This study investigates the adherence of Haemophilus influenzae type f (Hif) to epithelial cells mediated by vitronectin. The protocol outlines the steps for preparing epithelial cell monolayers and assessing bacterial adherence through colony counting.
Take a multi-well plate containing adherent epithelial cell monolayers. Replace media with serum-free media, preventing serum protein interference. Post-incubation, remove media. Wash the cells with buffer. Add chilled serum-free media containing vitronectin. Incubate at lower temperatures.
The N-terminal RGD-domain of vitronectin binds to the epithelial cell surface integrin receptors. Lower temperatures inhibit cellular internalization of the vitronectin-integrin complex.
Following integrin receptor saturation, remove media. Wash with buffer to remove excess vitronectin, which could impact the bacterial vitronectin-dependent integrin binding.
Add Haemophilus influenzae type f, or Hif, a gram-negative bacterial pathogen, suspension. Incubate.
Protein H on the Hif surface binds to the vitronectin's C-terminus, facilitating Hif adherence to the epithelial cell and subsequent internalization.
Remove media and wash with buffer to remove unbound Hif. Use a cell detachment enzyme to harvest the cells. Add serum-containing media to stop the enzymatic reaction. Transfer the cell suspension to a glass tube containing glass beads. Vortex to lyse the cells, releasing internalized and surface-attached bacteria.
Dilute the lysate with serum-free media and seed on a chocolate agar plate. Incubate for Hif to form colonies. Count the colonies.
A higher number of colonies indicates enhanced Hif adherence to the epithelial cells in the presence of vitronectin.
Culture A549 epithelial cells as described in the text protocol. After one wash and trypsinization of the cells at 37 degrees Celsius, dilute the cell suspension to 5,000 cells per milliliter using complete medium containing 5 micrograms per milliliter of gentamicin.
Prior to bacterial infection, replace the medium in the wells with F12 medium and incubate overnight at 37 degrees Celsius. Wash the cell monolayer three times with 1 milliliter of PBS at room temperature. Then, place the plate on ice and add 200 microliters of pre-chilled F12 medium, containing 10 micrograms per milliliter of vitronectin.
After incubating the plate at 4 degrees Celsius for 1 hour, discard the solution by pipetting, and wash the cell layer twice with 1 milliliter of PBS at room temperature. Add 100 microliters of freshly grown Hif wild-type in F12 medium to each well. Incubate the plate for 2 hours at 37 degrees Celsius. Then aspirate the medium with a pipette, and wash the A549 epithelial cells three times with PBS at 50 microliters per well of cell detachment solution and follow with a 5-minute incubation at 37 degrees Celsius.
Next, add 50 microliters of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells from each well to a 6-milliliter glass tube containing 4 glass beads. Lyse the cells at room temperature by vortexing for 2 minutes.
After diluting the cell lysate 100-fold, plate 10 microliters of the diluted sample onto a chocolate agar plate. Count the colonies following an overnight incubation at 37 degrees Celsius.
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