This study investigates the interaction between bacterial pathogens and mammalian epithelial cells using polymer beads that mimic bacterial surfaces. The experimental setup aims to reduce bacterial attachment to host cells, providing insights into bacterial-host interactions.
Take a multi-well plate containing a mammalian epithelial cell culture.
In the test wells, add a bacterial pathogen along with polymer beads coupled to bacterial surface proteins.
In the control wells, add the pathogen and beads lacking bacterial surface proteins. Incubate.
In the test wells, the beads mimic the bacterial surface and compete with the pathogen for binding to epithelial cell membrane phosphatidic acid, reducing bacterial attachment.
In the control wells, beads do not affect bacterial attachment.
Remove the media and wash with buffer to eliminate unbound components.
Incubate with a non-ionic detergent to lyse the cells, releasing membrane-attached bacteria and beads.
Pipette the lysate to create a homogeneous suspension, and serially dilute the homogenate using buffer.
Transfer the dilutions onto agar plates, incubate to allow colony formation, and count the colonies.
The test plate exhibits fewer colonies than the controls, indicating reduced bacterial-host interactions.
Prepare infection media by diluting bacterial cultures into colorless DMEM without additives.
Prewarmed to 37 degrees Celsius containing 10% volume to volume bead suspension. To give an MOI of 10, prepare one milliliter per well and 10 to 20%excess volume per sample. Remove the old medium from the wells and wash the cultured HeLa cells by adding to each well one milliliter of sterile PBS prewarm to 37 degrees Celsius. Remove the PBS and add one milliliter of infection medium per well. Add solutions containing control beads and bacteria as positive controls and adhesion beads and no bacteria as negative controls.
Add DMEM containing 0.1% Triton X 100 as lysis controls.
Incubate the plate in a tissue culture incubator at 37 degrees Celsius for the desired amount of time.
Measurements of bacterial adhesion are made one hour after infection. Remove the media from the cell layer and thoroughly wash the cell layer with sterile prewarmed PBS to remove any unattached cells. Wash at least three to four times with one milliliter of PBS each time. Lyse the host cells by adding to each well one milliliter of a sterile 1%volume to volume Triton X 100 solution in PBS. Incubate the plate at 37 degrees Celsius for five minutes.
Pipette each sample up and down several times before transferring the contents of each well to separate 1.5 milliliter tubes. Prepare tenfold serial dilutions of the samples into sterile PBS. Plate 100 microliters of each sample on marine LB agar and spread using a cell spreader. Optimize which dilution to plate depending on the bacterial strains and time point.
For this experimental setup a 10 to the fifth or 10 to the sixth fold dilution will give a suitable number of CFUs. Incubate the plates at 37 degrees Celsius overnight. On the following day, enumerate bacteria by colony counting.
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