The overall goal of this experiment is to measure the ability of bacterial strains to invade host cells. Host cells in culture are transferred onto glass cover slips in a 24 well plate and incubated until they reach co fluency. The cover slips are then washed and transferred to a new plate.
Bacteria harvested from a standard broth culture are washed by centrifugation and then added to the host cell culture. Once invasion has occurred, extracellular bacteria are killed using Gentamycin and the cover slips are washed and transferred to a new plate. Finally, the host cells are lysed and the lysates plated on agar plates to determine the number of intracellular bacteria.
Ultimately, results can be obtained that show the invasive ability of specific bacteria. This method can help answer key questions in the field of microbial pathogenesis, such as how bacterial invasion of host cells causes disease. It can also facilitate the identification and characterization of bacterial proteins involved in host pathogen interactions.
Demonstrating this procedure will be Dr.Andrew Edwards, a postdoc from a laboratory. For this assay we use human endothelial EAHY 9 26 cells cultured in T 75, flasks at 37 degrees Celsius. Harvest the cells once they've reached about 95%Co fluency.
Wash the cells with 10 milliliters of PBS To remove the growth media. Detach the cells by incubating in trypsin EDTA solution for five minutes. Then transfer to a chronicle tube.
Next centrifuge cells and Resus, suspend them in about 25 milliliters of complete DMEM medium or at a concentration of 1 million cells per milliliter using sterile forceps. Place a thermon cover slip in each well of a 24 well tissue culture plate. Be sure to place the opaque side of the cover slip on which cells can adhere facing upwards.
Next, add 500 microliters of cells to each well and incubate the plate at 37 degrees Celsius for 48 hours or until the cells have reached co fluency. Following the incubation, use clean forceps to carefully remove each cover slip and dip them in DMEM to remove soluble secreted proteins. Transfer the washed cover slips to individual wells of a fresh 24 well plate containing 490 microliters per well of complete DMEM.
Taking care to maintain the orientation of the cover slips so that the cells are facing upward. Prepare a 10 milliliter culture of staphylococcus aureus in brain heart infusion broth. Transfer the bacterial culture into a conical tube and centrifuge the bacteria 5, 000 times G for 10 minutes.
Once the cells have been pelleted, remove the supinate and Reese. Bend the pellet in 10 milliliters of sterile DMEM to wash the cells, centrifuge the cells once more and repeat the wash three times. Reese bend the bacteria in DMEM and measure the optical density of the suspension.
Adjust the concentration to obtain an OD 600 of 1.0, which corresponds to approximately 10 to the nine colony forming units per milliliter. Once the bacterial suspension is ready, add 10 microliters of bacteria to each well. Of the 24 well plate containing the cover slips.
Incubate the plate at 37 degrees Celsius for 15 to 90 minutes to allow the bacteria to attach to and invade the cells. Following this incubation, aspirate the supinate from each well containing the unbound bacteria and replace it with 10%F-B-S-D-M-E-M supplemented with Gentamycin. Incubate the plate at 37 degrees Celsius for 60 minutes to kill the extracellular bacteria.
Following this incubation dip. Wash each cover slip in DMEM three times to remove antibiotic. Then transfer them to a new 24 well plate containing 500 microliters of 0.5%Triton X 100 in PBS per well.
As previously, take care to keep the cell covered side facing up vigorously rinse each cover slip with Triton solution by pipetting the liquid in the well up and down directly onto the cover slip so as to ensure that all the cells will be lysed and the intracellular bacteria released. Perform serial dilutions of the lysate to quantify the number of infecting bacteria. Collect the lysate and plate serial dilutions on TSA plates.
Colony forming units can be determined on these plates after 24 hours of incubation at 37 degrees Celsius. This graph shows the number of wild type or fibrin connecting binding protein deficient staphylococcus aureus invading human endothelial cells compared to wild type bacteria. Fibronectin binding protein deficient, staphylococcus aureus are significantly impaired in their ability to invade endothelial cells.
Complementation of the mutant with a plasmid inod in fibronectin binding protein, A minus the fibronectin binding domain did not promote invasion. In contrast, complementation of the mutant with a plasmid encoding the entire fibronectin binding protein, aging restored invasion to wild type levels. In this experiment, lactococcus Lactus expressing the fibrin necking binding protein was shown to be able to invade host cells.
Whereas bacteria not expressing the protein were not the ability to adhere to cells was the same for both strains. Once mastered, this technique can be performed within three hours. After watching this video, you should have a good understanding of how to prepare bacteria and host cells to study cell invasion.
You should also be able to identify the stages of which additional questions can be asked to increase your understanding of this aspect of disease.
