We thank Dr. Bing-Yan Wang, Dr. Gena Tribble (The University of Texas, School of Dentistry), Dr. Richard Lamont (University of Louisville, School of Dentistry), and Dr. Samuel Shelburne (MD Anderson Cancer Center) for providing laboratory and clinical strains of the mitis group streptococci. We also thank Dr. Keith Blackwell (Department of Genetics, Harvard Medical School) for the C. elegans strains. Finally, we thank Dr. Danielle Garsin and her lab (The University of Texas, McGovern Medical School) for providing reagents and worm strains to conduct the study. Some worm strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).

1. Preparation of THY (Todd-Hewitt Yeast Extract) Agar Plates
<ol>
	<li>For 1 L of media, add 30 g of Todd-Hewitt powder, 2 g of yeast extract and 20 g of agar to a 2 L Erlenmeyer flask. Add 970 mL of deionized water to the contents of the flask and include a stir bar. Autoclave the media at a temperature of 121 &#176;C and pressure of 15 lb/inch2 for 30 min. Thereafter, set the media on a stir plate and allow for cooling with gentle stirring.</li>
	<li>Pour the media into appropriately sized sterile Petri dishes (100 mm x 15 mm dishes for growth and maintenance of bacteria, 35 mm x 10 mm dishes for killing assays) under a laminar flow. Allow the media to dry for 2 h under the laminar hood. Thereafter, the plates can be stored at 4 &#176;C for 1 month.</li>
</ol>
2. Preparation of Nematode Growth Medium (NGM) and RNAi Feeding Plates (NGM RNAi)
<ol>
	<li>Using a stir bar, dissolve 2.5 g of peptone and 3 g of NaCl in 970 mL of deionized water in a 2 L Erlenmeyer flask. Add 20 g of agar to the media. Autoclave the media at a temperature of 121 &#176;C and pressure of 15 lb/inch2 for 30 min. Set the media on a stir plate and allow for cooling with gentle stirring.</li>
	<li>Add the following solutions to the media for preparation of NGM plates: 25 mL of 1 M potassium phosphate buffer (pH = 6.0), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of (5 mg/mL in 95% ethanol) cholesterol, 1 mL of (10% v/w in ethanol) nystatin, and 1 mL of 25 mg/mL streptomycin.</li>
	<li>Add the following solutions to the media for preparation of NGM RNAi feeding plates: 25 mL of 1 M potassium phosphate buffer (pH = 6.0), 1 mL of 1 M MgSO4, 1 mL of 1 M CaCl2, 1 mL of (5 mg/mL in 95% ethanol) cholesterol, 1 mL of (10% v/w in ethanol) nystatin, 1 mL of 50 mg/mL carbenicillin, and 1 mL of IM IPTG.</li>
	<li>Pour the media into 60 mm x 15 mm sterile Petri dishes under laminar flow. Allow the media to dry for 2 h under the laminar hood. Subsequently, plates can be stored at 4 &#176;C for 1 month.</li>
</ol>
3. Maintenance of C. elegans
<ol>
	<li>Seed the NGM plates by spotting 50 &#181;L of overnight grown E. coli OP50 in the center of the plates. The E. coli culture is prepared previously in Luria-Bertani (LB) media and stored at 4 &#176;C for several months. Cover the plates and allow them to dry for 24 h under a laminar hood and thereafter store the plates in polystyrene container.</li>
	<li>Under a dissecting microscope, pick up 10 to 12 gravid adults using a sterile worm pick and transfer the worms to an E. coli OP50 seeded NGM plate. Incubate the plates at 20 &#176;C overnight.</li>
	<li>The next day, remove the adults using a sterile worm pick and allow the embryos to develop to L4 larvae at 20 &#176;C (~2.5 days).</li>
</ol>
4. Preparation of Age Synchronous Population of Worms
<ol>
	<li>Wash gravid adults from two to four NGM plates using M9W and collect them in a 15 mL conical tube.
	<ol>
		<li>To prepare M9W: combine 3 g of NaCl, 6 g of Na2HPO4, and 3 g of KH2PO4 and dissolve in a final volume of 1 L of deionized water. Autoclave the solution and add 1 mL of 1 M MgSO4.</li>
	</ol>
	</li>
	<li>Spin the tube at 450 x g for 1 min, then decant the supernatant while ensuring that the worm pellet stays intact.</li>
	<li>Add 400 &#181;L of 8.25% sodium hypochlorite (household bleach) and 100 &#181;L of 5 N NaOH to prepare the worm lysis solution. Add the lysis solution to the worm pellet and mix the contents by flicking the tube until 70% of the adult worms are lysed. Periodically observe the contents of the tube under a dissecting microscope to ensure there is no overbleaching of the eggs.</li>
	<li>Dilute the bleach mix by adding 10 mL of M9W to the contents of the conical tube.</li>
	<li>Spin the tube at 450 x g for 1 min. Decant supernatant, then add 10 mL of M9W.</li>
	<li>Repeat step 4.5 two more times.</li>
	<li>Resuspend the resulting egg pellet in 3-5 mL of M9W. Place the tube on a tube rotator. Allow the tubes to rotate at a speed of 18 rpm at room temperature (RT) overnight.</li>
	<li>The next day, spin the tube at 450 x g for 1 min to pellet the L1 larvae. Remove most of M9W by aspiration, leaving behind ~250 &#181;L of liquid in the tube. Resuspend the L1 larvae and place three 5 &#181;L drops of the worm suspension onto a Petri dish lid, then estimate the number of worms per &#181;L using a dissecting microscope.</li>
</ol>
5. Induction of RNAi in Worms
<ol>
	<li>Using a sterile loop or pick, streak out the desired strains of RNAi-containing E. coli from frozen stocks (Ahringer and Vidal libraries) onto 100 mm x 15 mm LB agar plates containing 50 &#181;g/mL carbenicillin and 15 &#181;g/mL of tetracycline. Incubate the plates at 37 &#176;C for 24 h.</li>
	<li>Pick and inoculate an isolated colony from the desired RNAi strain into sterile 15 mL conical tubes containing 2 mL of LB supplemented with 50 &#181;g/mL carbenicillin. Incubate the tubes at 37 &#176;C for 16 h in an orbital shaker at 150 rpm.</li>
	<li>The next day, spread 150 &#181;L of overnight grown culture onto 65 mm x 15 mm NGM RNAi feeding plates using a sterile spreader. Incubate the plates at 37 &#176;C for 24 h.</li>
	<li>Allow the plates to cool to RT after incubation at 37 &#176;C. Add an appropriate volume of M9W containing ~200 L1 larvae (obtained from the age synchronous population of worms steps) to the E. coli seeded NGM RNAi feeding plates. Incubate the plates at 20 &#176;C until the larvae reach the L4 stage (~2.5 days).</li>
</ol>
6. Preparation of Mitis Group Streptococci for Infection
<ol>
	<li>Streak out desired strains of streptococci on the 100 mm x 15 mm THY agar (if plates were stored at 4 &#176;C, pre-warm the plates to 37 &#176;C before streaking the respective strains), then incubate the plates at 37 &#176;C overnight (~18 h) in a candle jar providing a microaerophilic environment for the growth of the streptococci (the streaked plates can be stored for a week at 4 &#176;C).</li>
	<li>To propagate clinical isolates of streptococci, use tryptic soy blood agar. Incubate the plates at 37 &#176;C in a candle jar overnight (~18 h).</li>
	<li>The next day, remove the plates from the candle jar and pick isolated colonies using a sterile loop. Inoculate 15 mL sterile conical tubes containing 2 mL of THY broth. To propagate clinical isolates of streptococci, supplement the THY broth with 5% v/v of sheep blood. Close the caps tight and incubate the tubes at 37 &#176;C under static conditions.</li>
</ol>
7. Survival Assays
NOTE: The steps involved in this assay are depicted in Figure 1. To demonstrate that the H2O2 derived by the mitis group is responsible for the killing of the worm, supplement the media with catalase, or the mutant strain &#916;spxB and complement strain &#916;spxB;spxB+ of S. gordonii can be used. SpxB encodes for a pyruvate oxidase, which is responsible for the production of H2O2 in the mitis group.
<ol>
	<li>Pre-warm 35 mm x 10 mm THY plates to 37 &#176;C. Add 80 &#181;L of overnight grown cultures of the desired strains of streptococci and spread the bacteria completely across the agar surface using a sterile spreader. Incubate the plates at 37 &#176;C in a candle jar overnight (~18 h). As a control, seed two 35 mm x 10 mm NGM plates with 80 &#181;L of overnight grown cultures of E. coli OP50. Incubate the plates at 37 &#176;C overnight.</li>
	<li>To confirm that the H2O2 produced by the mitis group is responsible for killing of the worms, add 50 &#181;L containing 1,000 units of catalase c onto the THY plate. Spread the catalase solution using a sterile spreader and allow the plates to dry in the laminar flow for 30 min. Seed the plates thereafter with the respective streptococcus strains as described in step 7.1.</li>
	<li>The next day, remove the plates from the candle jar and allow the plates to cool to RT for 10-15 min. Using a sterile worm pick, transfer 30 L4 larvae from the NGM or NGM RNAi feeding plates to the streptococcus seeded THY plates. Use two seeded THY plates with a total of 60 worms per strain of streptococcus. Incubate the plates at 25 &#176;C.</li>
	<li>Using a dissecting microscope, count the number of live and dead L4 larvae on each plate at several timepoints. Initially, score the worms as dead or live every 30 min. Thereafter, when worms rapidly start to die, score them at 15 min intervals. Use the sterile worm pick to gently prod the worms and determine if they are dead or alive. A worm is considered dead if there is no movement in response to the prodding.</li>
	<li>The assay will take 5-6 h to complete. Repeat the experiment two more times. After completion of the assay, pool the data from the two plates. Input the data of each group, compare the survival curves, and perform Kaplan-Meier survival analysis using statistical software.</li>
</ol>
8. Preparation of Agarose Pads for Microscopy
<ol>
	<li>Dissolve 2% w/v of agarose in deionized water by heating the solution in a microwave. A volume of 5 mL of solution is adequate to prepare 20 slides.</li>
	<li>Stick lab tape lengthwise along two glass slides. This will determine the thickness of the agarose pads. Place a clean glass slide between the two taped slides.</li>
	<li>Place 100 &#181;L of molten agarose on the center of the clean slide. Immediately place another clean glass on top of the molten agarose and gently press down to make a pad. Allow the agarose to solidify and subsequently remove the top slide. The agarose pad is ready for use.</li>
</ol>
9. Observation of SKN-1 Localization in Response to Streptococcus Infection
NOTE: The steps involved in this assay are depicted in Figure 2. Localization of SKN-1 was determined using the SKN-1B/C::GFP transgenic worm strain. To demonstrate localization of SKN-1 due to the production of H2O2 by the mitis group, wild-type (WT), &#916;spxB, and the complement strain&#160;&#916;spxB;spxB+ of S. gordonii were used. Furthermore, the transgenic reporter strain SKN-1B/C::GFP and RNAi interference technique were used to demonstrate that components of the p38 MAPK pathway regulate the localization of SKN-1.
<ol>
	<li>Pre-warm 35 mm x 10 mm THY plates to 37 &#176;C. Add 80 &#181;L of overnight grown cultures of the desired strains of streptococcus and spread the bacteria completely across the agar surface using a sterile spreader. Incubate the plates at 37 &#176;C in a candle jar overnight (~18 h). As a control, seed three 35 mm x 10 mm NGM plates with 80 &#181;L of overnight grown cultures of E. coli OP50. Incubate these plates at 37 &#176;C for ~18 h.</li>
	<li>The next day, remove plates from the candle jar and allow the plates to cool to RT for 10-15 min. Wash L4 larvae using M9W from NGM and NGM RNAi feeding plates. Collect the worms in 15 mL conical tubes.</li>
	<li>Spin the tubes at 450 x g for 1 min. Decant the supernatant and add 10 mL of M9W.</li>
	<li>Repeat step 9.3 three more times.</li>
	<li>Resuspend the worms in ~250 &#181;L of M9W and place three 5 &#181;L drops of the worm suspension onto a clean Petri dish lid and estimate the number of worms per &#181;L using a dissecting microscope.</li>
	<li>Add ~100 L4 larvae to each THY streptococcus seeded and NGM E. coli seeded plates. Use three plates per strain of bacteria. Incubate the plates for 2 to 3 h at 25 &#176;C.</li>
	<li>Thereafter, remove the plates from the incubator, wash them with M9W, and collect the worms in 15 mL conical tubes.</li>
	<li>Wash the worms 3x as described in steps 9.3.</li>
	<li>Remove most of the M9W by aspiration and add 500 &#181;L of M9W containing 2 mM sodium azide or 2 mM tetramisole hydrochloride to the worm pellet. This will anesthetize the worms, ensuring that no movement occurs when imaged under the microscope. 
	CAUTION: Use personal protective equipment (PPE) when handling sodium azide. Prepare the azide solution under a chemical hood.</li>
	<li>Incubate the worm pellets at RT for 15 min. Then, spot 15 &#181;L of the worm suspension onto a prepared agarose pad. Gently place a no. 1.5 coverslip over the agarose pad containing the anesthetized worms.</li>
	<li>Using a fluorescent microscope, visualize the localization of SKN-1 utilizing FITC and DAPI filters.Image worms at 10x and 20x magnifications.</li>
	<li>Score the worms based on the level of localization of SKN-1. No nuclear localization, localization of SKN-1B/C::GFP in the anterior or posterior of the worm, and nuclear localization of SKN-1B/C::GFP in all intestinal cells are categorized as low, medium, and high levels of localization, respectively.</li>
	<li>After scoring the fluorescent micrographs, determine the statistical differences by chi- squared and Fisher&#39;s exact tests using statistical software.</li>
</ol>

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The authors declare that they have no competing financial interests.

The methods described can be used for other pathogenic bacteria such as Enterococcus faecium, which also produces H2O2 grown under anaerobic or microaerophilic conditions26. Typically, for most pathogenic organisms, it takes several days to weeks to complete the survival assays. However, due to the robust production of H2O2 by members of the mitis group, these assays could be completed within 5-6 h under the conditions described. This ensures th...

Mitis group streptococci are human commensals of the oropharyngeal cavity1. However, these organisms can escape this niche and cause a variety of invasive diseases2. The infections caused by these microorganisms include bacteremia, endocarditis, and orbital cellulitis2,3,4,5,6. Furthermore, they are emerging as causative agents of bloodstream infections in immunocompromised, neutropenic, and cancer patients that have undergone chemotherapy5,7,8,9.
The mechanisms underlying mitis group pathogenesis is obscure, because few virulence factors have been identified. The mitis group is known to produce H2O2, which has shown to play an important role in oral microbial communities10. More recently, several studies have highlighted a role for H2O2 as a cytotoxin that induces epithelial cell death11,12. S. pneumonia, which belongs to this group, has been shown to produce high levels of H2O2 that induces DNA damage and apoptosis in alveolar cells13. Using an acute pneumonia animal model, the same researchers demonstrated that production of H2O2 by the bacteria confers a virulence advantage. Studies on pneumococcal meningitis have also shown that pathogen-derived H2O2 acts synergistically with pneumolysin to trigger neuronal cell death14. These observations clearly establish that H2O2 produced by this group of bacteria is important for their pathogenicity.
Interestingly, it has also been shown that members of the mitis group S. mitis and S. oralis cause death of the nematode C. elegans via the production of H2O215,16. This free-living nematode has been used as a simple, genetically tractable model to study many biological processes. More recently, the worm has emerged as a model to study host-pathogen interactions17,18. In addition, several studies have highlighted the importance of studying oxidative stress using this organism19,20,21. Its short life cycle, ability to knockdown genes of interest by RNAi, and use of green fluorescent protein (GFP)-fused reporters to monitor gene expression are some of the attributes that make it an attractive model system. More importantly, the pathways that regulate oxidative stress and innate immunity in the worm are highly conserved with mammals20,22.
In this protocol, it is demonstrated how to use C. elegans to elucidate the pathogenicity caused by streptococcal-derived H2O2. A modified survival assay is shown, and members of the mitis group are able to kill the worms rapidly via the production of H2O2. Using members of the mitis group, a sustained biological source of reactive oxygen species (ROS) is provided, as opposed to chemical sources that induce oxidative stress in the worms. Furthermore, the bacteria are able to colonize the worms rapidly, which allows for H2O2 to be directly targeted to the intestinal cells (compared to other sources that have to cross several barriers). The assay is validated either 1) by determining the survival of the skn-1 mutant strain or 2) by knocking down skn-1 using RNAi in worms relative to the N2 wild-type and vector control treated worms. SKN-1 is an important transcription factor that regulates the oxidative stress response in C. elegans23,24,25. In addition to survival assays, a worm strain expressing a SKN-1B/C::GFP transgenic reporter is used to monitor activation of the oxidative stress response via the production of H2O2 by the mitis group.

<table><tbody><tr><td>&lt;strong&gt;Media and chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Agarose&amp;nbsp;</td><td>Sigma Aldrich</td><td>A9539-50G</td><td></td></tr><tr><td>Bacto peptone&amp;nbsp;</td><td>Fisher Scientific</td><td>DF0118-17-0</td><td></td></tr><tr><td>BD Bacto Todd Hewitt Broth</td><td>Fisher Scientific</td><td>DF0492-17-6</td><td></td></tr><tr><td>BD BBL Sheep Blood, Defibrinated&amp;nbsp;&amp;nbsp;</td><td>Fisher Scientific</td><td>B11947</td><td></td></tr><tr><td>BD Difco Agar&amp;nbsp;</td><td>Fisher Scientific</td><td>DF0145-17-0</td><td></td></tr><tr><td>BD Difco LB Broth</td><td>Fisher Scientific</td><td>DF0446-17-3</td><td></td></tr><tr><td>Blood agar (TSA with Sheep Blood)</td><td>Fisher Scientific</td><td>R01200</td><td></td></tr><tr><td>Calcium Chloride</td><td>Fisher Scientific</td><td>BP510-500</td><td></td></tr><tr><td>Carbenicillin</td><td>Fisher Scientific</td><td>BP26481</td><td></td></tr><tr><td>Catalase&amp;nbsp;</td><td>Sigma Aldrich</td><td>C1345-1G</td><td></td></tr><tr><td>Cholesterol</td><td>Fisher Scientific</td><td>ICN10138201</td><td></td></tr><tr><td>IPTG</td><td>Fisher Scientific</td><td>MP21021012</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Fisher Scientific</td><td>BP213-1</td><td></td></tr><tr><td>Nystatin</td><td>Acros organics</td><td>AC455500050</td><td></td></tr><tr><td>Potassium Phosphate Dibasic</td><td>Fisher Scientific</td><td>BP363-500</td><td></td></tr><tr><td>Potassium phosphate monobasic</td><td>Fisher Scientific</td><td>BP362-500</td><td></td></tr><tr><td>Sodium Azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Fisher Scientific</td><td>BP358-1</td><td></td></tr><tr><td>Sodium Hydroxide</td><td>Fisher Scientific</td><td>SS266-1</td><td></td></tr><tr><td>8.25% Sodium Hypochlorite</td><td></td><td></td><td></td></tr><tr><td>Sodium Phosphate Dibasic&amp;nbsp;</td><td>Fisher Scientific</td><td>BP332-500</td><td></td></tr><tr><td>Streptomycin Sulfate&amp;nbsp;</td><td>Fisher Scientific</td><td>BP910-50</td><td></td></tr><tr><td>Tetracyclin</td><td>Sigma Aldrich</td><td>87128-25G</td><td></td></tr><tr><td>(&amp;minus;)-Tetramisole hydrochloride</td><td>Sigma Aldrich</td><td>L9756</td><td></td></tr><tr><td>Yeast extract</td><td>Fisher Scientific</td><td>BP1422-500&amp;nbsp;</td><td></td></tr><tr><td>&lt;strong&gt;Consumables&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>15 mL Conical Sterile Polypropylene Centrifuge Tubes&amp;nbsp;</td><td>Fisher Scientific</td><td>12-565-269</td><td></td></tr><tr><td>Disposable Polystyrene Serological Pipettes 10 mL</td><td>Fisher Scientific</td><td>07-200-574</td><td></td></tr><tr><td>Disposable Polystyrene Serological Pipettes 25 mL</td><td>Fisher Scientific</td><td>07-200-575</td><td></td></tr><tr><td>Falcon Bacteriological Petri Dishes with Lid (35 mm x 10 mm)</td><td>Fisher Scientific</td><td>08-757-100A</td><td></td></tr><tr><td>No. 1.5&amp;nbsp; 18 mm x 18 mm Cover Slips</td><td>Fisher Scientific</td><td>12-541A</td><td></td></tr><tr><td>Petri Dish with Clear Lid (60 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875713A</td><td></td></tr><tr><td>Petri Dishes with Clear Lid (100 mm x 15 mm)</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Plain Glass Microscope Slides (75 mm x 25 mm)</td><td>Fisher Scientific</td><td>12-544-4</td><td></td></tr><tr><td>&lt;strong&gt;Software&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Prism</td><td>Graphpad</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Bacterial Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;S. oralis&lt;/em&gt; ATCC 35037</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;S. mitis&lt;/em&gt; ATCC 49456</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;S. gordonii&lt;/em&gt; DL1 Challis&amp;nbsp;&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; OP50</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; HT115</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Worm Strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Strain&lt;/strong&gt;</td><td>&lt;strong&gt;Genotype&lt;/strong&gt;</td><td>&lt;strong&gt;Transgene&lt;/strong&gt;</td><td>&lt;strong&gt;Source&lt;/strong&gt;</td></tr><tr><td>N2</td><td>C. elegans wild isolate</td><td></td><td>CGC</td></tr><tr><td>EU1</td><td>skn-1(zu67) IV/nT1 [unc-?(n754) let-?] (IV;V)</td><td></td><td>CGC</td></tr><tr><td>LD002</td><td>IdIs1</td><td>SKN-1B/C::GFP + rol-6(su1006)</td><td>Keith Blackwell</td></tr></tbody></table>

studying oxidative stress caused by the mitis group streptococci in caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented protocol uses this model to determine the pathogenicity caused by the mitis group streptococci via the production of H2O2. The mitis group streptococci are an emerging threat that cause many human diseases such as bacteremia, endocarditis, and orbital cellulitis. Described here is a protocol to determine the survival of these worms in response to H2O2 produced by this group of pathogens. Using the gene skn-1 encoding for an oxidative stress response transcription factor, it is shown that this model is important for identifying host genes that are essential against streptococcal infection. Furthermore, it is shown that activation of the oxidative stress response can be monitored in the presence of these pathogens using a transgenic reporter worm strain, in which SKN-1 is fused to green fluorescent protein (GFP). These assays provide the opportunity to study the oxidative stress response to H2O2 derived by a biological source as opposed to exogenously added reactive oxygen species (ROS) sources.

Members of the mitis group S. mitis, S. oralis, and S. gordonii rapidly killed the worms, as opposed to S. mutans, S. salivarius, and non-pathogenic E. coli OP50 (Figure 3A). The median survival for S. mitis, S. oralis, and S. gordonii was 300 min, 300 min, and 345 min, respectively. To determine if the killing was mediated by H2O2, catalase...

Watch this Scientific Journal Video about Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans at JoVE.com

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

The nematode Caenorhabditis elegans is an excellent model to dissect host-pathogen interactions. Described here is a protocol to infect the worm with members of the mitis group streptococci and determine activation of the oxidative stress response against H2O2 produced by this group of organisms.

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

Caenorhabditis elegans (C. elegans), a free-living nematode, has emerged as an attractive model to study host-pathogen interactions. The presented ...

studying-oxidative-stress-caused-mitis-group-streptococci

Research

JoVE Journal

Developmental Biology

7.1K Views.  University of Texas Health Science Center. In dit protocol laten we zien hoe je een vrije leefmethode c. elegans gebruiken om de pathogeniteit te verduidelijken die wordt veroorzaakt door waterstofperoxide geproduceerd via Streptococci van de Mitis Group. Het belangrijkste voordeel van deze techniek is dat we pathogeniteit en de activering van stressreacties in de worm kunnen bestuderen met behulp van een duurzame biologische bron van reactieve zuurstofsoorten in tegenstelling tot chemische bronnen Voor een liter media voeg 30 g Todd ...

Video: Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

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.

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.

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

Genetics

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in cellular and mammalian models. Here, we describe detailed methods to perform stable isotopic profiling of intermediary metabolism and metabolic flux in the nematode, Caenorhabditis elegans. Methods are described for profiling whole worm free amino acids, labeled carbon dioxide, labeled organic acids, and labeled amino acids in animals exposed to stable isotopes either from early development on nematode growth media agar plates or beginning as young adults while exposed to various pharmacologic treatments in liquid culture. Free amino acids are quantified by high performance liquid chromatography (HPLC) in whole worm aliquots extracted in 4% perchloric acid. Universally labeled 13C-glucose or 1,6-13C2-glucose is utilized as the stable isotopic precursor whose labeled carbon is traced by mass spectrometry in carbon dioxide (both atmospheric and dissolved) as well as in metabolites indicative of flux through glycolysis, pyruvate metabolism, and the tricarboxylic acid cycle. Representative results are included to demonstrate effects of isotope exposure time, various bacterial clearing protocols, and alternative worm disruption methods in wild-type nematodes, as well as the relative extent of isotopic incorporation in mitochondrial complex III mutant worms (isp-1(qm150)) relative to wild-type worms. Application of stable isotopic profiling in living nematodes provides a novel capacity to investigate at the whole animal level real-time metabolic alterations that are caused by individual genetic disorders and/or pharmacologic therapies.

Stable Isotopic Profiling of Intermediary Metabolic Flux in Developing and Adult Stage Caenorhabditis elegans

Stable isotopic profiling by gas chromatography mass spectrometric analysis of intermediary metabolic flux is described in the nematode, Caenorhabditis elegans. Methods are detailed for assessing isotopic enrichment in carbon dioxide, organic acids, and amino acids following isotope exposure either during development on agar plates or during adulthood in liquid culture.

Stable isotopic profiling has long permitted sensitive investigations of the metabolic consequences of genetic mutations and/or pharmacologic therapies in ...

Biology

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed under anoxia for 20 hr&#160;(simulated ischemia), and subsequently moved to a normal atmosphere with food (simulated reperfusion). This experimental paradigm results in increased death and neuronal damage, and techniques are presented to assess organism viability, alterations to the morphology of touch neuron processes, as well as touch sensitivity, which represents the behavioral output of neuronal function. Finally, a method for constructing hypoxic incubators using common kitchen storage containers is described. The addition of a mass flow control unit allows for alterations to be made to the gas mixture in the custom incubators, and a circulating water bath allows for both temperature control and makes it easy to identify leaks. This method provides a low cost alternative to commercially available units.

An Anoxia-starvation Model for Ischemia/Reperfusion in C. elegans

A protocol is described that uses anoxia/starvation in C. elegans to model ischemia/reperfusion. Functional outcomes include increased mortality, visible abnormalities in GFP-labeled neuronal processes, and impaired behavioral responses that require neuronal function.

Protocols for anoxia/starvation in the genetic model organism C. elegans simulate ischemia/reperfusion. Worms are separated from bacterial food and placed ...

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for the conversion and replication of their benign cellular isoform. Accumulating evidence suggests that many protein aggregates can act as self-propagating templates and corrupt the folding of cognate proteins. Although aggregates can be functional under certain circumstances, this process often leads to the disruption of the cellular protein homeostasis (proteostasis), eventually leading to devastating diseases such as Alzheimer&#8217;s disease (AD), Parkinson&#8217;s disease (PD), Amyotrophic lateral sclerosis (ALS), or transmissible spongiform encephalopathies (TSEs). The exact mechanisms of prion propagation and cell-to-cell spreading of protein aggregates are still subjects of intense investigation. To further this knowledge, recently a new metazoan model in Caenorhabditis elegans, for expression of the prion domain of the cytosolic yeast prion protein Sup35 has been established. This prion model offers several advantages, as it allows direct monitoring of the fluorescently tagged prion domain in living animals and ease of genetic approaches. Described here are methods to study prion-like behavior of protein aggregates and to identify modifiers of prion-induced toxicity using C. elegans.

Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

Prion-like propagation of protein aggregates has recently emerged as being implicated in many neurodegenerative diseases. The goal of this protocol is to describe, how to use the nematode C. elegans as a model system to monitor protein spreading and to investigate prion-like phenomena.

Prions are unconventional self-propagating proteinaceous particles, devoid of any coding nucleic acid. These proteinaceous seeds serve as templates for ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and experimentally tractable. It is economical and easy to culture and dispense in liquid medium which makes it well suited for medium-throughput screening. We have previously validated the use of transgenic luciferase expressing C. elegans strains to provide rapid in vivo assessment of the nematode&#8217;s ATP levels.1-3 Here we present the required materials and procedure to carry out bioassays with the bioluminescent C. elegans strains PE254 or PE255 (or any of their derivative strains). The protocol allows for in vivo detection of sublethal effects of drugs that may identify mitochondrial toxicity, as well as for in vivo detection of potential beneficial drug effects. Representative results are provided for the chemicals paraquat, rotenone, oxaloacetate and for four firefly luciferase inhibitory compounds. The methodology can be scaled up to provide a platform for screening drug libraries for compounds capable of modulating mitochondrial function. Pre-clinical evaluation of drug toxicity is often carried out on immortalized cancerous human cell lines which derive ATP mostly from glycolysis and are often tolerant of mitochondrial toxicants.4,5 In contrast, C. elegans depends on oxidative phosphorylation to sustain development into adulthood, drawing a parallel with humans and providing a unique opportunity for compound evaluation in the physiological context of a whole live multicellular organism.

A Screenable In Vivo Assay for Mitochondrial Modulators Using Transgenic Bioluminescent Caenorhabditis elegans

A protocol is described for in vivo detection of effects of mitochondrial inhibitors in the model organism Caenorhabditis elegans and for identification of potential enhancing compounds. This protocol can be used to screen drug libraries for compounds modulating mitochondrial function.

The multicellular model organism Caenorhabditis elegans is a small nematode of approximately 1 mm in size in adulthood that is genetically and ...

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous system and muscle. Consistent with this, mitochondrial dysfunction has been associated with a myriad of neurodegenerative diseases and aging in general. Caenorhabditis elegans have been a powerful model system for elucidating the many intricacies of mitochondrial function. Mitochondrial respiration is a strong indicator of mitochondrial function and recently developed respirometers offer a state-of-the-art platform to measure respiration in cells. In this protocol, we provide a technique to analyze live, intact C. elegans. This protocol spans a period of ~7 days and includes steps for (1) growing and synchronization of C. elegans, (2) preparation&#160;of compounds to be injected and hydration of probes, (3) drug loading and cartridge equilibration, (4) preparation of worm assay plate and assay run, and (5) post-experiment data analysis.

Measurement of Oxygen Consumption Rates in Intact Caenorhabditis elegans

Mitochondrial respiration is critical for organismal survival; therefore, oxygen consumption rate is an excellent indicator of mitochondrial health. In this protocol, we describe the use of a commercially available respirometer to measure basal and maximal oxygen consumption rates in live, intact, and freely-motile Caenorhabditis elegans.

Optimal mitochondrial function is critical for healthy cellular activity, particularly in cells that have high energy demands like those in the nervous ...

Measurements of Physiological Stress Responses in C. Elegans

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and physiology. Thus, organisms have evolved targeted and specific stress responses dedicated to repair damage and maintain homeostasis. These mechanisms include the unfolded protein response of the endoplasmic reticulum (UPRER), the unfolded protein response of the mitochondria (UPRMT), the heat shock response (HSR), and the oxidative stress response (OxSR). The protocols presented here describe methods to detect and characterize the activation of these pathways and their physiological consequences in the nematode, C. elegans. First, the use of pathway-specific fluorescent transcriptional reporters is described for rapid cellular characterization, drug screening, or large-scale genetic screening (e.g., RNAi or mutant libraries). In addition, complementary, robust physiological assays are described, which can be used to directly assess sensitivity of animals to specific stressors, serving as functional validation of the transcriptional reporters. Together, these methods allow for rapid characterization of the cellular and physiological effects of internal and external proteotoxic perturbations.

Measurements of Physiological Stress Responses in C. Elegans

Here, we characterize cellular proteotoxic stress responses in the nematode C. elegans by measuring the activation of fluorescent transcriptional reporters and assaying sensitivity to physiological stress.

Organisms are often exposed to fluctuating environments and changes in intracellular homeostasis, which can have detrimental effects on their proteome and ...

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a growing number of age-associated diseases. For instance, proteins with polyglutamine expansions are prone to aggregation, as exemplified with the huntingtin protein and concomitant onset of Huntington&#8217;s disease. The age-associated deterioration of the proteome has been widely studied through the use of transgenic Caenorhabditis elegans expressing polyQ repeats fused to a yellow fluorescent protein (YFP). This polyQ::YFP transgenic animal model facilitates the direct quantification of the age-associated decline of the proteome through imaging the progressive formation of fluorescent foci (i.e., protein aggregates) and subsequent onset of locomotion defects that develop as a result of the collapse of the proteome. Further, the expression of the polyQ::YFP transgene can be driven by tissue-specific promoters, allowing the assessment of proteostasis across tissues in the context of an intact multicellular organism. This model is highly amenable to genetic analysis, thus providing an approach to quantify aging that is complementary to lifespan assays. We describe how to accurately measure polyQ::YFP foci formation within either neurons or body wall muscle during aging, and the subsequent onset of behavioral defects. Next, we highlight how these approaches can be adapted for higher throughput, and potential future applications using other emerging strategies for C. elegans genetic analysis.

Quantifying Tissue-Specific Proteostatic Decline in Caenorhabditis elegans

Proteostatic decline is a hallmark of aging, facilitating the onset of neurodegenerative diseases. We outline a protocol to quantifiably measure proteostasis in two different Caenorhabditis elegans tissues through heterologous expression of polyglutamine repeats fused to a fluorescent reporter. This model allows rapid in vivo genetic analysis of proteostasis.

The ability to maintain proper function and folding of the proteome (protein homeostasis) declines during normal aging, facilitating the onset of a ...

Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans

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