This study investigates the interaction between Staphylococcus aureus and epithelial cells, focusing on the mechanisms of bacterial internalization. The experiment utilizes lysostaphin to selectively target extracellular bacteria while preserving intracellular ones.
Begin with a monolayer of epithelial cells in a suitable culture medium. These cells express integrins - membrane-spanning surface receptors. The culture media contains epithelial cell-secreted fibronectin - a glycoprotein.
Add a suspension of Staphylococcus aureus - a gram-positive pathogenic bacteria - to the culture plate. These bacteria contain fibronectin-binding proteins, or FnBPs, anchored in their cell wall.
Incubate the plate to initiate bacterial infection, wherein the fibronectin binds to the bacteria's FnBP and the integrins on the epithelial host cells. This tripartite FnBP-fibronectin-integrin interaction mediates bacterial internalization in the host.
Treat the cells with lysostaphin - an endopeptidase enzyme - and incubate. Lysostaphin selectively enters the cell walls of extracellular bacteria. It cleaves the pentaglycine bridges in the peptidoglycans, thereby lysing them while the intracellular bacteria remain protected from lysostaphin.
Supplement the culture with a proteolytic enzyme to deactivate the remaining lysostaphin, preventing it from killing the intracellular bacteria in the steps.
Add a lysis buffer to the culture, which ruptures the host epithelial cells and releases intracellular bacteria in the cell lysate. Subsequently, plate the cell lysate containing bacteria on a suitable culture plate and incubate.
The bacteria grow and multiply to form bacterial colonies, which can be analyzed to estimate the extent of bacterial internalization in the host cells.
Before cell inoculation, observe every well of the 24-well plate by low magnification microscopy to ensure that the cells are healthy and growing as expected. Then, remove and discard the spent cell culture medium from the 24-well plate, and add 500 microliters of the bacterial suspension to each well containing 100% confluent cells. Incubate the cells for 2 hours at 36 degrees Celsius and 5% carbon dioxide.
To quantify the intracellular bacteria with the improved enzyme protection assay, prepare 4X lysis buffer, 2% Triton X-100, trypsin-EDTA, and lysostaphin stock and working solutions as mentioned in the text manuscript.
Next, prepare 6.25 milliliters of complete infection medium supplemented with lysostaphin, by adding 6 milliliters of complete infection medium to 250 microliters of the lysostaphin working solution. Then, add 250 microliters of the complete infection medium supplemented with lysostaphin into each well, and gently agitate the plate by swiveling the plate by hand.
To allow the lysostaphin to kill the extracellular bacteria, incubate the cells for 1 hour at 36 degrees Celsius and 5% carbon dioxide. Then, inactivate the lysostaphin by adding 10 microliters of proteinase K at 20 micrograms per milliliter into each well, and incubating the cells for 2 minutes at room temperature.
Next, add 250 microliters of 4X lysis buffer to lyse the cells by osmotic shock, and incubate the cells for 10 minutes at 36 degrees Celsius. After the incubation, pipette the cell lysate up and down several times, to ensure that the cells are fully lysed and homogenized.
Then, use an automatic spiral plater to determine the Staphylococcus aureus load of each well, and incubate the agar plates for 18 to 24 hours at 36 degrees Celsius. The next day, count the number of colonies with a colony counter to calculate the intracellular Staphylococcus aureus load of each well.
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