A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions

The following protocols will describe methods for culturing and studying the invasion by the anaerobic species, P. gingivalis; however, these protocols may be used for a number of anaerobic pathogens. Although HUVECs are used, this protocol may be used for other eukaryotic cells both immune and non-immune.

1. Anaerobic Chamber Use and Maintenance

Note: P. gingivalis is an anaerobe sensitive to normal levels of oxygen encountered in ambient air. A controlled anaerobic environment is vital for the cultivation of P. gingivalis.

1. Here, maintain an artificial atmosphere designated as mixed anaerobic gas (80% N₂, 10% H₂, 10% CO₂) in a vinyl anaerobic chamber (Figure 1A). Use an airlock (Figure 1B) for transferring items from the laboratory environment to the anaerobic chamber. The airlock operates manually, twice purging with N₂ gas before introducing the mixed anaerobic gas.
2. Use a hydrogen sulfide removal column (Figure 1C) for maintenance-free removal of the undesirable hydrogen sulfide. Place a dehumidifier within the chamber to remove H₂O created by the catalyst and to avoid aerosols that facilitate the spread of contamination.
   Note: Hydrogen sulfide is a natural metabolic byproduct of many anaerobic bacteria and its accumulation is toxic to bacteria and can result in damage to electronics and decrease the lifetime of a catalyst.
3. Use a fan box to circulate the chamber's atmosphere through a palladium catalyst, which removes oxygen in the presence of hydrogen (Figure 1D).
   Note: A recirculating atmospheric (high-efficiency particulate air or HEPA) filter removes airborne contaminants with a size of 0.22 µm or larger.
4. Culture anaerobic bacteria in a 37 °C incubator that is located inside the anaerobic chamber. Use standard aseptic techniques when working inside the anaerobic chamber.

2. Preparation of Anaerobic Bacteria

Note: P. gingivalis is aerotolerant and can be stored in aerobic conditions but it will not grow in the presence of oxygen at levels higher than 6%. An anaerobic chamber is necessary for the proper cultivation of P. gingivalis and other anaerobic species (Figure 1). Proper training and education on anaerobic chamber use are required before working with micro anaerobes.

1. Equilibrate all liquid media and plates to anaerobic conditions for at least 12 hr prior to experimentation to remove residual oxygen.
2. Transfer P. gingivalis from a -80 °C freezer to an anaerobic chamber, and let thaw.
3. Streak P. gingivalis on trypticase soy blood agar plates (TSA II with 5% sheep blood). Wrap plates in parafilm and store at 37 °C in an anaerobic incubator for 4-7 days.
4. Inoculate P. gingivalis into 3 ml brain heart infusion (BHI) broth supplemented with hemin and menadione, an enriched non-selective liquid media for the isolation and culture of anaerobic and fastidious microorganisms, using sterile loops.
   Note: For long-term storage, mix bacterial cultures prepared in BHI with glycerol or DMSO (10-20% final concentration) and place in a -80 °C freezer.
5. Prepare a starter culture of P. gingivalis by making a 1:10 dilution and allowing bacteria to grow until the mid-log phase.

Note: The optical density of the bacterial suspension is determined and the bacterial concentration for each strain to be examined is adjusted. For P. gingivalis a suspension at OD₆₆₀ of 0.7 corresponds to mid-log phase and ~7 x 10⁸ cells/ml. Growth conditions described in the protocol above are specific for P. gingivalis and may need to be adapted for other bacterial strains.

3. Endothelial Cell Culture

Note: Purchase pooled primary HUVECs and culture in basal medium containing vascular endothelial growth factors (VEGF) at 37 °C in 5% CO₂ according to manufacturer's instructions.

1. Seed HUVECs in T-75 flasks at 2.5 x 10⁵ cells/flask in 15 ml VEGF media.
   Note: Check viability via a 1:1 dilution with 4.0% trypan blue. Cells with a compromised membrane will retain trypan blue, and healthy cells with intact membranes will appear white when viewed under a binocular light microscope. Count 100 cells, and ensure that over 80% of the cells are viable.
2. Replace media every 2 days with pre-warmed fresh VEGF media until cells reach ~80% confluency.
3. Wash cells once with pre-warmed phosphate-buffered saline (PBS). Liberate cells from the T75 flask by incubating with 2 ml trypsin-EDTA (0.25%) for 5 min followed by 2 ml trypsin neutralizing solution.
4. Collect suspended HUVECs in a 50 ml conical tube. Wash out any extra cells from T-75 flasks with PBS and transfer to 50 ml conical tubes.
5. Centrifuge cells at 200 x g for 10 min.
6. Remove supernatant, and suspend cell pellet in 10 ml pre-warmed VEGF media.
7. Determine cell concentration using a hemocytometer or similar cell counting device.
8. Calculate the amount of cell suspension to add to either a 6-well plate (400,000/well) or a 12-well plate with coverslips (50,000/well). HUVECs will be ready for experimentation the next day.

4. Survival Assay Invasion/Interaction (Plating)

Note: When performing this assay, prepare two 6-well plates of endothelial cells seeded at 400,000 cells/well. One plate will be used to assess bacteria attached to and internalized by host cells. The other plate will account for intracellular bacteria. The 6-well plate allows for triplicates of two samples to be performed in one experiment. For an outline of this protocol please refer to the survival assay flowchart (Figure 2).

1. Prepare anaerobic bacteria as described above (see section 1) until they reach mid-log growth (OD₆₆₀ 0.5-0.7).
2. Centrifuge bacteria at 5,000 x g for 10 min.
   Note: If the centrifuge is outside an anaerobic chamber, carry bacterial samples in a tightly sealed 15 ml tube, and wrap the cap with parafilm to prevent oxygen leakage.
3. Place pelleted P. gingivalis back in the chamber, and discard supernatant. Wash with PBS, and pellet bacteria again before resuspending in VEGF media. Prepare suspensions for all bacterial strains to be tested at OD₆₆₀ of 0.7 which corresponds to the mid-log phase (~7 x 10⁸ cells/ml). The bacteria are now ready for infection.
4. Transfer 6-well plates containing HUVECs from the tissue culture incubator into the anaerobic chamber. Remove media and wash three times with anaerobic PBS. Add 2 ml of anaerobic VEGF media to each well and place the plates at 37 °C in the anaerobic incubator for 20 min to equilibrate the temperature for infection.
   Note: Plate bacteria on blood agar plates to ensure the ones used for infection are homogenous and not contaminated upon infection.
5. Infect host cells with bacteria at a multiplicity of infection (MOI bacteria: host) of 100:1.
   Note: HUVEC cell number is determined by performing a trypan exclusion test on a single well before infection. Bacterial cell number is determined via optical density (e.g., OD of 0.5 = 5 x 10⁸ cells/ml). Bacterial concentration is adjusted to proper MOI based on HUVEC's concentration.
6. Place 6-well plates with infected HUVECs into the anaerobic incubator and allow bacteria to interact with host cells for 30 min.
7. Prepare saponin in BHI (1.0% w/v) inside the anaerobic chamber and filter through a 0.2 µm filter.
8. Survival of both attached and internalized bacteria.
   1. Remove plates from the incubator, aspirate media, wash three times with anaerobic PBS, and add 2 ml filtered 1.0% saponin (prepared as described in step 4.8). Incubate for 15 min to allow host cell lysis.
   2. Scrape the bottom of each well with a cell scraper. Collect the cell mixture from each well and make a 1:1 dilution in BHI.
   3. Proceed to make serial dilutions of the sample. Depending on bacterial species and concentration, adjust serial dilutions. Start with 1:100 or 1:1,000 dilutions.
   4. Plate 200 µl of desired dilution on blood agar plates. Wrap the plates in parafilm and place them in the anaerobic incubator at 37 °C.
   5. Following seven days of incubation at 37 °C, remove the plates and count colony forming units (CFUs) using a lightbox to manually count colonies.
      Note: CFUs are enumerated. For larger quantities of CFUs, images may be taken and computer software can be used to facilitate the enumeration of CFUs.