1. Preparation of reagents and materials
NOTE: All volumes are consistent with an assay using two 6-well plates.
2. Preparation of bacteria
NOTE: All Shigella laboratory cultivation and storage protocols are adapted from Payne, S. M.43.
CAUTION: Shigella spp. are Risk Group 2 pathogens44. Perform all laboratory work in a BSL-2 environment, with additional safety measures undertaken to limit accidental exposures due to the low infectious dose of Shigella spp.
3. Preparation of HT-29 eukaryotic cells
NOTE: All volumes are consistent with an assay using two 6-well plates. HT-29 cell lines were acquired from the American Type Culture Collection (ATCC). HT-29 maintenance protocols are adapted from ATCC recommendations46. All media should be pre-warmed in a water bath at 37 °C prior to use. All HT-29 maintenance protocols should be performed in a biosafety cabinet. Refrain from producing bubbles when mixing/working with HT-29 cells in media to avoid dramatic changes in pH.
4. Adherence assay
NOTE: All volumes are consistent with an assay using two 6-well plates.
5. Invasion assay
NOTE: All volumes are consistent with an assay using two 6-well plates.
6. Intracellular replication assay
NOTE: All volumes are consistent with an assay using two 6-well plates.
Adherence, invasion, and intracellular replication assays were performed comparing S. flexneri 2457T wild type (WT) to S. flexneri ΔVF (ΔVF), a mutant hypothesized to negatively regulate Shigella virulence. Since Shigella uses bile salts as a signal to regulate virulence17,18,47, experiments were performed after bacterial subculture in TSB media as well as TSB supplemented with 0.4% (w/v) bile salts18. Bile salts exposure during the subculture step acts as a pre-treatment to replicate small intestinal transit prior to colonic infection17,18,47. Figure 1 analyzes the effect of the ΔVF mutation on the ability of S. flexneri to adhere to HT-29 colonic epithelial cells. Percent adherence is plotted on the y-axis and represents the ratio of recovered bacteria following HT-29 lysis standardized to the number of input bacteria. As expected, both S. flexneri WT and ΔVF strains had a significant increase in adherence when subcultured with bile salts supplementation in comparison to TSB without bile salts supplementation18. However, there was no difference in adherence to HT-29 cells between WT and ΔVF mutant strains within each subculture condition. These data indicate that the ΔVF mutation has no effect on the ability of S. flexneri to adhere to HT-29 epithelial cells with or without the bile salts pre-treatment.
In Figure 2, the effect of the ΔVF mutation on the ability of S. flexneri to invade (Figure 2A) and replicate (Figure 2B) inside of HT-29 colonic epithelial cells with or without bile salts pre-treatment was analyzed. Percent recovery is plotted on the y-axis and represents the ratio of recovered bacterial cells following HT-29 cell lysis standardized to the number of input bacteria. In Figure 2A, there was an expected significant increase in the ability of WT S. flexneri 2457T to invade HT-29 cells after pre-exposure to bile salts48, while the S. flexneri ΔVF mutant displayed a smaller increase in invasion following bile salts pre-exposure compared to the WT strain. The ΔVF mutant had increased invasion rates of HT-29 cells compared to the WT subcultured in TSB, but had similar invasion rates as WT when subcultured in TSB supplemented with bile salts (Figure 2A). These results suggest that the ΔVF mutation enhances the ability of S. flexneri to invade HT-29 cells, which lessens the effect of the bile salts pre-exposure even though the invasion ability of the ΔVF mutant did increase further following bile salts subculture.
Overall, 10-fold more bacteria were recovered following overnight incubation (Figure 2B) compared to the 90 min incubation (Figure 2A), which demonstrates the differences in monitoring intracellular growth versus invasion, respectively. When infected HT-29 cells were incubated for 18 h to allow for intracellular replication of the bacteria (Figure 2B), the impact of the bile salts pre-treatment decreased for both the WT and ΔVF strains. However, the reduced effect of bile salts pre-treatment during intracellular replication was more dramatic for the ΔVF mutant. Since the increase in intracellular replication of both strains when pre-exposed to bile salts was smaller than the increase in invasion rates in the same conditions, we hypothesize that bile salts have a greater impact on the early steps in S. flexneri pathogenesis. The ΔVF mutant displayed an increase in percent recovery from overnight replication inside HT-29 cells compared to WT (Figure 2B) following both subculture conditions. However, the percent recoveries of the ΔVF mutant were similar regardless of bile salts pre-exposure. These data trends suggest that the ΔVF mutant replicates more efficiently inside HT-29 cells compared to WT, and that bile salts pre-exposure does not impact the ability of the ΔVF mutant to replicate intracellularly, as observed for WT (Figure 2B). Since the difference between the mutant and WT strains in the bile salts pre-exposure condition was not observed during the 90 min invasion assay, we hypothesize that the product encoded by the deleted VF gene may also regulate S. flexneri replication inside HT-29 cells. Combined, both analyses demonstrate that the ΔVF mutant is more virulent relative to WT, which suggests that the VF gene product is a negative regulator of S. flexneri virulence.
Figure 1: Bile salts pre-exposure induced adherence of S. flexneri to HT-29 cells. S. flexneri 2457T WT and ΔVF mutant cells were subcultured in either TSB or TSB supplemented with 0.4% (w/v) bile salts (TSB+BS) media. The bacteria were then applied to HT-29 cells at a multiplicity of infection (MOI) of 100 and incubated for 3 h to examine adherence. After incubation, infected HT-29 cells were washed and lysed, and serial dilutions of recovered bacteria were plated to enumerate colony-forming units per mL (CFU/mL). The number of adherent bacteria is plotted relative to the input bacteria titers to establish the percent adherence. Data are representative of one biological replicate with three technical replicates (individual dots). Error bars indicate the standard error of the mean (SEM). Statistical significance was determined by a Student's t-test (*p < 0.05; ***p < 0.001). Please click here to view a larger version of this figure.
Figure 2: Bile salts pre-exposure increased WT S. flexneri invasion and intracellular replication. S. flexneri 2457T WT and ΔVF mutant cells were subcultured in either TSB or TSB supplemented with bile salts (TSB+BS) media. The bacteria were then applied to HT-29 cells at an MOI of 100, centrifuged onto the cells, and incubated at 37 °C with 5% CO2 for 45 min. Cells were washed with PBS, and extracellular bacteria were lysed by the addition of gentamicin to the DMEM to exclusively recover intracellular bacteria. After 90 min (A, bacterial invasion) or 18 h (B, intracellular replication) incubations, infected HT-29 cells were washed and lysed, and serial dilutions of recovered bacteria were plated to enumerate colony-forming units per mL (CFU/mL). The number of intracellular bacteria is plotted relative to the input bacterial titers to establish the percent recovery. Data are representative of one biological replicate, each with three technical replicates (individual dots). Error bars indicate SEM. Statistical significance was determined by a Student's t-test (*p < 0.05). Please note the differences in the y-axis scales between panels (A) and (B). Please click here to view a larger version of this figure.
0.22 μm PES filter | Millipore-Sigma | SCGP00525 | Sterile, polyethersulfone filter for sterilizing up to 50 mL media |
14 mL culture tubes | Corning | 352059 | 17 mm x 100 mm polypropylene test tubes with cap |
50 mL conical tubes | Corning | 430829 | 50 mL clear polypropylene conical bottom centrifuge tubes with leak-proof cap |
6-well tissue culture plates | Corning | 3516 | Plates are treated for optimal cell attachment |
Bile salts | Sigma-Aldrich | B8756 | 1:1 ratio of cholate to deoxycholate |
Congo red dye | Sigma-Aldrich | C6277 | A benzidine-based anionic diazo dye, >85% purity |
Countess cell counting chamber slide | Invitrogen | C10283 | To be used with the Countess Automated Cell Counter |
Dimethyl sulfoxide (DMSO) | Sigma-Aldrich | D8418 | A a highly polar organic reagent |
Dulbecco’s Modified Eagle Medium (DMEM) | Gibco | 10569-010 | DMEM is supplemented with high glucose, sodium pyruvate, GlutaMAX, and Phenol Red |
Fetal Bovine Serum (FBS) | Sigma-Aldrich | F4135 | Heat-inactivated, sterile |
Gentamicin | Sigma-Aldrich | G3632 | Stock concentration is 50 mg/mL |
HT-29 cell line | ATCC | HTB-38 | Adenocarcinoma cell line; colorectal in origin |
Paraffin film | Bemis | PM999 | Laboratory sealing film |
Petri dishes | Thermo Fisher Scientific | FB0875713 | 100 mm x 15 mm Petri dishes for solid media |
Phosphate-buffered saline (PBS) | Thermo Fisher Scientific | 10010049 | 1x concentration; pH 7.4 |
Select agar | Invitrogen | 30391023 | A mixture of polysaccharides extracted from red seaweed cell walls to make bacterial plating media |
T75 flasks | Corning | 430641U | Tissue culture flasks |
Triton X-100 | Sigma-Aldrich | T8787 | A common non-ionic surfactant and emulsifier |
Trypan blue stain | Invitrogen | T10282 | A dye to detect dead tissue culture cells; only live cells can exclude the dye |
Trypsin-EDTA | Gibco | 25200-056 | Reagent for cell dissociation for cell line maintenance and passaging |
Tryptic Soy Broth (TSB) | Sigma-Aldrich | T8907 | Bacterial growth media |
The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.
The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.
The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.