A Permeable Membrane Insert-Based Infection System to Study the Impact of Bacterial Toxins

1. Bacterial Culture

1. Generate isogenic mutant strains to be used for the study of the specific secreted factor of interest.
   Note: The GAS (Group A Streptococcus) M1T1 5448 strains utilized for these experiments included WT (Wild type) GAS', a sagAΔcat SLS-deficient (sag = Streptolysin S associated gene, SLS = Streptolysin S) mutant (abbreviated in text as ΔsagA), and a sagA complemented strain (abbreviated in text as ΔsagA+sagA).
2. Prepare overnight liquid cultures of bacterial strains of interest.  Grow GAS M1T1 5448 strains in 10 ml of Todd-Hewitt broth at 37 °C for 16-20 hr prior to infecting human cells.
3. Centrifuge overnight bacterial cultures (2,400 rcf for 10 min) and resuspend in fresh bacterial medium.
   Note: Resuspension in the same volume as the overnight cultures were grown in (5-10 ml) generally produces a desirable initial optical density.
4. Normalize bacterial cultures to equal starting concentrations by diluting the re-suspended cultures in additional bacterial medium until the same optical density at 600 nm (OD₆₀₀) is obtained for all strains to be tested (OD₆₀₀ of approximately 1.0 is recommended for convenience).
   Note: In these studies, stationary phase bacterial cultures were used to infect host cells immediately following normalization. However, as some strains of bacteria exit stationary phase nonsynchronously it may be more appropriate to begin host infections with log phase cultures if this is found to be the case for the strains of interest.
5. Prior to further analysis, perform a colony counting assay to determine the colony forming units (CFU) per milliliter present in the starting cultures so that the multiplicity of infection (MOI), or ratio of bacteria per host cell, can be determined.
   1. Prepare 1 ml serial dilutions of bacterial cultures that have been normalized to the desired optical density in phosphate-buffered saline (PBS) or appropriate bacterial growth medium (Todd-Hewitt broth was used in these studies). Prepare dilutions ranging from 10^-1 to 10^-6 to produce at least one set of agar plates with countable colonies (30-300 colonies). Perform all dilutions in triplicate.
   2. Transfer 100 µl of each serial dilution onto an agar plate containing the appropriate medium for the bacterial species being analyzed.
      Note: Todd-Hewitt agar was used in these studies.
   3. Use a glass or plastic bacterial spreader to evenly distribute the 100 µl aliquot across the entire surface of the agar plate, and continue spreading until the liquid has been absorbed into the agar. Perform under flame using a sterile technique.
   4. Incubate the agar plates at 37 °C overnight or until countable colonies appear.
   5. Count the colonies that grow on each plate and calculate the CFU/ml in the original culture by the following formula: number of colonies x inverse of the serial dilution/culture volume plated in milliliters.
      e.g. (200 colonies x 1/10^-5 dilution) / 0.1 ml plated = 2.0 x 10⁸ CFU/ml.

2. Host Cell Culture

1. Obtain an appropriate host cell line for the desired analyses.
   Note: HaCaT human epithelial keratinocytes, a kind gift from V. Nizet, were used for these studies.
   1. Maintain HaCaT cells in 100 mm culture dishes in Dulbecco's Modified Eagle's Medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS). Incubate at 37 °C with 5% CO₂.

3. Host Cell Infection with Permeable Membrane Insert System

1. Plate cells in appropriate tissue culture dishes and allow them to grow until they reach 90% confluency.
   Note: The HaCaT cells used in these studies typically reach confluency within 2-3 days after plating.
   1. For lysate collection (e.g. signaling analysis and adenosine triphosphate (ATP) determination assays), plate HaCaT cells in 6 well dishes at a seeding density of approximately 3 x 10⁵ cells/well.
   2. For immunofluorescence imaging experiments, plate cells on sterile glass coverslips within 6 well dishes at a seeding density of approximately 3 x 10⁵ cells/well.
   3. For ethidium homodimer membrane permeabilization and lactate dehydrogenase (LDH) release assays, plate HaCaT cells in 24 well dishes at a seeding density of approximately 5 x 10⁴ cells/well.
2. Immediately prior to treatment, wash cells with 1x sterile PBS.
3. Apply fresh growth medium to cells. For the conditions described here, apply 2 ml medium per well for 6 well plates or 0.5 ml medium per well for 24 well plates. If pharmacological treatments are being tested, mix with fresh medium and apply during this step.
   Note: Some assays may require alternative media. For example, as phenol red and high serum concentrations interfere with the LDH release assay, phenol red-free Dulbecco's modified eagle's medium (DMEM) supplemented with 1% bovine serum albumin (BSA) (w/v), 2 mM L-glutamine, and 1 mM sodium pyruvate was utilized for these experiments.
4. After fresh medium has been applied, carefully place a sterile 0.4 µm permeable membrane insert in each well. Perform this step in a laminar flow hood, and use sterile forceps to transfer the permeable membrane insert into each well.
5. Apply fresh cell culture medium to the upper chamber of each well according to the manufacturer's instructions. For the conditions described here, apply 1 ml medium per well for 6 well plates or 0.1 ml medium per well for 24 well plates.
6. Apply an appropriate volume of normalized bacterial cultures (see section 1) to the upper chamber of the permeable membrane insert system. Use a bacterial medium for uninfected control treatments. For the results described here, add 25 µl of the normalized cultures per chamber for 24 well plates and 100 µl of the cultures per chamber for 6 well plates.
   Note: In these studies, wild-type or mutant GAS was applied to host cells at a MOI of 10. MOI was calculated based on the final eukaryotic cell numbers (e.g. 2 x 10⁶ cells/well), such that 10 times as many bacteria were added per host cell at the beginning of the infection period (e.g. 2 x 10⁷ CFU per well). Appropriate MOI will vary with different bacterial species and with the desired follow-up analyses.
   Note: In addition to using bacterial medium for uninfected controls, other useful controls for certain applications of this technique may include heat-killed bacteria or spent bacterial medium for comparison.
7. Incubate infected cells at 37 °C with 5% CO₂.

4. Sample Collection

1. To collect cell culture medium, host cell lysates, or glass coverslips with intact cells for follow-up analyses, begin by carefully removing the permeable membrane insert with sterile forceps.
2. For Assessment of Secreted Host Proteins:
   1. Collect the medium from the lower chamber into 1.5 ml tubes. Avoid disturbing the monolayer during collection.
   2. Centrifuge samples at 14,000 rcf for 10 min at 4 °C to remove cellular debris.
   3. Remove all but 50 µl of the supernatant to a fresh 1.5 ml tube and store at -20 °C or use immediately.