of Aeromonas were consistent. A. dhakensis was shown to be the most toxic among the 4 major Aeromonas species causing clinical infections. These methods are shown to be a convenient way to evaluate the toxicity among and within Aeromonas species and contribute to our understanding of the pathogenesis of Aeromonas infection.

Evaluating Virulence and Pathogenesis of Aeromonas Infection in a Caenorhabditis elegans Model

Here, we introduce three different experiments to study Aeromonas infection in C. elegans. Using these convenient methods, it is easy to evaluate the toxicity among and within Aeromonas species.

The human pathogen Aeromonas has been clinically shown to cause gastroenteritis, wound infections, septicemia, and urinary tract infections. Most human ...

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the context of the intestines. Recently, ecological sampling of wild Caenorhabditis nematodes has discovered a diverse array of associated microbes, including bacteria, viruses, fungi, and microsporidia. Many of these microbes have interesting colonization or infection phenotypes that warrant further study, but they are often unculturable. This protocol presents a method to enrich the desired intestinal microbes in C. elegans and related nematodes and reduce the presence of the many contaminating microbes adhering to the cuticle. This protocol involves forcing animals into the dauer stage of development and using a series of antibiotic and detergent washes to remove external contamination. As dauer animals have physiological changes that protect nematodes from harsh environmental conditions, any intestinal microbes will be protected from these conditions. But, for enrichment to work, the microbe of interest must be maintained when animals develop into dauers. When the animals leave the dauer stage, they are singly propagated into individual lines. F1 populations are then selected for desired microbes or infection phenotypes and against visible contamination. These methods will allow researchers to enrich unculturable microbes in the intestinal lumen, which make up part of the natural microbiome of C. elegans and intracellular intestinal pathogens. These microbes can then be studied for colonization or infection phenotypes and their effects on the host fitness.

Selective Cleaning of Wild Caenorhabditis Nematodes to Enrich for Intestinal Microbiome Bacteria

Wild Caenorhabditis nematodes are associated with many microbes, often in the gut lumen or infecting the intestine. This protocol details a method to enrich unculturable microbes colonizing the intestine, taking advantage of the resistance of the dauer cuticle.

Caenorhabditis elegans (C. elegans)&#160;has proven to be an excellent model for studying host-microbe interactions and the microbiome, especially in the ...

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

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.

Studying Inherited Immunity in a Caenorhabditis elegans Model of Microsporidia Infection

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&#160;model.

Inherited immunity describes how some animals can pass on the "memory" of a previous infection to their offspring. This can boost pathogen resistance in ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on host physiology. It also fluoresces in blue when dying, providing a convenient means of pinpointing death. This property has been exploited to develop high-throughput label-free C. elegans survival assays (LFASS). These involve time-lapse fluorescence recording of worm populations set in multiwell plates, from which population median time of death can be derived. The present study adopts the LFASS approach to screen multiple microbial isolates at once for the effects on C. elegans susceptibility to severe heat and oxidative stresses. Such microbial screening pipeline, which can notably be used to prescreen probiotics, using severe stress resistance as a proxy for host health is reported here. The protocol describes how to grow both C. elegans gut microbiota isolate collections and synchronous worm populations in multiwell arrays before combining them for the assays. The example provided covers the testing of 47 bacterial isolates and one control strain on two worm strains, in two stress assays in parallel. However, the approach pipeline is readily scalable and applicable to the screening of many other modalities. Thus, it provides a versatile setup to rapidly survey a multiparametric landscape of biological and biochemical conditions that impact C. elegans health.

High-Throughput Screening of Microbial Isolates with Impact on Caenorhabditis elegans Health

Gut microbes may positively or negatively impact the health of their host via specific or conserved mechanisms. Caenorhabditis elegans is a convenient platform to screen for such microbes. The present protocol describes high-throughput screening of 48 bacterial isolates for impact on nematode stress resistance, used as a proxy for worm health.

With its small size, short lifespan, and easy genetics, Caenorhabditis elegans offers a convenient platform to study the impact of microbial isolates on ...

Biology

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather than individual worm samples to quantify bacterial load in this organism. Here it is argued that the large inter-individual variability seen in bacterial colonization of the C. elegans intestine is informative, and that batch digest methods discard information that is important for accurate comparison across conditions. As describing the variation inherent to these samples requires large numbers of individuals, a convenient 96-well plate protocol for disruption and colony plating of individual worms is established.

Using Single-Worm Data to Quantify Heterogeneity in Caenorhabditis elegans-Bacterial Interactions

This protocol describes a 96-well disruption of individual bacterially colonized Caenorhabditis elegans following cold paralysis and surface bleaching&#160;to remove external bacteria. The resulting suspension is plated on agar plates to allow accurate, medium-throughput quantification of bacterial load in large numbers of individual worms.

The nematode Caenorhabditis elegans is a model system for host-microbe and host-microbiome interactions. Many studies to date use batch digests rather ...

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant matter, especially rotten fruits like apples or on compost heaps. It is also associated with certain invertebrate hosts such as slugs and woodlice. These habitats are rich in microbes, which serve as food for C. elegans and which can also persistently colonize the nematode gut. To date, the exact diversity and consistency of the native C. elegans microbiota across habitats and geographic locations is not fully understood. Here, we describe a suitable approach for isolating C. elegans from nature and characterizing the microbiota of worms. Nematodes can be easily isolated from compost material, rotting apples, slugs, or attracted by placing apples on compost heaps. The prime time for finding C. elegans in the Northern Hemisphere is from September until November. Worms can be washed out of collected substrate material by immersing the substrate in buffer solution, followed by the collection of nematodes and their transfer onto nematode growth medium or PCR buffer for subsequent analysis. We further illustrate how the samples can be used to isolate and purify the worm-associated microorganisms and to process worms for 16S ribosomal RNA analysis of microbiota community composition. Overall, the described methods may stimulate new research on the characterization of the C. elegans microbiota across habitats and geographic locations, thereby helping to obtain a comprehensive understanding of the diversity and stability of the nematode's microbiota as a basis for future functional research.

Isolation and Characterization of the Natural Microbiota of the Model Nematode Caenorhabditis elegans

Caenorhabditis elegans is one of the main model species in biology, yet almost all research is performed in the absence of its naturally associated microbes. The methods described here will help to improve our understanding of the diversity of associated microbes as a basis for future functional C. elegans research.

The nematode Caenorhabditis elegans interacts with a large diversity of microorganisms in nature. In general, C. elegans is commonly found in rotten plant ...

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their gut microbiomes. While experiments with well-characterized bacteria or defined bacterial communities can facilitate the analysis of molecular mechanisms, studying nematodes in their natural microbial context is essential for exploring the diversity of such mechanisms. At the same time, the isolation of worms from the wild is not always feasible, and, even when possible, sampling from the wild restricts the use of the genetic toolkit otherwise available for C. elegans research. The following protocol describes a method for microbiome studies utilizing compost microcosms for the in-lab&#160;growth in microbially diverse and natural-like environments.
Locally sourced soil can be enriched with produce to diversify the microbial communities in which worms are raised and from which they are harvested, washed, and surface-sterilized for subsequent analyses. Representative experiments demonstrate the ability to modulate the microbial community in a common soil by enriching it with different produce and further demonstrate that worms raised in these distinct environments assemble similar gut microbiomes distinct from their respective environments, supporting the notion of a species-specific core gut microbiome. Overall, compost microcosms provide natural-like in-lab environments for microbiome research as an alternative to synthetic microbial communities or to the isolation of wild nematodes.

Compost Microcosms as Microbially Diverse, Natural-like Environments for Microbiome Research in Caenorhabditis elegans

Compost microcosms bring the microbial diversity found in nature into the laboratory to facilitate microbiome research in Caenorhabditis elegans. Provided here are protocols for setting up microcosm experiments, with the experiments demonstrating the ability to modulate environmental microbial diversity to explore the relationships between environmental microbial diversity and worm gut microbiome composition.

The nematode Caenorhabditis elegans is emerging as a useful model for studying the molecular mechanisms underlying interactions between hosts and their ...

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

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