An Assay to Study the Role of Vitronectin in Bacterial Adherence to Host Epithelial Cells

1. Study of Vn-dependent adherence of bacteria to epithelial cells

1. Culture A549 cells (type II alveolar epithelial cells) in a 75-cm² tissue culture flask with F12 medium supplemented with 10% (vol/vol) fetal calf serum (FCS) (complete medium) and 5 µg/mL gentamicin. Incubate the flask in an incubator with 5% CO₂ at 37 °C for 3 days until 80% confluent (confluency can be estimated by visualizing surface coverage by the cells using an inverted microscope). The following four steps describe how to prepare the epithelial cells for the assay.
   1. Wash the cell monolayer in the flask twice with 20 mL of PBS by gentle swirling. To detach the cells from the flask surface, add 2 mL of cell detachment enzyme solution and incubate the flask for 5 min at 37 °C. Tap the flask with your palm to detach all the cells from the plastic surface. Pipette up and down a few times to disperse cell clumps.
   2. Add 18 mL of F12 complete medium to the flask and transfer the entire cell suspension to a 50-mL sterile Falcon tube. Centrifuge the cell suspension for 5 min at 200 x g at RT and discard the supernatant. Resuspend the cell pellet in 10 mL of F12 complete medium.
   3. Remove 10 µL of the cell suspension and place it in an Eppendorf tube, then add 90 µL of Trypan blue solution. Load the sample into a hemocytometer (depth 0.1 mm) after placing the coverslip.
   4. Count all viable cells in areas A, B, C, and D (each field consists of 16 squares, and each square has an area of 0.0025 mm²), then calculate the average number of cells ([A+B+C+D]/4). Calculate the number of cells per milliliter using the following equation:
      Viable cells/mL = average cell count × 10⁴ × dilution factor.
      NOTE: Here, the dilution factor is 10.
   5. Dilute the cell suspension to 5.0 x 10³ cells/mL using a complete medium containing 5 µg/mL gentamicin. Dispense 500 μL of cell suspension into each well of a 24-well cell culture plate. Incubate the plate at 37 °C in 5% CO₂ until the cells are 90% confluent.
2. Prior to bacterial infection, remove the medium from the wells and add F12 medium (without FCS), then incubate overnight at 37 °C.
3. Wash the cell monolayer three times with 500 µL of PBS at RT. Place the plate on ice and add 100 µL of pre-chilled F12 medium containing 10 µg/mL of Vn. Incubate plate at 4 °C for 1 h. For control wells, add only F12 medium.
4. After incubation, discard the solution by pipetting and wash the cell layer twice with 1 mL of PBS at RT.
5. Culture Hif clinical isolates in brain-heart infusion (BHI) medium supplemented with 10 µg/mL NAD and hemin at 37 °C with shaking at 200 rpm. Use Luria-Bertani medium to culture E. coli. Harvest Hif at mid-log phase (OD600 = 0.3) and resuspend the bacteria in phosphate-buffered saline (PBS), pH 7.2, containing 1% (w/v) bovine serum albumin (BSA) (blocking buffer; PBS-BSA). Adjust the suspension to 10⁹ CFU/mL. Resuspend a culture of freshly grown Hif M10 in F12 medium (2×10⁸ CFU/mL). Add 100 µL of this bacterial suspension to each well and incubate the plate for 2 h at 37 °C.
   NOTE: Each well contains approximately 2 x 10⁵ A549 cells. For infection, 2 x 10⁷ bacterial CFU were added, corresponding to a multiplicity of infection of 100.
6. Remove the medium by pipetting and wash the A549 epithelial cells three times with PBS. Add 50 µL/well of cell detachment solution and incubate the plate for 5 min at 37 °C.
7. Next, add 50 µL of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells (≈100 μL [i.e., the entire volume]) from each well to a 6-mL glass tube containing four glass beads. Lyse the cells at RT by vortexing for 2 min.
8. Dilute 10 µL of the cell lysate 100-fold by adding 10 µL of lysate to 990 µL of F12 medium. Plate 10 µL of the diluted sample onto a chocolate agar plate.
9. Incubate the chocolate agar plate at 37 °C overnight, then count the colonies. Each colony represents one CFU.