Porphyromonas gingivalis as a Model Organism for Assessing Interaction of Anaerobic Bacteria with Host Cells

The overall goal of this video is to demonstrate protocols to assess the interaction of anaerobic bacteria with host cells. The conditions used to cultivate ary cells are toxic to anaerobic bacteria and vice versa. This poses a challenge in studying their interactions and conditions optimal for both must be achieved for them to remain metabolically active during the period of study.

Such conditions will more accurately reflect the scenario of host pathogen interaction found in vivo. The method presented here will help answer questions regarding mechanisms involved in microbial survival as well as binding and internalization of anaerobic bacterial cells during infection. Generally, individuals new to this method will struggle because working with anaerobic bacteria requires a knowledge of an anaerobic chamber and good septic techniques.

Visual demonstration of this method is critical as the steps involving the use of an anaerobic chamber are difficult to learn. Also, caution needs to be applied when experimenting with anaerobic chambers For culture and experimentation with por ominous gingiss. A vinyl anaerobic chamber is maintained with an artificial atmosphere containing 80%nitrogen, 10%hydrogen and 10%carbon dioxide place 30 blood agar plates, 500 milliliters of PBS and 30 milliliters of vascular endothelial growth factor or VEGF medium inside the anaerobic chamber.

24 hours in advanced to equilibrate the reagents seed four times 10 to the five human umbilical vein endothelial cells or VE per well in six well plates in VEGF medium and leave them in the cell culture incubator overnight for attachment. Prepare porro gingis cultures in brain heart infusion, or BHI medium as described in the accompanying text and allow the cultures to reach log phase while the optical density of the culture at 660 nanometers ranges between 0.5 to 0.7. Transfer the bacteria to a 15 milliliter tube and wrap a piece of paraform around the cap to prevent aerobic closure.

Then centrifuge the bacteria at 5, 000 times G for 10 minutes inside the anaerobic chamber. Wash the pelleted bacteria with anaerobic PBS and spin down again at 5, 000 times G for 10 minutes. Reese has the bacteria in VEGF medium to infect the HU X with por ominous transfer.

The six well plates from the incubator to the anaerobic chamber aspirate the medium from the test wells and wash three times with anaerobic PBS Determine the cell number from one representative well by trian blue exclusion assay. Then add two milliliters per well of anaerobic VEGF medium and place the plates at 37 degrees Celsius in the anaerobic incubator for 20 minutes. To equilibrate, determine the optical density of the bacteria and resus.

Suspend them in an adequate volume of VEGF medium to infect cells at a multiplicity of infection of 100 to one bacteria to cells ratio. Add the bacterial suspension to the cells and incubate for 30 minutes at 37 degrees Celsius for infection for cell lysis inside the anaerobic chamber. Prepare a 1%weight per volume saponin solution in BHI medium and filter through a 0.2 micron filter to study bacterial interactions with host cells.

Remove the six well plate from the incubator and aspirate the infection medium. Wash three times with anaerobic PBS. Then add two milliliters of saponin solution to each well and incubate for 15 minutes to allow cell lysis.

Next, scrape the cells from the bottom of the well and dilute the cell lysate one to one with BHI medium. Then make serial dilutions starting at one to 100, plate 200 microliters of a suitable dilution on blood agar plates. Wrap the plates with perfil and incubate them in a 37 degrees Celsius anaerobic incubator for seven days to allow colony formation.

Then using a light box count the colony forming units or CFUs on each plate following infection of HU E cells. Wash the cells three times with anaerobic PBS. Next, add two milliliters of VEGF medium supplemented with pre optimized concentrations of antibiotics to the HU E cells.

Incubate the cells for one hour, aspirate the medium and add two milliliters of 1%saponin solution in BHI to each well incubate for 15 minutes. Next, scrape the lysed cells and collect the lysate in a centrifuge tube. Dilute the lysate one to one with BHI medium, and then prepare serial dilutions starting at one to 100.

Plate 200 microliters of the suitable concentrations on blood. Agar plates. Allow the bacteria to grow for seven days in an anaerobic chamber and then count the colonies.

The plating method described accounts for only the live bacteria that interact with the cells. In order to overcome this drawback, we use microscopy where both live and dead bacteria in association of the cell can be visualized To visualize internalized bacteria. First aseptically transfer autoclave.

The glass cover slips inside each well of a 12 well plate plate, five times 10 to the four hve per well on the cover. Slip and incubate for 24 hours to label bacteria, centrifuge, midlock, phase bacteria, and resuspend the pellet with anaerobic PBS centrifuge again and repeat the washing step. Add 20 microliters of 0.2 millimolar B-C-E-C-F-A-M to two milliliters of anaerobic PBS containing five to seven times 10 to the eighth.

Bacteria per milliliter incubate at 37 degrees Celsius for 30 minutes in the dark. The fluorescent dye we use can stain most anaerobic bacteria, thus no other reagents such as a specific antibody are required. Centrifuge the bacterial suspension at 5, 000 times G for 10 minutes to remove the unbound labeling reagent and resuspend the bacteria in VEGF medium.

Then infect the UIC cells at a ratio of 100 to one bacteria to cells and incubate at 37 degrees Celsius for 30 minutes. Following incubation, remove the infection medium and wash the cells three times with PBS. Then add 4%paraldehyde and incubate at room temperature for 10 minutes.

To fix the cells in the anaerobic chamber, remove the fixed cells from the chamber and then wash the cover slips with PB S3 times Incubate the cover slips in one milliliter of 0.2%tritton X 100 for 10 minutes to perme the cells. Then wash the cover slips with PB S3 times now. Add 50 microliters of 50 micrograms per milliliter of T-R-I-T-C foid in to each cover, slip and incubate for 45 minutes at room temperature to stain cellular oligomeric actin.

After incubation, wash the cover slip with PB S3 times. Invert the cover slip on a glass slide with soft set mounting medium containing one microgram per milliliter dappy for nuclear staining. Then seal the periphery of the cover slip with nail polish and observe under confocal microscopy interaction of the anaerobic bacterium.

Porous gingis with uve is shown in these results. Serial dilution of the cell, lysate containing cell attached or internalized bacteria to 100 times yielded countable number of CFUs on blood agar plates. Seven days after incubation comparison of CFU yield on agar plates between infection with wildtype porus vallis W 83 and deletion mutant V 315 infections is shown here.

The mutation affects the ability of the bacterium to infect. VE shown here is a graphical representation of the bacterial colonies obtained from infected cells. When interaction was assessed, fewer mutant bacteria were recovered from infected cells compared to the wild type strain.

Results obtained after killing the extracellular bacteria by antibiotic treatment are shown in the right hand columns. Again, the mutant strain was inefficient at invading uve cells live. Bacteria are observed upon labeling with BCE CFAM.

Additional staining of the actin filaments and cellular nucleus are visible in red and blue fluorescence respectively. Once mastered, each of these techniques can be done in six hours if performed properly. While attempting this procedure, it's important to remember to maintain the bacteria under anaerobic conditions as well as to equate all media and supplies in the anaerobic chamber for up to 24 hours before experimentation.

Don't forget that working with anaerobic pathogens can be extremely hazardous and precautions such as wearing gloves, washing hands, and using a septic techniques should always be taken while performing this procedure. So this method can provide insight into predental pathogens. It can also be applied to other systems such as aspiration, pneumonia, intraabdominal infections, gynecological infections, and many more anaerobic infections found on various sites of the human body.

The implications of this technique extend to our drug therapy for various anaerobic pathogens because inhibitory measures can easily be added to the protocol and internalization directly measured following treatment. After watching this video, you should have a good understanding of how to culture anaerobic bacteria and assess their ability to interact with host cells.