This study investigates the interaction between Shigella flexneri and cancerous epithelial cells, focusing on the mechanisms of bacterial uptake and the formation of stress granules. The research highlights the role of virulence proteins in modulating cellular processes and the subsequent cellular response.
Begin with a multi-well plate containing adherent cancerous epithelial cells on a coverslip.
Add Shigella flexneri, a pathogenic bacterium.
Centrifuge the plate to promote bacterial contact with the cells, then incubate.
Using a specialized secretion system, Shigella injects virulence proteins into the epithelial cells.
These virulence proteins modulate the epithelial signaling system, facilitating bacterial uptake.
Inside the cells, the bacteria continue secreting virulence proteins, which disrupt cellular translation and accumulate untranslated mRNAs.
RNA-binding and signaling proteins aggregate with the untranslated mRNAs, forming membrane-less cytoplasmic granules known as stress granules.
Discard the media and wash to remove unattached bacteria.
Next, add pre-warmed culture media supplemented with gentamicin to eliminate surface-attached bacteria.
Add a fixative to preserve cellular structures and stress granules.
Discard the fixative and wash with gentle shaking to remove residual fixative.
The cells are ready for immunofluorescence staining to visualize Shigella-induced stress granules.
Replace the wash with 500 microliters of infection medium per well, and centrifuge the 24-well plate to spin the bacteria onto the cells.
Transfer the settled bacterial cocultures into a 37 degree Celsius tissue culture incubator for 30 minutes to facilitate the host cell infection.
At the end of the incubation, remove the exogenous bacteria from the cells with three rinses in 1 milliliter of fresh 37 degree Celsius HeLa culture medium per wash.
Then, cover the infected cells with 1 milliliter of fresh culture medium containing gentamicin and return the plate to the cell culture incubator.
After one hour, aspirate the supernatant completely, and fix the samples in 0.5 milliliters of room temperature 4% paraformaldehyde in PBS for 30 minutes.
Next, discard the fixative into the appropriate waste container, and wash the coverslips with 1 milliliter of tris-buffered saline for two minutes with gentle shaking.
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