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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

Abstract

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.

Introduction

Diarrheal diseases caused by enteric bacterial pathogens are a significant global health burden. In 2016, diarrheal diseases were responsible for 1.3 million deaths worldwide and were the fourth leading cause of death in children younger than five years of age1,2. The Gram-negative, enteric bacterial pathogen Shigella is the causative agent of shigellosis, a major cause of diarrheal deaths worldwide3. Shigellosis causes significant morbidity and mortality each year in children from lower- and middle-income countries4,5, while infections in high-income countries are linked to daycare center, foodborne, and waterborne outbreaks6,7,8,9. Ineffective vaccine development10 and rising rates of antimicrobial resistance (AMR)11,12 have complicated the management of large-scale Shigella outbreaks. Recent Centers for Disease Control and Prevention data show that nearly 46% of Shigella infections in the United States displayed drug resistance in 202013,14, while the World Health Organization has declared Shigella as an AMR priority pathogen for which new therapies are urgently needed15.

Shigella infections are easily transmitted via the fecal-oral route upon ingestion of contaminated food or water, or through direct human contact. Shigella has evolved to be an efficient, human-adapted pathogen, with an infectious dose of 10-100 bacteria sufficient to cause disease16. During small intestinal transit, Shigella is exposed to environmental signals, such as elevated temperature and bile17. Detection of these signals induces transcriptional changes to express virulence factors that enhance the ability of the bacteria to infect the human colon17,18,19. Shigella does not invade the colonic epithelium from the apical surface, but rather transits across the epithelial layer following uptake into specialized antigen-presenting microfold cells (M cells) within the follicle-associated epithelium20,21,22. Following transcytosis, Shigella cells are phagocytosed by resident macrophages. Shigella rapidly escapes the phagosome and triggers macrophage cell death, resulting in the release of pro-inflammatory cytokines5,23,24. Shigella then invades colonic epithelial cells from the basolateral side, lyses the macropinocytic vacuole, and establishes a replicative niche in the cytoplasm5,25. Pro-inflammatory cytokines, particularly interleukin-8 (IL-8), recruit polymorphonuclear neutrophil leukocytes (PMNs) to the site of infection, which weakens epithelial tight junctions, and enables bacterial infiltration of the epithelial lining to exacerbate basolateral infection5. The PMNs destroy the infected epithelial lining to contain the infection, which results in the characteristic symptoms of bacillary (bloody) dysentery5. Although invasion and intracellular replication mechanisms have been thoroughly characterized, new research is demonstrating important new concepts in Shigella infection, including virulence regulation during gastrointestinal (GI) transit17, adherence19, improved basolateral access through barrier permeability26, and asymptomatic carriage in malnourished children27.

The ability of Shigella spp. to cause diarrheal disease is restricted to humans and non-human primates (NHP)28. Shigella intestinal infection models have been developed for zebrafish29, mice30, guinea pigs31, rabbits21,32,33, and pigs34,35. However, none of these model systems can accurately replicate the disease characteristics observed during human infection36. Although NHP models of shigellosis have been established to study Shigella pathogenesis, these model systems are expensive to implement and require artificially high infectious doses, up to nine orders of magnitude higher than the infectious dose of humans37,38,39,40,41,42. Thus, the remarkable adaptation of Shigella for infection of human hosts necessitates the use of human-derived cell cultures to recreate physiologically relevant models for accurate interrogation of Shigella pathogenesis.

Here, detailed procedures are described to measure the rates of Shigella adherence to, invasion of, and replication within HT-29 colonic epithelial cells. Using these standardized protocols, the molecular mechanisms by which bacterial virulence genes and environmental signals impact each step of Shigella infection can be interrogated to better understand the dynamic host-pathogen interaction relationship.

Protocol

1. Preparation of reagents and materials

NOTE: All volumes are consistent with an assay using two 6-well plates.

  1. TSB medium: Add 0.5 L of deionized (DI) water to 15 g of Tryptic Soy Broth (TSB, see Table of Materials) medium and autoclave. Store at room temperature.
  2. Bile salts medium (TSB + BS): To prepare TSB containing 0.4% (w/v) bile salts, resuspend 0.06 g of bile salts (BS, see Table of Materials) in 15 mL of autoclaved TSB. Filter sterilize using a 0.22 µm PES filter.
    NOTE: The bile salts consist of a 1:1 mixture of sodium cholate and sodium deoxycholate. Prepare fresh media immediately before use.
  3. DMEM + 10% (v/v) FBS: Add 5 mL of fetal bovine serum (FBS) to 45 mL of Dulbecco's Modified Eagle Medium (DMEM). Store at 4 °C.
  4. DMEM + gentamicin: To a 50 mL tube, add 50 mL of DMEM and 50 µL of 50 mg/mL gentamicin (see Table of Materials).
    NOTE: Make fresh aliquot and warm in a 37 °C water bath prior to each experiment.
  5. PBS + 1% (v/v) Triton X-100: Add 150 µL of Triton X-100 to 15 mL of Phosphate-buffered saline (PBS).
    NOTE: Make fresh aliquot and warm in a 37 °C water bath prior to each experiment.
  6. TSB + Congo red indicator plates: Add 15 g of TSB, 7.5 g of select agar, and 0.125 g of Congo red dye (see Table of Materials) to a 1 L bottle. Add 0.5 L of DI water and autoclave. Pour 10-20 mL of media into individual sterile Petri dishes (100 mm x 15 mm) and let solidify.
    CAUTION: Congo red is carcinogenic and a reproductive toxin. Ensure that handling of Congo red is performed using the appropriate personal protective equipment. Consult the product safety data sheet for additional information.
    NOTE: Approximately 20 plates are made from 0.5 L Congo red media. Plates can be prepared 2-3 days in advance and left inverted at room temperature until use. For long-term storage, place inverted plates in plastic sleeves at 4 °C for up to 3 months.
  7. DMEM + 10% (v/v) FBS and 5% (v/v) dimethyl sulfoxide (DMSO): Add 42.5 mL of DMEM, 5 mL of FBS, and 2.5 mL of DMSO to a 50 mL tube. Store at 4 °C.

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.

  1. Growth of Shigella from frozen stocks
    1. Transfer a small amount of frozen culture from the cryogenic vial to a TSB + Congo red agar plate using a sterile applicator.
    2. Flame sterilize an inoculating loop and allow it to cool. Streak inoculum back and forth across one quadrant of the plate. Flame the loop, allow it to cool, then streak from the first quadrant onto the second quadrant of the plate. Repeat to streak inoculum into the third and fourth quadrants of the plate.
      NOTE: Alternatively, streak inoculum using a fresh sterile applicator between each quadrant.
    3. Invert the plate and incubate at 37 °C overnight.
      NOTE: Incubation at temperatures ≥37 °C is required for expression of Shigella virulence factors necessary for observation of Congo red-positive (CR+) phenotype45. Avirulent colonies will have a white appearance and will not be invasive.
    4. Seal the plate with paraffin film and store refrigerated at 4 °C.
      NOTE: Bacterial colonies will remain viable on agar plates for 1-2 weeks.
  2. Overnight growth of Shigella in liquid culture
    1. Aliquot 3 mL of TSB media into sterile 14 mL culture tubes.
    2. Pick a single, well-isolated red (CR+) colony using a sterile applicator and resuspend in liquid media.
    3. Incubate cultures overnight (16-18 h) at 37 °C with shaking at 250 rotations per minute (rpm).

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.

  1. Thawing HT-29 cells from frozen stock
    1. Thaw the vial of HT-29 cells in a 37 °C water bath.
      NOTE: Ensure the cap stays fully above the water to avoid contamination. Thawing should take less than 2 min.
    2. Remove the vial from the water immediately after the culture is fully thawed and decontaminate with 70% ethanol. Ensure that all steps from this point are performed using aseptic techniques.
    3. Add all the contents of the vial to a 15 mL centrifuge tube containing 9 mL of DMEM + 10% FBS. Centrifuge at 125 x g for 5 min at room temperature.
    4. Decant the supernatant into a waste container and resuspend the pellet in 10 mL of warm DMEM + 10% FBS. Transfer resuspended cells to a 75 cm2 tissue culture flask (T75) containing 10 mL of warm DMEM + 10% FBS (total volume of 20 mL).
    5. Incubate cells at 37 °C with 5% CO2 until the cells reach 90% confluency (approximately 6-7 days).
      NOTE: Confluency is estimated through visual approximation.
  2. Seeding HT-29 cells
    1. Pre-warm 20 mL of PBS and 50 mL of DMEM + 10% FBS in a 37 °C water bath and pre-warm 3 mL of 0.25% (w/v) Trypsin-EDTA to room temperature.
    2. Once HT-29 cells (from step 3.1) reach 90% confluency, decant HT-29 cell culture media from the T75 flask into a waste container. Pour ~10 mL of warm PBS into the flask and swirl gently to wash. Decant the PBS into a waste container. Wash with warm PBS again and decant.
    3. Add 2-3 mL of 0.25% (w/v) Trypsin-EDTA and gently swirl across the entire surface area. Incubate at 37 °C with 5% CO2 for 4 min.
    4. Remove the flask from the incubator and gently swirl the Trypsin-EDTA, visually ensuring that all cells detach from the surface.
    5. Immediately add 6 mL of warm DMEM + 10% FBS to deactivate the Trypsin. Pipette up and down to thoroughly mix.
    6. Transfer all the contents to a 15 mL centrifuge tube and spin it at 500 x g for 5 min at room temperature.
    7. Gently decant supernatant into a waste container and resuspend the pellet in 6 mL of warm DMEM + 10% FBS.
    8. Immediately after resuspension, transfer 10 µL of suspended HT-29 cells from the middle of the culture to a 0.2 mL PCR tube. Add 10 µL of Trypan blue dye to the PCR tube and mix.
    9. Add 10 µL of HT-29 cell/Trypan blue mix to a disposable Countess cell counter chamber slide (see Table of Materials). Enumerate the number of live cells and calculate cell viability.
      NOTE: When documenting the number of cells in the sample, read the number under the "live" cell count, not the total cell count. Alternatively, cell enumeration can be performed manually using a hemocytometer.
    10. Seed resuspended HT-29 cells into a fresh T75 flask or 6-well plate.
      1. For T75 flask:
        1. Pipet gently to mix, then transfer 2.5 x 106 cells to a fresh T75 flask according to the equation below:
          figure-protocol-8114
        2. Add warm DMEM + 10% FBS media to a final volume of 20 mL (final concentration of 1.25 x 105 cells/mL).
        3. Disperse cells evenly across the flask by gently rocking back and forth.
        4. Incubate at 37 °C with 5% CO2 until cells reach 80% confluency.
          NOTE: For optimal growth, replace the DMEM + 10% FBS media in the T75 flask every ~3 days. Decant media into a waste container and add 10 mL of warm PBS to the flask. Swirl the PBS around gently and decant it into the waste container. Then add 20 mL of fresh, warm DMEM + 10% FBS to the flask and return to the 37 °C, 5% CO2 incubator.
      2. For 6-well plate:
        1. Pipet gently to mix, then transfer 5.85 x 106 cells to a fresh 50 mL conical tube according to the equation below:
          figure-protocol-9075
        2. Add warm DMEM + 10% FBS media to a final volume of 26 mL (final concentration of 2.25 x 105 cells/mL).
        3. Pipet gently to mix, then dispense 2 mL (4.5 x 105 cells) into individual wells of 6-well plates.
        4. Disperse cells evenly across the well by gently rocking up/down and left/right 2-3x.
        5. Incubate at 37 °C with 5% CO2 until cells reach 80%-95% confluency (approximately 3-4 days).
          NOTE: 85% confluency is recommended for invasion and intracellular replication assays, while 90%-95% confluency is recommended for adherence assays. Cells should reach ~85% confluency after 48 h incubation with a final concentration of approximately 1 x 106 cells/well. Adjustments to the number of cells seeded and length of incubation may be required.
  3. Making frozen HT-29 stocks
    1. Aliquot 1 mL of DMEM + 10% FBS + 5% DMSO media into individual cryogenic vials.
    2. Add 1 x 106 HT-29 cells from step 3.2.7 to each vial. Calculate the volume of cells according to the formula below:
      figure-protocol-10335
    3. Store HT-29 cells long-term below -130 °C in liquid nitrogen vapor storage freezer.

4. Adherence assay

NOTE: All volumes are consistent with an assay using two 6-well plates.

  1. Subculture overnight Shigella cultures via 1:50 dilution into fresh media.
    1. Vortex, then add 100 µL of each overnight culture to 5 mL of fresh TSB or TSB + BS in a suitably sized culture tube.
      NOTE: Limit culture volume to <20% of culture flask or tube volume to ensure proper aeration.
    2. Incubate at 37 °C with shaking at 250 rpm until cells reach an optical density (OD600) of 0.7 (mid-log phase of Shigella growth); about 2-2.5 h.
      NOTE: During the subculture, aliquot 50 mL of DMEM and a sufficient volume of PBS for all washing steps and place in a 37 °C water bath. Allow media to reach 37 °C before use.
  2. Transfer 2 x 108 colony forming units (CFUs) subcultured Shigella to individual 2 mL microcentrifuge tubes.
    NOTE: 2 x 108 CFUs corresponds to approximately 1 mL of bacterial cells at an OD600 of 0.7. Use OD600 readings to approximate CFU/mL according to the calibration of each individual spectrophotometer.
  3. Wash each Shigella sample 2x with PBS.
    1. Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature. Aspirate the supernatant, then add 1 mL of warm PBS and resuspend the pellet well, gently pipetting the sample up and down until the mixture is fully homogeneous (8-10x).
    2. Repeat step 4.3.1 1x additional time.
    3. Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature, aspirate the supernatant, and resuspend the pellets in 2 mL of warm DMEM.
      NOTE: The final concentration of resuspended bacteria will be 1 x 108 CFU/mL.
  4. Vortex, then add 1 mL (1 x 108 CFUs) of resuspended Shigella to each well of the prepared HT-29 colonic epithelial monolayers in 6-well plates (from step 3.2.10.2).
    NOTE: Infections are normally performed at a multiplicity of infection (MOI; ratio of bacterial to epithelial cells) of 100. To test different MOIs, dilute resuspended Shigella in warm DMEM to the desired concentration, then add 1 mL of diluted bacteria to HT-29 monolayers. For example, to test an MOI of 10, dilute bacteria 1:10 by adding 150 µL of 1 x 108 CFU/mL bacteria to 1.35 mL of warm DMEM, then apply 1 mL (1 x 107 CFUs) to HT-29 cells.
  5. Incubate the 6-well plates at 37 °C with 5% CO2 for 3 h.
  6. During the incubation, determine the bacterial infection titer.
    1. Prepare 10-fold serial dilutions of resuspended Shigella cells (from step 4.3.3) into PBS.
    2. Plate 100 µL of the 1 x 10-5 and 1 x 10-6 dilutions onto TSB + Congo red plates and incubate overnight at 37 °C.
      NOTE: Plating 100 µL from the 1 x 10-5 and 1 x 10-6 dilutions corresponds to a final dilution factor of 1 x 10-6 and 1 x 10-7, respectively.
  7. After incubation, wash the monolayers 4-5x with PBS.
    1. Aspirate media from each well.
      NOTE: When aspirating media from 6-well plates, guide the tip of the aspirator along the bottom side of the wells, trying to avoid contact with the HT-29 cells.
    2. Add 1 mL of warm PBS to each well and wash gently.
      NOTE: To gently wash 6-well monolayers with PBS, move the plate up and down and side to side on the benchtop. Washing plates in a circular motion and/or removing the plate from the benchtop surface can cause the mechanical removal of cells from plastic.
    3. Repeat steps 4.7.1 and 4.7.2 4x additional times.
  8. Remove PBS by aspiration and lyse HT-29 cells by adding 1 mL of PBS + 1% Triton X-100 to each well.
  9. Incubate 6-well plates at 37 °C for 5 min.
  10. Use a cell scraper or a bent pipette tip to scrape the lysed cells from the bottom of the well and transfer the full 1 mL into a fresh 1.7 mL microcentrifuge tube.
  11. Determine the number of cell-associated bacteria.
    1. Vortex each tube (from step 4.10) for at least 30 s to further displace Shigella from the lysed eukaryotic cells.
    2. Prepare 10-fold serial dilutions of lysates into PBS.
    3. Plate 100 µL of the 1 x 10-2, 1 x 10-3, and 1 x 10-4 dilutions onto TSB + Congo red plates and incubate overnight at 37 °C.
      NOTE: Plating 100 µL from the 1 x 10-2, 1 x 10-3, and 1 x 10-4 dilutions corresponds to a final dilution factor of 1 x 10-3, 1 x 10-4, and 1 x 10-5, respectively.

5. Invasion assay

NOTE: All volumes are consistent with an assay using two 6-well plates.

  1. Subculture overnight Shigella cultures via 1:50 dilution into fresh media.
    1. Vortex, then add 100 µL of each overnight culture to 5 mL of fresh TSB or TSB + BS in a suitably sized culture tube.
      NOTE: Limit culture volume to <20% of culture flask or tube volume to ensure proper aeration.
    2. Incubate at 37 °C with shaking at 250 rpm until cells reach an OD600 of 0.7 (mid-log phase of Shigella growth); about 2-2.5 h.
      NOTE: During the subculture, aliquot 50 mL of DMEM + 50 mg/mL gentamicin and a sufficient volume of PBS for all washing steps and place in a 37 °C water bath. Allow media to reach 37 °C before use.
  2. Transfer 2 x 108 CFUs subcultured Shigella to individual 2 mL microcentrifuge tubes.
    NOTE: 2 x 108 CFUs corresponds to approximately 1 mL of bacterial cells at an OD600 of 0.7. Use OD600 readings to approximate CFU/mL according to the calibration of each individual spectrophotometer.
  3. Wash Shigella samples 1x with PBS.
    1. Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature. Aspirate the supernatant, then add 1 mL of warm PBS and resuspend the pellet well, gently pipetting the sample up and down until the mixture is fully homogeneous (8-10x).
    2. Repeat step 5.3.1. 1x additional time.
    3. Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature, aspirate the supernatant, and resuspend the pellets in 2 mL of warm DMEM.
      NOTE: The final concentration of resuspended bacteria will be 1 x 108 CFU/mL.
  4. Vortex, then add 1 mL (1 x 108 CFUs) of resuspended Shigella plus 1 mL of DMEM to each well of the prepared HT-29 colonic epithelial monolayers in 6-well plates (from step 3.2.10.2).
    NOTE: Infections are normally performed at a multiplicity of infection (MOI; ratio of bacterial to epithelial cells) of 100. To test different MOIs, dilute resuspended Shigella in DMEM to the desired concentration, then add 1 mL of diluted bacteria to HT-29 monolayers. For example, to test an MOI of 10, dilute bacteria 1:10 by adding 150 µL of 1 x 108 CFU/mL bacteria to 1.35 mL of DMEM, then add 1 mL (1 x 107 CFUs) to HT-29 cells.
  5. To promote bacterial contact with the HT-29 cells, centrifuge the 6-well plates at 2,000 x g for 10 min at room temperature or 37 °C if the temperature setting can be adjusted.
    NOTE: Centrifugation promotes bacterial contact with the HT-29 cells, which bypasses the need for adherence factors and allows the bacteria to quickly invade the cells.
  6. Incubate 6-well plates at 37 °C with 5% CO2 for 45 min.
  7. During the incubation, determine the bacterial infection titer.
    1. Prepare 10-fold serial dilutions of resuspended Shigella cells (from step 5.3.3) into PBS.
    2. Plate 100 µL of the 1 x 10-5 and 1 x 10-6 dilutions onto TSB + Congo red plates and incubate overnight at 37 °C.
      NOTE: Plating 100 µL from the 1 x 10-5 and 1 x 10-6 dilutions corresponds to a final dilution factor of 1 x 10-6 and 1 x 10-7, respectively.
  8. Thoroughly wash infected HT-29 cells 3x with 1 mL of PBS.
    1. Aspirate media from each well.
      NOTE: When aspirating media from 6-well plates, guide the tip of the aspirator along the bottom side of the wells, trying to avoid contact with the HT-29 cells.
    2. Add 1 mL of warm PBS to each well and wash gently.
      NOTE: To gently wash 6-well monolayers with PBS, move the plate up and down and side to side on the benchtop. Washing plates in a circular motion and/or removing the plate from the benchtop surface can cause the mechanical removal of cells from plastic.
    3. Repeat steps 5.8.1 and 5.8.2 2x additional times.
  9. Remove PBS by aspiration, then add 2 mL of warm DMEM supplemented with 50 µg/mL gentamicin to each well and incubate for 30 min at 37 °C with 5% CO2.
  10. Thoroughly wash infected HT-29 cells 3x with 1 mL of PBS.
    1. Repeat washing step 5.8.
  11. Remove PBS by aspiration, then add 2 mL of warm DMEM supplemented with 50 µg/mL gentamicin to each well and incubate for 60 min at 37 °C with 5% CO2.
  12. Thoroughly wash infected HT-29 cells 3x with 1 mL of PBS.
    1. Repeat washing step 5.8.
  13. Remove PBS by aspiration and lyse HT-29 cells by adding 1 mL of PBS + 1% Triton X-100 to each well.
  14. Incubate 6-well plates at 37 °C for 5 min.
  15. Use a cell scraper or a bent pipette tip to scrape the lysed cells from the bottom of the well and transfer the full 1 mL into a fresh 1.7 mL microcentrifuge tube.
  16. Determine the number of intracellular bacteria.
    1. Vortex each tube (from step 5.15) for at least 30 s to further displace Shigella from the lysed eukaryotic cells.
    2. Prepare 10-fold serial dilutions of lysates into PBS.
    3. Plate 100 µL of the 1 x 10-2 and 1 x 10-3 dilutions onto TSB + Congo red plates and incubate overnight at 37 °C.
      NOTE: Plating 100 µL from the 1 x 10-2 and 1 x 10-3 dilutions corresponds to a final dilution factor of 1 x 10-3 and 1 x 10-4, respectively.

6. Intracellular replication assay

NOTE: All volumes are consistent with an assay using two 6-well plates.

  1. Subculture overnight Shigella cultures via 1:50 dilution into fresh media.
    1. Vortex, then add 100 µL of each overnight culture to 5 mL of fresh TSB or TSB + BS in a suitably sized culture tube.
      NOTE: Limit culture volume to <20% of culture flask or tube volume to ensure proper aeration.
    2. Incubate at 37 °C with shaking at 250 rpm until cells reach an OD600 of 0.7 (mid-log phase of Shigella growth); about 2-2.5 h.
      NOTE: During the subculture, aliquot 50 mL of DMEM + 50 mg/mL gentamicin and a sufficient volume of PBS for all washing steps and place in a 37 °C water bath. Allow media to reach 37 °C before use.
  2. Transfer 2 x 108 CFUs subcultured Shigella to individual 2 mL microcentrifuge tubes.
    NOTE: 2 x 108 CFUs corresponds to approximately 1 mL of bacterial cells at an OD600 of 0.7. Use OD600 readings to approximate CFU/mL according to the calibration of each individual spectrophotometer.
  3. Wash Shigella samples 1x with PBS.
    1. Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature. Aspirate the supernatant, then add 1 mL of warm PBS and resuspend the pellet well, gently pipetting the sample up and down until the mixture is fully homogeneous (8-10x).
    2. Repeat step 6.3.1. 1x additional time.
    3. Pellet cells by centrifugation at 17,000 x g for 2 min at room temperature, aspirate the supernatant, and resuspend the pellets in 2 mL of warm DMEM.
      NOTE: The final concentration of resuspended bacteria will be 1 x 108 CFU/mL.
  4. Vortex, then add 1 mL (1 x 108 CFUs) of resuspended Shigella plus 1 mL of DMEM to each well of prepared HT-29 colonic epithelial monolayers in 6-well plates (from step 3.2.10.2).
    NOTE: Infections are normally performed at a multiplicity of infection (MOI; ratio of bacterial to epithelial cells) of 100. To test different MOIs, dilute resuspended Shigella in DMEM to the desired concentration, then add 1 mL of diluted bacteria to HT-29 monolayers. For example, to test an MOI of 10, dilute bacteria 1:10 by adding 150 µL of 1 x 108 CFU/mL bacteria to 1.35 mL of DMEM, then apply 1 mL (1 x 107 CFUs) to HT-29 cells.
  5. To promote bacterial contact with the HT-29 cells, centrifuge the 6-well plates at 2,000 x g for 10 min at room temperature or 37 °C if the temperature setting can be adjusted.
    NOTE: Centrifugation promotes bacterial contact with the HT-29 cells, which bypasses the need for adherence factors and allows the bacteria to quickly invade the cells.
  6. Incubate 6-well plates at 37 °C with 5% CO2 for 45 min.
  7. During the incubation, determine the bacterial infection titer.
    1. Prepare 10-fold serial dilutions of resuspended Shigella cells (from step 6.3.3) into PBS.
    2. Plate 100 µL of the 1 x 10-5 and 1 x 10-6 dilutions onto TSB + Congo red plates and incubate overnight at 37 °C.
      NOTE: Plating 100 µL from the 1 x 10-5 and 1 x 10-6 dilutions corresponds to a final dilution factor of 1 x 10-6 and 1 x 10-7, respectively.
  8. Thoroughly wash infected HT-29 cells 3x with 1 mL of PBS.
    1. Aspirate media from each well.
      NOTE: When aspirating media from 6-well plates, guide the tip of the aspirator along the bottom side of the wells, trying to avoid contact with the HT-29 cells.
    2. Add 1 mL of warm PBS to each well and wash gently.
      NOTE: To gently wash 6-well monolayers with PBS, move the plate up and down and side to side on the benchtop. Washing plates in a circular motion and/or removing the plate from the benchtop surface can cause the mechanical removal of cells from plastic.
    3. Repeat steps 6.8.1 and 6.8.2 2x additional times.
  9. Remove PBS by aspiration, then add 2 mL of warm DMEM supplemented with 50 µg/mL gentamicin to each well and incubate for 30 min at 37 °C with 5% CO2.
  10. Thoroughly wash infected HT-29 cells 3x with 1 mL of PBS.
    1. Repeat washing step 6.8.
  11. Remove PBS by aspiration, then add 2 mL of warm DMEM with 50 µg/mL gentamicin to each well of the 6-well plates and incubate at 37 °C with 5% CO2 for the desired length of time to allow for intracellular replication (up to 24 h).
  12. Thoroughly wash cells 2x with 1 mL of PBS.
    1. Repeat washing step 6.8.
  13. Remove PBS by aspiration and lyse HT-29 cells by adding 1 mL of PBS + 1% Triton X-100 to each well.
  14. Incubate 6-well plates at 37 °C for 5 min.
  15. Use a cell scraper or bent pipette tip to scrape the lysed cells from the bottom of the well and transfer the full 1 mL into a fresh 1.7 mL microcentrifuge tube.
  16. Determine the number of intracellular bacteria.
    1. Vortex each tube (from step 6.15) for at least 30 s to further displace Shigella from the lysed eukaryotic cells.
    2. Prepare 10-fold serial dilutions of lysates into PBS.
    3. Plate 100 µL of the 1 x 10-2, 1 x 10-3, and 1 x 10-4 dilutions onto TSB + Congo Red plates and incubate overnight at 37 °C.
      NOTE: Plating 100 µL from the 1 x 10-2, 1 x 10-3, and 1 x 10-4 dilutions corresponds to a final dilution factor of 1 x 10-3, 1 x 10-4, and 1 x 10-5, respectively.

Results

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 w...

Discussion

This protocol describes a set of three standardized assays to study Shigella adherence, invasion, and intracellular replication of intestinal epithelial cells. Although these methods are merely modified versions of classical gentamicin assays used to study the invasion and intracellular replication of various bacterial pathogens within host cells49,50,51, special considerations must be applied when studying Shigella...

Disclosures

The authors declare no conflicts of interest.

Acknowledgements

Support for the authors includes Massachusetts General Hospital's Department of Pediatrics, the Executive Committee on Research Interim Support Funding (ISF) award 2022A009041, the National Institute of Allergy and Infectious Diseases grant R21AI146405, and the National Institute of Diabetes and Digestive and Kidney Diseases grant Nutrition Obesity Research Center at Harvard (NORCH) 2P30DK040561-26. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Materials

NameCompanyCatalog NumberComments
0.22 μm PES filterMillipore-SigmaSCGP00525Sterile, polyethersulfone filter for sterilizing up to 50 mL media
14 mL culture tubesCorning35205917 mm x 100 mm polypropylene test tubes with cap
50 mL conical tubesCorning43082950 mL clear polypropylene conical bottom centrifuge tubes with leak-proof cap
6-well tissue culture platesCorning3516Plates are treated for optimal cell attachment
Bile saltsSigma-AldrichB87561:1 ratio of cholate to deoxycholate
Congo red dyeSigma-AldrichC6277A benzidine-based anionic diazo dye, >85% purity
Countess cell counting chamber slideInvitrogenC10283To be used with the Countess Automated Cell Counter
Dimethyl sulfoxide (DMSO)Sigma-AldrichD8418A a highly polar organic reagent
Dulbecco’s Modified Eagle Medium (DMEM)Gibco10569-010DMEM is supplemented with high glucose, sodium pyruvate, GlutaMAX, and Phenol Red
Fetal Bovine Serum (FBS)Sigma-AldrichF4135Heat-inactivated, sterile
GentamicinSigma-AldrichG3632Stock concentration is 50 mg/mL
HT-29 cell lineATCCHTB-38Adenocarcinoma cell line; colorectal in origin
Paraffin filmBemisPM999Laboratory sealing film
Petri dishesThermo Fisher ScientificFB0875713100 mm x 15 mm Petri dishes for solid media
Phosphate-buffered saline (PBS)Thermo Fisher Scientific100100491x concentration; pH 7.4
Select agarInvitrogen30391023A mixture of polysaccharides extracted from red seaweed cell walls to make bacterial plating media
T75 flasksCorning430641UTissue culture flasks
Triton X-100Sigma-AldrichT8787A common non-ionic surfactant and emulsifier 
Trypan blue stainInvitrogenT10282A dye to detect dead tissue culture cells; only live cells can exclude the dye
Trypsin-EDTAGibco25200-056Reagent for cell dissociation for cell line maintenance and passaging
Tryptic Soy Broth (TSB)Sigma-AldrichT8907Bacterial growth media

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