Lysostaphin-Based Enzyme Protection Assay: An In Vitro Method to Quantify Intracellular Staphylococcus aureus Load by Enzyme-Mediated Selective Killing of Extracellular Bacteria

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.