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

Summary

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.

Abstract

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.

Introduction

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 cellulitis^2,3,4,5,6. Furthermore, they are emerging as causative agents of bloodstream infections in immunocompromised, neutropenic, and cancer patients that have undergone chemotherapy⁵

Protocol

1. Preparation of THY (Todd-Hewitt Yeast Extract) Agar Plates

1. 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 °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.
2. 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....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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	BP1422-500
Consumables
15 mL Conical Sterile Polypropylene Centrifuge Tubes	Fisher Scientific	12-565-269
Disposable Polystyrene Serological Pipettes 10 mL	Fisher Scientific	07-200-574
Disposable Polystyrene Serological Pipettes 25 mL	Fisher Scientific	07-200-575
Falcon Bacteriological Petri Dishes with Lid (35 mm x 10 mm)	Fisher Scientific	08-757-100A
No. 1.5  18 mm x 18 mm Cover Slips	Fisher Scientific	12-541A
Petri Dish with Clear Lid (60 mm x 15 mm)	Fisher Scientific	FB0875713A
Petri Dishes with Clear Lid (100 mm x 15 mm)	Fisher Scientific	FB0875712
Plain Glass Microscope Slides (75 mm 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