Name: studying oxidative stress caused by the mitis group streptococci in caenorhabditis elegans
Uploaded: 2019-03-23T15:00:00
Description: Watch this Scientific Journal Video about Studying Oxidative Stress Caused by the Mitis Group Streptococci in Caenorhabditis elegans at JoVE.com

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 d...

<ol>
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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.

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			<td height="20" style="height:20px;width:381px;">Media and chemicals</td>
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			<td height="20" style="height:20px;width:381px;">Agarose&#160;</td>
			<td style="width:179px;">Sigma Aldrich</td>
			<td style="width:119px;">A9539-50G</td>
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			<td height="20" style="height:20px;width:381px;">Bacto peptone&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">DF0118-17-0</td>
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			<td height="20" style="height:20px;width:381px;">BD Bacto Todd Hewitt Broth</td>
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			<td style="width:119px;">DF0492-17-6</td>
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			<td height="20" style="height:20px;width:381px;">8.25% Sodium Hypochlorite</td>
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			<td style="width:179px;">Fisher Scientific</td>
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			<td height="20" style="height:20px;width:381px;">15mL Conical Sterile Polypropylene Centrifuge Tubes&#160;</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">12-565-269</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Disposable Polystyrene Serological Pipettes 10mL</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-574</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Disposable Polystyrene Serological Pipettes 25mL</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-575</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Falcon Bacteriological Petri Dishes with Lid (35 x 10 mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">08-757-100A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">No. 1.5&#160; 18 mm X 18 mm Cover Slips</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">12-541A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Petri Dish with Clear Lid (60 x 15 mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875713A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Petri Dishes with Clear Lid (100X15mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">FB0875712</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Plain Glass Microscope Slides (75 x 25 mm)</td>
			<td style="width:179px;">Fisher Scientific</td>
			<td style="width:119px;">12-544-4</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Software&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Prism</td>
			<td style="width:179px;">Graphpad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Bacterial Strains</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. oralis ATCC 35037</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. mitis ATCC 49456</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">S. gordonii DL1 Challis&#160;&#160;</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">E. coli OP50</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">E. coli HT115</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Worm Strains</td>
			<td style="width:179px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">Strain</td>
			<td style="width:179px;">Genotype</td>
			<td style="width:119px;">Transgene</td>
			<td>Source</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">N2</td>
			<td style="width:179px;">C. elegans wild isolate</td>
			<td style="width:119px;"></td>
			<td>CGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">EU1</td>
			<td style="width:179px;">skn-1(zu67) IV/nT1 [unc-?(n754) let-?] (IV;V)</td>
			<td style="width:119px;"></td>
			<td>CGC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:381px;">LD002</td>
			<td style="width:179px;">IdIs1</td>
			<td style="width:119px;">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 ...

Research

JoVE Journal

Developmental Biology

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