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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.
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.
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
1. Preparation of THY (Todd-Hewitt Yeast Extract) Agar Plates
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.......
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.......
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....
Name | Company | Catalog Number | Comments |
Media and chemicals | |||
Agarose | Sigma Aldrich | A9539-50G | |
Bacto peptone | Fisher Scientific | DF0118-17-0 | |
BD Bacto Todd Hewitt Broth | Fisher Scientific | DF0492-17-6 | |
BD BBL Sheep Blood, Defibrinated  | Fisher Scientific | B11947 | |
BD Difco Agar | Fisher Scientific | DF0145-17-0 | |
BD Difco LB Broth | Fisher Scientific | DF0446-17-3 | |
Blood agar (TSA with Sheep Blood) | Fisher Scientific | R01200 | |
Calcium Chloride | Fisher Scientific | BP510-500 | |
Carbenicillin | Fisher Scientific | BP26481 | |
Catalase | Sigma Aldrich | C1345-1G | |
Cholesterol | Fisher Scientific | ICN10138201 | |
IPTG | Fisher Scientific | MP21021012 | |
Magnesium sulfate | Fisher Scientific | BP213-1 | |
Nystatin | Acros organics | AC455500050 | |
Potassium Phosphate Dibasic | Fisher Scientific | BP363-500 | |
Potassium phosphate monobasic | Fisher Scientific | BP362-500 | |
Sodium Azide | Sigma Aldrich | S2002-25G | |
Sodium chloride | Fisher Scientific | BP358-1 | |
Sodium Hydroxide | Fisher Scientific | SS266-1 | |
8.25% Sodium Hypochlorite | |||
Sodium Phosphate Dibasic | Fisher Scientific | BP332-500 | |
Streptomycin Sulfate | Fisher Scientific | BP910-50 | |
Tetracyclin | Sigma Aldrich | 87128-25G | |
(−)-Tetramisole hydrochloride | Sigma Aldrich | L9756 | |
Yeast extract | Fisher Scientific | BP1422-500Â | |
Consumables | |||
15mL Conical Sterile Polypropylene Centrifuge Tubes | Fisher Scientific | 12-565-269 | |
Disposable Polystyrene Serological Pipettes 10mL | Fisher Scientific | 07-200-574 | |
Disposable Polystyrene Serological Pipettes 25mL | Fisher Scientific | 07-200-575 | |
Falcon Bacteriological Petri Dishes with Lid (35 x 10 mm) | Fisher Scientific | 08-757-100A | |
No. 1.5Â 18 mm X 18 mm Cover Slips | Fisher Scientific | 12-541A | |
Petri Dish with Clear Lid (60 x 15 mm) | Fisher Scientific | FB0875713A | |
Petri Dishes with Clear Lid (100X15mm) | Fisher Scientific | FB0875712 | |
Plain Glass Microscope Slides (75 x 25 mm) | Fisher Scientific | 12-544-4 | |
Software | |||
Prism | Graphpad | ||
Bacterial Strains | |||
S. oralis ATCC 35037 | |||
S. mitis ATCC 49456 | |||
S. gordonii DL1 Challis  | |||
E. coli OP50 | |||
E. coli HT115 | |||
Worm Strains | |||
Strain | Genotype | Transgene | Source |
N2 | C. elegans wild isolate | CGC | |
EU1 | skn-1(zu67) IV/nT1 [unc-?(n754) let-?] (IV;V) | CGC | |
LD002 | IdIs1 | SKN-1B/C::GFP + rol-6(su1006) | Keith Blackwell |
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