Streptococci/epithelial cell coculture assay

Purpose

To assess the intracellular bacteriolytic efficacy of endolysins against Group A Streptococcus (GAS), this protocol establishes a co-culture model to quantify GAS adherence, invasion, and intracellular survival in epithelial cells.

Materials and reagents

• Group A Streptococcus (GAS) strain D471
• Human epithelial cells (e.g., HEp-2, A549, or other relevant cell line)
• 24-well tissue culture plates
• Dulbecco’s Modified Eagle Medium (DMEM) or other appropriate cell culture medium
• Fetal bovine serum (FBS)
• Sterile phosphate-buffered saline (PBS)
• Trypsin-EDTA (0.25% trypsin, 0.02% EDTA)
• Triton X-100 solution (0.025% in PBS)
• Penicillin (10 μg/mL)
• Gentamicin (200 μg/mL)
• Endolysin solution (concentration optimized as per experimental design)
• Todd-Hewitt Yeast extract (THY) agar or blood agar plates
• Sterile microcentrifuge tubes and pipette tips
• Sterile serological pipettes

Equipment

• CO₂ incubator (37°C, 5% CO₂)
• Centrifuge
• Biosafety cabinet
• Light microscope (optional for cell morphology observation)
• Colony counter

Detailed protocol:

A. Preparation of epithelial cell monolayer

1. Seed epithelial cells into 24-well tissue culture plates at a density of ~2 × 10⁵ cells/well.
2. Incubate cells at 37°C in 5% CO₂ until they reach ~80% confluence (typically 24–48 hours).

B. Preparation of bacterial inoculum

     3. Grow GAS strain D471 overnight in THY broth.

     4. Harvest bacterial cells by centrifugation at 10,000 × g for 5 min.

     5. Wash pellet once with sterile PBS.

     6. Resuspend in serum-free DMEM and adjust the concentration to ~2 × 10⁷ CFU/mL.

C. Co-culture infection

     7. Infect epithelial cell monolayers at a multiplicity of infection (MOI) of 100:1 (bacteria:epithelial cells).

     8. Incubate for 1 hour at 37°C in 5% CO₂ to allow bacterial adhesion and invasion.

     9. Following incubation, aspirate media and wash wells 3 times with sterile PBS to remove non-adherent bacteria.

D. Total adhered and internalized bacteria enumeration

    10. Add 100 μL of 0.25% trypsin–0.02% EDTA to each well and incubate at 37°C for 5 minutes to detach cells.

    11. Add 400 μL of 0.025% Triton X-100 in PBS to lyse epithelial cells.

    12. Monitor cell lysis under a microscope; complete lysis typically occurs within 5–10 minutes.

    13. Collect lysate, serially dilute in PBS, and plate on THY agar for CFU enumeration. These represent both adhered and internalized bacteria.

E. Enumeration of internalized bacteria only

    14. To differentiate internalized bacteria, treat co-cultures (after Step 8) with antibiotics:

    15. Add 10 μg/mL penicillin and 200 μg/mL gentamicin in serum-free medium. Incubate for 1 hour at 37°C to eliminate extracellular (non-adherent and adherent) bacteria.

    16. Wash wells 3 times with PBS.

    17. Proceed with trypsinization and lysis as in Steps 10–13.

    18. Plate serial dilutions on blood agar plates and incubate overnight to enumerate internalized GAS.

F. Endolysin treatment assay

    19. After antibiotic treatment (Step 14), wash co-cultures 3 times with PBS.

    20. Add endolysin-containing medium to each well and incubate for 1 hour at 37°C.

    21. Proceed with cell detachment and lysis as in Steps 10–13.

    22. Serially dilute and plate lysates to enumerate viable intracellular bacteria post-endolysin treatment.

Data analysis

• Calculate CFUs per well based on dilution factors for each condition.
• Compare internalized CFUs pre- and post-endolysin treatment to determine intracellular bacterial killing efficacy.
• Statistical analysis can be performed using either Student’s t-test or ANOVA, depending on experimental design.