This method can help answer key questions in host-pathogen interaction field such as how bacterial pathogens cause disease in humans by utilizing human proteins. The main advantage of this technique is it can answer if any pathogen can utilize Vitronectin for establishing infection of the host. We will focus upon Vitronectin, a host protein that takes part in the extracellular matrix, but also functions as a complement inhibitor in determinant pathway of complement activation.
These facts are very important and highly attractive for pathogens causing disease in humans. To prepare for flow cytometry, resuspend the bacterial pellets with 50 microliters of blocking buffer containing 250 nanomolar Vitronectin. Then, incubate the samples for one hour at room temperature without shaking.
After incubation, pellet the bacteria by centrifugation at 3, 500 g for five minutes. Then, wash the pellets three times using PBS and similar centrifugation steps. Following wash, add 50 microliters of primary sheep antihuman Vitronectin polyclonal antibodies to the bacterial pellet at a one to 100 dilution in PBS BSA.
Incubate the suspension for one hour at room temperature. Then, wash the bacteria three times with PBS to remove unbound antibodies. Next, add 50 microliters of PBS BSA containing fluorescein isothiocyanate conjugated donkey anti-sheep polyclonal antibodies to the pellet.
Incubate at room temperature for one hour in the dark. Finally, after three washing steps, resuspend the bacterial pellet with 300 microliters of PBS and analyze by flow cytometry. To prepare for ELISA, dispense 100 microliters of protein solution into each well of a PolySorp microtiter plate.
Close the plate with the lid and store at four degrees Celsius overnight to facilitate the immobilization of protein onto microtiter plate wells. Discard the solution from the microtiter plate by tilting upside down over the sink. Then, wash the wells three times with 300 microliters of PBS per well.
Block the coated wells for one hour at room temperature with PBS containing 2.5 weight volume percent BSA. After removing the blocking solution, wash the wells three times with 300 microliters of PBST per well. Now, add 100 microliters of 50 nanomolar recombinant his-tagged PH to each sample.
In the control wells, add only 100 microliters of PBS BSA. Incubate the plate for one hour at room temperature. Following incubation, discard the protein solution and remove the unbound proteins by washing the wells three times with 300 microliters of PBST per well.
Then, add 100 microliters of PBS BSA containing horse radish peroxidase conjugated anti-his polyclonal antibodies. After incubating for one hour at room temperature, wash the wells three times with 300 microliters of PBST per well. Detect the antigen-antibody complexes by adding 100 microliters of ELISA detection reagent to each well.
Then, add 50 microliters of one molar sulfuric acid per well to stop the reaction. Measure the optical density of the wells at 450 nanometers using a microplate reader. Immobilize human Vitronectin on amine reactive sensors using the amine coupling method according to the manufacturer's guidelines.
Using PBS, serially dilute the recombinant PH ligand from zero to four micromolar and transfer the resulting solutions to a 96-well black flat-bottom microtiter plate. Run the experiment at 30 degrees Celsius using a bio-layer interferometry instrument. Pipette 10 microliters of a two microgram per milliliter solution of Vitronectin in PBS onto a glass microscope slide as a single drop.
Allow the drop to dry on the slide for 30 minutes at room temperature. Wash the protein-coated glass slides three times by dipping them in a beaker containing PBS for two seconds to remove excess uncoated protein. Now, add 10 milliliters of a fresh Hif culture into a sterile plastic Petri dish and submerge the coated glass slides in the culture medium.
Incubate the dishes at 37 degrees Celsius for one hour with shaking at 20 RPM. After incubation, remove any unbound bacteria by submerging the slides three times in a beaker filled with PBS before visualizing the bound bacteria by Gram staining as described in the text protocol. Culture A549 epithelial cells as described in the text protocol.
After one wash and trypsinization of the cells at 37 degrees Celsius, dilute the cell suspension to 5, 000 cells per milliliter using complete medium containing five micrograms per milliliter of gentamycin. Prior to bacterial infection, replace the medium in the wells with F12 medium and incubate overnight at 37 degrees Celsius. Wash the cell monolayer three times with one milliliter of PBS at room temperature.
Then, place the plate on ice and 200 microliters of pre-chilled F12 medium containing 10 micrograms per milliliter of Vitronectin. After incubating the plate at four degrees Celsius for one hour, discard the solution by pipetting and wash the cell layer twice with one milliliter of PBS at room temperature. Add 100 microliters of freshly grown Hif wild type in F12 medium to each well.
Incubate the plate for two hours at 37 degrees Celsius. Then, aspirate the medium with a pipette and wash the A549 epithelial cells three times with PBS. Add 50 microliters per well of cell detachment solution and follow with a five-minute incubation at 37 degrees Celsius.
Next, add 50 microliters of F12 complete medium per well to stop the enzymatic reaction. Transfer the epithelial cells from each well to a six milliliter glass tube containing four glass beads. Lyse the cells at room temperature by vortexing for two minutes.
After diluting the cell lysate 100-fold, plate 10 microliters of the diluted sample onto a chocolate agar plate. Count the colonies following an overnight incubation at 37 degrees Celsius. To perform the analysis of Vitronectin-dependent resistance to the bactericidal activity of human serum, first culture and pellet the bacteria as described in the text protocol.
Following centrifugation, resuspend the bacterial pellet with one volume of Dextrose-Gelatin-Veronal buffer. Now, add 1, 500 CFU of bacteria to 100 microliters of DGVB plus plus containing 5%serum. Incubate the sample at 37 degrees Celsius for 15 minutes with shaking at 300 RPM.
Remove a 10 microliter aliquot from the reaction mixture at zero minutes and 15 minutes. Place the aliquot on chocolate agar and incubate the plate at 37 degrees Celsius overnight. After incubation of the chocolate agar plates, count the colonies appearing on the plates and calculate the percentage of bacteria killed as described in the text protocol.
All Hif clinical isolates tested in this study recruited Vitronectin to the cell surface as determined by flow cytometry. Binding of Vitronectin by the wild type Hif strain caused a shift in the population whereas the mutant did not bind Vitronectin. Protein-protein interactions between major Vitronectin-binding protein PH and Vitronectin were estimated by ELISA.
The results indicate a significant interaction between PH and both Vitronectin and the positive control relative to the negative control. Interaction between PH and Vitronectin were estimated in real-time using bio-layer interferometry to have an affinity of 2.2 micromolar. Furthermore, bacterial lacking expression of PH on the cell surface exhibited reduced adherence to Vitronectin-coated glass surfaces in comparison to wild type cells.
In addition, the presence of Vitronectin on the surface of epithelial cells significantly enhanced the adherence of Hif. To estimate the complement inhibiting activity of Vitronectin, serum-mediated killing was examined. Wild type Hif cells exhibited higher serum resistance than mutant cells.
No bacteria were killed in the presence of heat-inactivated serum. Interestingly, wild type Hif exhibited reduced survival in Vitronectin-depleted serum and replenishment of Vitronectin increased bacterial survival. However, the mutant strain did not respond to Vitronectin depletion because it could not recruit Vitronectin to the cell surface.
Once mastered, this technique it can be done in three to four days for any pathogen and remember to use the fresh culture of the bacterial pathogens. After watching this video, you should have good understanding on how to analyze the Vitronectin binding to a pathogen. In addition to this technique, Vitronectin binding surface proteins of bacteria can be identified by using proteomics and western blotting.
This technique is important for the researchers in the field of host-pathogen interaction and also to explore the mechanism of virulence in bacteria.
