An Assay for Bacterial Extracellular Protein-Mediated Cytotoxicity on PMNs
Transcription
Take a culture of Staphylococcus aureus, a pathogenic bacteria that secretes leukocidin — a pore-forming toxin — comprising monomeric S and F components.
Pellet the cells and serially dilute the supernatant. Transfer the leukocidin-containing supernatant into a microplate having polymorphonuclear leukocytes, or PMNs, and incubate.
The S component binds to specific transmembrane receptors on PMNs, followed by hetero-oligomerization with the F component to insert into the cell membrane, resulting in pore formation.
The pore causes a decrease in intracellular potassium ions, activating NLR family pyrin domain-containing 3 or NLRP3 protein.
NLRP3 binds to apoptosis-associated speck-like protein containing a caspase recruitment domain, or ASC, which recruits pro-caspase-1, forming an inflammasome.
Auto-proteolysis converts pro-caspase-1 to active caspase-1, triggering cell death.
Harvest the cells at defined intervals. Add propidium iodide or PI, which enters dead cells through damaged membranes to bind DNA, emitting fluorescence.
Using flow cytometry, quantify PI-stained cells to assess the cytotoxic effect of both leukocidin concentration and incubation time.
