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 chemotherapy5,7,8,9.
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.
1. Preparation of THY (Todd-Hewitt Yeast Extract) Agar Plates
2. Preparation of Nematode Growth Medium (NGM) and RNAi Feeding Plates (NGM RNAi)
3. Maintenance of C. elegans
4. Preparation of Age Synchronous Population of Worms
5. Induction of RNAi in Worms
6. Preparation of Mitis Group Streptococci for Infection
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 ΔspxB and complement strain ΔspxB;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.
8. Preparation of Agarose Pads for Microscopy
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), ΔspxB, and the complement strain ΔspxB;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.
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 was supplemented to THY agar. The killing of the worms was abolished in the presence of catalase (Figure 3B). To further confirm whether streptococcal derived H2O2 mediated killing of the worms, survival on the ΔspxB mutant strain, WT strain, and complement strain ΔspxB;spxB+ of S. gordonii was analyzed. Death of the worms was not observed on the ΔspxB mutant strain compared to the wild-type and complement strains (Figure 3C). These data suggest that the H2O2 produced by the mitis group mediates killing of the worms. We also observed similar killing kinetics when the worms were exposed to clinical isolates of the mitis group streptococci obtained from the blood of cancer patients (Figure 3D). Based on the data, the pathogenicity caused by H2O2 produced by the mitis group streptococci was assessed.
To identify host genes that are essential against streptococcal infections, skn-1 was knocked down, which encodes for the oxidative stress response transcription factor in C. elegans. Then, survival relative to the vector control treated worms was compared. A significant decrease in the survival of the skn-1 knockdown worms was observed compared to the vector control treated worms (Figure 4A). This data was further validated using a skn-1 mutant strain, and its survival was compared to that of the N2 wild-type worms. We observed a similar killing phenotype as the skn-1 mutant, as seen with the skn-1 knockdown, demonstrating that SKN-1 influenced the survival of the worms on the mitis group (Figure 4B).
Next, it was determined whether the H2O2 produced by the mitis group caused localization of SKN-1B/C::GFP in the worms. Localization of SKN-1B/C::GFP was observed in worms exposed to the wild-type and complement stains and not in response to the ΔspxB mutant strain of S. gordonii (Figure 5A,B). Furthermore, to determine the activation of SKN-1, components of the p38 MAPK pathway were knocked down. Reduced localization of SKN-1B/C::GFP in nsy-1, sek-1, pmk-1, and skn-1 knockdown worms relative to the vector control treated worms was observed. The data suggests the p38 MAPK is required for the activation of SKN-1 in response to H2O2 produced by the mitis group (Figure 5C,D).
Figure 1: Flowchart depicting the steps involved in preparation of the survival assays. Please click here to view a larger version of this figure.
Figure 2: Flowchart depicting the steps involved in localization of SKN-1 during infection. Please click here to view a larger version of this figure.
Figure 3: H2O2-mediated killing of C. elegans by mitis group streptococci. Kaplan-Meier survival curves of L4 larvae exposed to (A) S. gordonii, S. oralis, S. mitis, S. salivarius, S. mutans, and E. coli OP50. (B) S. gordonii, S. oralis, S. mitis, S. salivarius, S. mutans, and E. coli OP50 on THY plates in the presence of 1,000 U of catalase. (C) S. gordonii WT, ΔspxB mutant, and ΔspxB;spxB+ complement strains on N2 L4 larvae. (D) S. oralis (VGS#3), S. oralis (VGS#4), S. mitis (VGS#10), S. mitis (VGS#13), and E. coli OP50. The data are representative of experiments repeated two or more times, with n = 60 worms for each condition. Kaplan-Meier log rank analysis was used to compare survival curves and calculate the median survival. P values < 0.05 were considered to be statistically significant. This figure has been modified and adapted with permission15. Please click here to view a larger version of this figure.
Figure 4: SKN-1 is required for survival of the worms on S. gordonii. (A) Survival of vector control treated and skn-1 knockdown worms exposed to S. gordonii. (B) Survival of N2 and skn-1(zu67) mutant worms fed on S. gordonii. The data are representative of experiments repeated two or more times, with n = 60 worms for each condition. Kaplan-Meier log rank analysis was used to compare survival curves and calculate the median survival. P values < 0.05 were considered to be statistically significant. This figure has been modified and adapted with permission15. Please click here to view a larger version of this figure.
Figure 5: Streptococcal H2O2 mediated activation of SKN-1 is dependent on the p38 MAPK pathway. (A) Representative images of the localization of SKN-1B/C::GFP in worms exposed to the WT, ΔspxB mutant, and ΔspxB;spxB+ complement strains of S. gordonii. Closeups are shown in the upper righthand corners of each image. Scale bar = 100 µm. (B) The degree of nuclear localization of SKN-1B/C::GFP and percentage of worms in each category fed on WT, ΔspxB mutant, and ΔspxB;spxB+ complement strains of S. gordonii. Significantly low levels of nuclear localization of SKN-1B/C::GFP were observed in the ΔspxB mutant (p < 0.0001) compared to the WT and ΔspxB;spxB+ complement strains of S. gordonii. (C) Representative images of the localization of SKN-1B/C::GFP in nsy-1, sek-1, pmk-1, skn-1 knockdown, and vector control treated worms on S. gordonii. Closeups are shown in the upper righthand corners of each image. Scale bar = 100 µm. (D) The degree of SKN-1B/C::GFP nuclear localization and percentage of worms in each category fed on nsy-1, sek-1, pmk-1, skn-1 knockdown, and vector control treated worms on S. gordonii. Significantly low levels of nuclear localization of SKN-1B/C::GFP were observed in the nsy-1 (p < 0.01), sek-1 (p < 0.001), pmk-1 (p < 0.0001),and skn-1 knockdown (p < 0.0001) compared to the vector control treated worms on S. gordonii. Greater than 100 worms exposed to each strain were imaged, and the experiment was repeated three times. This figure has been modified and adapted with permission15. Please click here to view a larger version of this figure.
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 the capability to screen several gene candidates involved in host immunity and the oxidative stress response over a short time period.
In this protocol, H2O2 produced by the bacteria is in direct contact with the intestinal cells of the worm, as opposed to other exogenous ROS sources that must cross several barriers. This ensures that the H2O2 is delivered to the intestinal cells; hence, a more robust killing response is observed in the worm. Using fluorescently labeled bacteria, it was determined that the worms must be exposed to pathogens for 30 min for complete colonization of the intestinal tract15. It is advised to use less than 1 week old streak plates of streptococcal strains to ensure their viability and ability to produce H2O2. In addition, the streptococcal strains must be grown under microaerophilic conditions for optimal production of H2O2. L4 larvae or 1 day old adults can be used for this assay. It is critical that all worms used in an experiment are the same age and sex. Younger worms tend to die more slowly compared to older hermaphrodites. L4 animals are more easily distinguished because their developing vulva is visible at mid-body as a clear patch that contrasts with the rest of the body. It is also important that no E. coli OP50 are transferred to the streptococcus seeded plates. Contamination of killing plates with E. coli can cause the attenuation or abrogation of killing of the worms. To avoid contamination, it is necessary to pick worms away from the E. coli lawn. When scoring the survival assays, it is advised to observe the pharyngeal pumping, foraging behavior of the head, and body movement. To ensure that the worm is dead, it is recommended to gently prod the nose, side of the body, or tail and observe any movement.
RNAi feeding and the survival of the worms on the mitis group was combined to identify candidates that are involved in the defense against H2O2. Using the gene skn-1 that encodes for an oxidative stress response transcription factor, its requirement for survival of the worm in response to H2O2 is demonstrated here. Hence, this assay can be adapted to screen for several genes and identify potential candidates required for oxidative stress response and immunity. RNAi feeding of the worms is achieved by adding age synchronized L1 larvae to the RNAi expressing E. coli lawns. During the worm synchronization protocol, it is essential to monitor lysis of the worm cuticles in the presence of bleach and sodium hydroxide. The cuticle of adults and larvae will continually dissolve, while the embryos are partially protected by the thick eggshell. However, prolonged incubation in the presence of the blead sodium hydroxide mix may cause the embryos to die. Therefore, it is important to observe the tube containing the worms under a dissecting microscope periodically during bleaching. Another step to consider in the synchronization protocol is the maintenance of arrested L1 larvae. The arrested larvae can be maintained on the tube rotator for 5 days at room temperature. It is recommended to use the larvae for RNAi feeding within 1 to 2 days after hatching. Prolonged maintenance in M9W can result in the formation of the dauer stage.
Lastly, a transgenic worm expressing SKN-1 fused to GFP was used to monitor activation of the oxidative stress response in the presence of the mitis group. It is shown by RNAi that the components of the p38 MAPK are required for localization of SKN-1B/C::GFP to the nuclei of the intestinal cells. It is important to use the L3 or L4 stages of this strain to observe localization of SKN-1B/C::GFP, as localization of SKN-1 tends to diminish in the adult stage. To better observe the localization of SKN-1B/C::GFP, it is advised to overlap the obtained images using the FITC and DAPI filter settings. Autofluorescence generated by the lipofuscins help provide contrast for observation of SKN-1 localization in the worm. However, it has also been shown that the signal from weakly expressed GFP reporters is masked by autofluorescence emitted by various sources in the gut of the worm. Autofluorescence has been shown to increase with age and is highest in the intestine and uterus of the C. elegans27. To overcome this problem, a recent study utilized a triple band GFP filter setup to monitor the localization of SKN-1B/C::GFP in C. elegans28. This setup separates the GFP signal from autofluoresence, displaying the GFP and autofluoresence in the green and yellow channels, respectively.
C. elegans is used in this protocol to study host-pathogen interactions and ascertain how H2O2 produced by the mitis group causes pathogenicity. More importantly, by using this model, the effects of H2O2 on endoplasmic reticular stress, mitochondrial damage, mitophagy, autophagy, and oxidative stress can be studied. Furthermore, mechanisms by which H2O2 acts as a virulence factor to elicit immune responses by disrupting core processes of the cell can be identified. Hence, this worm is recognized as a powerful model system for discovering new insights into host-pathogen interactions.
The authors have nothing to disclose.
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).
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 | |||
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. 1.5 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 |