Investigating Autophagy-Mediated Clearance of Intracellular Bacteria In Vitro

Take pig kidney cell cultures, with one culture pre-treated with an inhibitor that increases lysosomal pH, impairing autophagic-lysosomal degradation.

Add pathogenic bacteria and centrifuge to enhance host-pathogen contact.

Incubate to allow bacterial invasion and internalization into endocytic vesicles.

The internalized bacteria consume host nutrients to replicate, triggering metabolic stress.

This stress activates autophagy, where an autophagic membrane expands around the vesicle, forming an autophagosome.

In the untreated cells, the autophagosome targets bacteria for lysosomal degradation.

In the inhibitor-treated cells, autophagic clearance is impaired, leading to higher bacterial loads.

Post-infection, wash the cells and apply an antibiotic to eliminate the extracellular bacteria.

Add an enzyme to digest the extracellular matrix and dislodge the cells.

Mechanically lyse the cells to release the intracellular bacteria.

Plate the lysates to allow bacterial growth.

Fewer colonies from the untreated cells confirm the role of the autophagy-lysosomal degradation pathway in clearing intracellular bacteria.