This study investigates the interaction between intracellular pathogenic bacteria and human epithelial cells, focusing on the remodeling of the endo-lysosomal system. The methodology involves using fluorescently labeled endosomes to visualize the effects of bacterial infection on cellular structures.
Take a chambered slide containing human epithelial cells with fluorescently labeled endosomes.
Introduce intracellular pathogenic bacteria and incubate, allowing them to adhere to the cells.
The bacteria inject proteins that reorganize the host cytoskeleton, forming membrane protrusions that internalize the bacteria into vacuoles.
These vacuoles extend tubular projections connecting to the endo-lysosomal system, a network of vesicles and organelles regulating intracellular trafficking.
These projections redirect the vesicle transport and remodel the endo-lysosomal system.
Wash to remove extracellular bacteria, then incubate with an antibiotic-containing medium to eliminate any remaining bacteria.
Replace with a serum-free imaging medium containing the antibiotic, and add nanoparticles labeled with a contrasting fluorophore.
Incubate to allow nanoparticle entry via endocytosis and accumulation in the remodeled vesicles.
Wash to remove non-internalized nanoparticles, then incubate in a serum-supplemented imaging medium.
The fluorescent nanoparticles and labeled endosomes enable visualization of the bacteria-induced endo-lysosomal remodeling.
To begin this procedure, measure the OD 600 of the subcultured bacteria and dilute to an OD 600 of 0.2 in one milliliter of PBS, add appropriate amounts of bacteria to the HeLa cells in the 8 well chamber slide to achieve a multiplicity of infection of 100. Incubate for 30 minutes in the cell incubator.
Wash the HeLa cells three times with PBS to remove non internalized bacteria. This time point is set as zero hour post-infection or zero hour p.i. Add 300 microliters of fresh culture medium containing 100 micrograms per milliliter of gentamicin to the cells and maintain for one hour.
After one hour, replace the medium with imaging medium containing 10 micrograms per milliliter of gentamicin. Add gold-BSA-rhodamine NPs to the HeLa cells to obtain a final concentration of OD 520 of 0.1.
Incubate for one hour. After one hour, remove the medium and wash three times with PBS. Finally, add 300 microliters of fresh imaging medium containing 10 percent FCS and 10 micrograms per milliliter of gentamicin for the rest of the incubation time.
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