This study investigates the adherence of fluorescent protein-tagged Pseudomonas aeruginosa to epithelial cell monolayers. The methodology allows for quantification of host-pathogen interactions through a bacterial adherence assay.
Take a multi-well plate containing adherent epithelial cell monolayer susceptible to bacterial adherence.
Add varying numbers of fluorescent protein-tagged Pseudomonas aeruginosa, a pathogenic bacterium, to achieve the desired bacteria-to-epithelial cell ratio.
Centrifuge to bring the bacteria closer to the epithelial cells, facilitating interactions.
Type IV pili, proteinaceous fibers on the bacteria, play a pivotal role by binding to specific glycolipids on the epithelial cells, mediating bacterial adherence to the host cells.
Post-incubation, wash with a buffer to remove non-adherent bacteria. Fix the cells to preserve the cell structure.
Add DAPI, a fluorescent stain that binds strongly to A-T-rich DNA regions, staining the nucleus. Cover the cells with buffer to prevent cell drying.
Acquire images of the GFP-labeled bacteria attached to epithelial cells with DAPI-stained nucleus. Enumerate the adherent bacteria on a single epithelial cell.
Increased adherent bacteria per host cell in a dose-dependent manner, enables quantification of host-pathogen interactions.
For bacterial adherence assay, wash the previously cultured A549 cell monolayers three times by adding 100 microliters of warm PBS to each well, and gently pipette up and down. Then, discard PBS. Alternatively, after adding PBS, wait for ten seconds and then, remove PBS using vacuum.
To determine the kinetics of bacterial association, overlay the cells with 100 microliters of desired concentrations of bacterial suspension with different multiplicity of infection. Spin down the bacteria and incubate the infected A549 cells at 37 degrees Celsius and 5% carbon dioxide for an additional one hour. Remove the unbound bacteria by washing the monolayers five times with warm PBS as demonstrated.
Next, fix the cells by adding 100 microliters of 4% formaldehyde in all the wells of a 96-well plate on the ice. After 15 minutes, wash the plate three times with PBS to remove the fixation solution, staining the nuclei with 50 nanograms per milliliter DAPI stain for 10 minutes at room temperature.
After washing the cells with PBS, cover the infected A549 cells with 100 microliters of PBS to avoid drying, and then, store the plate at 4 degrees Celsius for up to two days in the dark or proceed for imaging.
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