A Bacterial Infection Assay for Studying Anaerobic Bacteria-Host Cell Interactions
Transkript
Begin with a culture of endothelial cells.
Replace the media with anaerobic media. Incubate in an anaerobic chamber for temperature equilibration.
Add Porphyromonas gingivalis, a pathogenic anaerobic bacterium, and incubate briefly.
The bacteria possess proteinaceous projections, known as fimbriae, which bind to specific adhesion molecules on the endothelial cells, facilitating bacterial adherence.
This binding triggers intracellular signaling cascades, resulting in actin cytoskeleton rearrangement and bacterial internalization into phagosomes.
Post-incubation, remove media. Wash with an anaerobic buffer to remove non-adhered extracellular bacteria.
Add a mild detergent to permeabilize the host cell membranes and induce cell lysis, releasing internalized and surface-attached bacteria without causing damage.
Dilute the lysate-containing bacteria with media and prepare serial dilutions.
Plate these dilutions on blood agar plates. Incubate under anaerobic conditions to allow Porphyromonas gingivalis to proliferate and form colonies.
Post-incubation, count the colony-forming units (CFUs) on each plate to quantify surface-attached and internalized bacteria, indicating bacteria-host cell interactions.
