Take a multi-well plate containing adhered epithelial cells and treat it with green fluorescent protein-tagged Coxiella mutants; some with mutations in the type IV secretion system gene, or T4SS gene — crucial for replication.
Replicative mutants interact with epithelial cells, triggering Coxiella engulfment, forming Coxiella-containing vacuole, or CCV, and recruiting lysosome-associated membrane glycoprotein-1, or LAMP1 to CCV.
This facilitates effector proteins' secretion into the cell cytoplasm via the type IV secretion system, promoting bacterial replication.
Mutants with T4SS gene mutations remain unreplicated.
Post-incubation, stain the cells with a fluorescent dye, fix them, and permeabilize the cell membrane.
Introduce LAMP1-specific primary antibodies interacting with the LAMP1 proteins on CCV.
Overlay with red-fluorophore-labeled secondary antibodies, interacting with primary antibodies, while Hoechst dye stains the cells' nuclei.
Under an epifluorescence microscope, red-fluorescence vacuoles containing fewer green Coxiella near the nucleus indicate defective replication of Coxiella mutants.
However, vacuoles containing many green Coxiella inside the cells indicate the successful intracellular growth of replicative Coxiella mutants.
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