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&#39;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.

Watch this Scientific Journal Video about The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions at JoVE.com

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 ...

the-nematode-caenorhabditis-elegans-versatile-vivo-model-to-study

Nanyang Technological University

Research

JoVE Journal

Genetics

9.3K Views.  Worcester Polytechnic Institute. Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

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

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the replacement of the entire gene with a selectable marker via homologous recombination. During homologous recombination, exchange of DNA takes place between sequences with high similarity. Therefore, linear genomic sequences flanking a target gene can be used to specifically direct a selectable marker to the desired integration site. Blunt ends of the deletion construct activate the cell&#39;s DNA repair systems and thereby promote integration of the construct either via homologous recombination or by non-homologous-end-joining. In organisms with efficient homologous recombination, the rate of successful gene deletion can reach more than 50% making this strategy a valuable gene disruption system. The smut fungus Ustilago maydis is a eukaryotic model microorganism showing such efficient homologous recombination. Out of its about 6,900 genes, many have been functionally characterized with the help of deletion mutants, and repeated failure of gene replacement attempts points at essential function of the gene. Subsequent characterization of the gene function by tagging with fluorescent markers or mutations of predicted domains also relies on DNA exchange via homologous recombination. Here, we present the U. maydis strain generation strategy in detail using the simplest example, the gene deletion.

Genetic Manipulation of the Plant Pathogen Ustilago maydis to Study Fungal Biology and Plant Microbe Interactions

We describe a robust gene replacement strategy to genetically manipulate the smut fungus Ustilago maydis. This protocol explains how to generate deletion mutants to investigate infection phenotypes. It can be extended to modify genes in any desired way, e.g., by adding a sequence encoding a fluorescent protein tag.

Gene deletion plays an important role in the analysis of gene function. One of the most efficient methods to disrupt genes in a targeted manner is the ...

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation became a valuable experimental tool to investigate the DNA damage response (DDR) in vivo. It allows real-time high-resolution single-cell analysis of macromolecular dynamics in response to laser-induced damage confined to a submicrometer region in the cell nucleus. However, various laser conditions have been used without appreciation of differences in the types of damage induced. As a result, the nature of the damage is often not well characterized or controlled, causing apparent inconsistencies in the recruitment or modification profiles. We demonstrated that different irradiation conditions (i.e., different wavelengths as well as different input powers (irradiances) of a femtosecond (fs) near-infrared (NIR) laser) induced distinct DDR and repair protein assemblies. This reflects the type of DNA damage produced. This protocol describes how titration of laser input power allows induction of different amounts and complexities of DNA damage, which can easily be monitored by detection of base and crosslinking damages, differential poly (ADP-ribose) (PAR) signaling, and pathway-specific repair factor assemblies at damage sites. Once the damage conditions are determined, it is possible to investigate the effects of different damage complexity and differential damage signaling as well as depletion of upstream factor(s) on any factor of interest.

Laser Microirradiation to Study In Vivo Cellular Responses to Simple and Complex DNA Damage

The goal of this protocol is to describe how to use laser microirradiation to induce different types of DNA damage, including relatively simple strand breaks and complex damage, to study DNA damage signaling and repair factor assembly at damage sites in vivo.

DNA damage induces specific signaling and repair responses in the cell, which is critical for protection of genome integrity. Laser microirradiation ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus allowing a single investigator to screen more treatments or conditions over the same amount of time without loss of data quality. The method requires common equipment found in most laboratories working with C. elegans and is thus simple to adopt. The approach centers on assaying independent samples of a population at each observation point, rather than a single sample over time as with traditional longitudinal methods. Scoring entails adding liquid to the wells of a multi-well plate, which stimulates C. elegans to move and facilitates quantifying changes in healthspan. Other major benefits of the Replica Set method include reduced exposure of agar surfaces to airborne contaminants (e.g. mold or fungus), minimal handling of animals, and robustness to sporadic mis-scoring (such as calling an animal as dead when it is still alive). To appropriately analyze and visualize the data from a Replica Set style experiment, a custom software tool was also developed. Current capabilities of the software include plotting of survival curves for both Replica Set and traditional (Kaplan-Meier) experiments, as well as statistical analysis for Replica Set. The protocols provided here describe the traditional experimental approach and the Replica Set method, as well as an overview of the corresponding data analysis.

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

Here we describe the Replica Set method, an approach to quantitatively measure C. elegans lifespan/survival and healthspan in a high-throughput and robust manner, thus allowing screening of many conditions without sacrificing data quality. This protocol details the strategy and provides a software tool for analysis of Replica Set data.

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human genetics research. Although a number of in silico algorithms have been developed to predict the pathogenicity of variants identified in these datasets, functional studies are critical to determining how specific genomic variants affect protein function, especially for missense variants. In the Undiagnosed Diseases Network (UDN) and other rare disease research consortia, model organisms (MO) including Drosophila, C. elegans, zebrafish, and mice are actively used to assess the function of putative human disease-causing variants. This protocol describes a method for the functional assessment of rare human variants used in the Model Organisms Screening Center Drosophila Core of the UDN. The workflow begins with gathering human and MO information from multiple public databases, using the MARRVEL web resource to assess whether the variant is likely to contribute to a patient&#39;s condition as well as design effective experiments based on available knowledge and resources. Next, genetic tools (e.g., T2A-GAL4 and UAS-human cDNA lines) are generated to assess the functions of variants of interest in Drosophila. Upon development of these reagents, two-pronged functional assays based on rescue and overexpression experiments can be performed to assess variant function. In the rescue branch, the endogenous fly genes are &#34;humanized&#34;&#160;by replacing the orthologous Drosophila gene with reference or variant human transgenes. In the overexpression branch, the reference and variant human proteins are exogenously driven in a variety of tissues. In both cases, any scorable phenotype (e.g., lethality, eye morphology, electrophysiology) can be used as a read-out, irrespective of the disease of interest. Differences observed between reference and variant alleles suggest a variant-specific effect, and thus likely pathogenicity. This protocol allows rapid, in vivo assessments of putative human disease-causing variants of genes with known and unknown functions.

In Vivo Functional Study of Disease-associated Rare Human Variants Using Drosophila

The goal of this protocol is to outline the design and performance of in vivo experiments in Drosophila melanogaster to assess the functional consequences of rare gene variants associated with human diseases.

Advances in sequencing technology have made whole-genome and whole-exome datasets more accessible for both clinical diagnosis and cutting-edge human ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The large body of work on C. elegans makes it an ideal candidate to integrate into large-population, whole-animal studies to dissect the complex biological components and their relationships with another organism. In order to use C. elegans in collaborative -omics research, a method is needed to generate large populations of animals where a single sample can be split and assayed across diverse platforms for comparative analyses.
Here, a method to culture and collect an abundant mixed-stage C. elegans population on a large-scale culture plate (LSCP) and subsequent phenotypic data is presented. This pipeline yields sufficient numbers of animals to collect phenotypic and population data, along with any data needed for -omics experiments (i.e., genomics, transcriptomics, proteomics, and metabolomics). In addition, the LSCP method requires minimal manipulation to the animals themselves, less user preparation time, provides tight environmental control, and ensures that handling of each sample is consistent throughout the study for overall reproducibility. Lastly, methods to document population size and population distribution of C. elegans life stages in a given LSCP are presented.

Culture and Assay of Large-Scale Mixed-Stage Caenorhabditis elegans Populations

To use Caenorhabditis elegans (C. elegans) in omics research, a method is needed to generate large populations of worms where a single sample can be measured across platforms for comparative analyses. Here, a method to culture C. elegans populations on large-scale culture plates (LSCPs) and to document population growth is presented.

Caenorhabditis elegans (C. elegans) has been and remains a valuable model organism to study developmental biology, aging, neurobiology, and genetics. The ...