This study investigates the interaction between Pseudomonas aeruginosa and rat pulmonary endothelial cells, focusing on the effects of bacterial effector proteins on cellular structures. The research highlights the mechanisms leading to cytoskeletal disruption and the release of cytotoxic amyloid oligomers.
Take a monolayer of rat pulmonary endothelial cells.
Wash the cells with buffer. Add a suspension of Pseudomonas aeruginosa and incubate.
During incubation, the bacteria contact the cells and inject effector proteins into the cytoplasm.
These effector proteins induce microtubule breakdown and cytoskeletal disruption.
The cytoskeletal disruption activates cellular stress responses, leading to the formation of misfolded, cytotoxic protein aggregates-amyloid oligomers.
The effector proteins also create pores in the cell membrane, allowing amyloid oligomer release into the extracellular space.
In addition, the cytoskeletal disruption alters cellular morphology, creating intercellular gaps in the endothelial monolayer.
Next, observe the monolayer under a light microscope. Once the intercellular gaps become evident, terminate the incubation.
Then, collect the supernatant containing cytotoxic amyloids, and centrifuge to pellet the debris.
Collect the supernatant and filter it to remove any remaining bacteria.
The harvested supernatant containing cytotoxic amyloids is ready for further analysis.
To produce cytotoxic supernatant, wash a 150 centimeter dish of endothelial cells with HBSS, and dilute a P. aeruginosa colony to an absorbance of 0.25 at 540 nanometers. Further dilute the bacterial cells to allow a multiplicity of infection of 20 to 1 in 20 milliliters of HBSS and seed the bacteria onto the plate of endothelial cells. Then place the infected cells in a 37 degrees Celsius and 5% CO2 incubator for four to five hours.
It is essential that the incubation of the bacteria and the endothelial cells occur for an appropriate length of time. If the incubation is too short, the amyloids will not be released into the supernatant. If the incubation is too long, the cells will actually lyse and release all of their contents into the supernatant. The indicator that we use for an appropriate duration of incubation is the formation of gaps in the endothelial monolayer.
When gaps can be observed in the cell monolayer by light microscopy, harvest the supernatant for centrifugation to remove any cellular debris. Decant the supernatant into a syringe that is equipped with a 0.2 micron filter and pass the supernatant through the filter to remove any contaminating bacteria.
Then set aside 1.5 milliliters of sterile supernatant for cytotoxicity testing and freeze the rest of the sample at minus 80 degrees Celsius.
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