Isolation of Salmonella typhimurium-containing Phagosomes from Macrophages

The overall goal of this procedure is to isolate intact and enriched bacteria containing Phagosomes. This method can help us answer key questions in the field of Immunology such as changes in Phagosome maturation and antigen processing when pathogenic microorganisms invade host cells. The main advantage of the technique is that it produces high quality bacteria containing Phagosomes using simple steps and without a requirement of specialized equipment.

Individuals new to the method would find it technically approachable in comparison to the non methodologies. We first had the idea to develop this method when we were studying the mechanisms by which intracellular pathogens hijack the Phagosomes to their advantage. Demonstrating this procedure will be Saray Gutierrez, a post-doc from my laboratory.

To start Salmonella Typhimurium culture, use a bacteria loop to inoculate a single bacterial colony into 5 milliliters of brain heart infusion broth. Then incubate the suspension in a 37 degrees Celsius incubator overnight while shaking. Transfer one milliliter of this bacterial suspension into 19 milliliters BHI broth in a conical flask on the following day.

Incubate again in 37 degrees Celsius incubator while shaking. When the OD600 reaches one, remove the flask from the incubator and transfer the culture into a 50 milliliter tube. Then centrifuge the tube at 5400 x G at 4 degrees Celsius for 15 minutes.

After removing the supernatant, re-suspend the bacterial pellet in 10 milliliters sterile PBS. After repeating the centrifugation once, re-suspend the pellet in 4.9 milliliters of PBS. Next, add 100 microliters of freshly prepared Biotin linking solution to the bacterial suspension.

After splitting this suspension into five 1.5 milliliter tubes, incubate in a thermal block at room temperature with constant shaking at 350 rpm for two hours. After centrifuging the tubes at 15, 000 x G at room temperature for ten minutes, discard the supernatant. After two more centrifugation steps and complete removal of the Biotin linking solution, re-suspend this Biotin-coded bacteria in one milliliter sterile PBS in a single 1.5 milliliter tube.

Transfer 100 microliters of streptavidin-conjugated magnetic beads solution into a fresh 1.5 milliliter tube and place it in a magnetic rack for five minutes. After removing the solvent from the tube, remove the tube from the magnetic rack. Then re-suspend the beads that remained in the tube with one milliliter of Biotin coded bacterial suspension.

Incubate the tube on a thermo block at room temperature for one hour while shaking at 350 rpm. Next, place the tube on the magnetic rack for five minutes. Use a pipette to remove the bacteria that did not adhere to the wall and transfer to a new tube labeled non-coded bacteria.

The tube with magnetic beads adhering to the wall contains Biotin Streptavidin coded bacteria. Remove the tube with Biotin Streptavidin coded bacteria from the magnetic rack and re-suspend in one milliliter sterile PBS. After the tube has been on the magnetic rack for five minutes, remove the PBS.

Finally, re-suspend the washed Biotin Streptavidin coded bacteria in 500 microliters of sterile PBS and label the tube as coded bacteria. On the day prior to infection, plate fully differentiated BMDMs in a six centimeter dish in RPMIs supplemented with ten percent FBS. On the following day, centrifuge the coded bacterial suspension at 15, 000 x G at four degrees Celsius for five minutes.

After removing the supernatant, re-suspend the bacterial pellet in RPMI medium supplemented with ten percent FBS. Remove the medium from the BMDMs and add five milliliters of coded bacterial suspension in RPMI medium in order to infect the cells. To synchronize Phagocytosis, incubate at room temperature for ten minutes.

Then to initiate Phagocytosis, incubate at 37 degrees Celsius in a five percent Carbon Dioxide incubator for 30 minutes. After incubation, remove the medium from the dishes and wash the cells three times at RPMI to remove non internalized bacteria. To kill any non-Phagocytized bacteria at ten milliliters of RPMI containing ten percent FBS in 50 micrograms per milliliter Gentamicin.

Finally, incubate the infected BMDMs at 37 degrees Celsius in a five percent Carbon Dioxide incubator until the desired time point. First, prepare the volume of required Phagosome Isolation Buffer A by adding DTT and Cytochalasin B just before use and Protease and Phosphatase Inhibitors as recommended by the manufacturer. Remove the medium from the infected cells at the desired time point and wash the cells with sterile PBS pre-warmed to room temperature.

Then add 750 microliters of Phagosome Isolation Buffer A to the dish and incubate on ice for 20 minutes. Remember to rock the plate occasionally. Next, add 250 microliters of Phagosome Isolation Buffer B and then rock the plates to ensure that the buffer completely covers the surface.

Gently scrape the cells using a rubber policeman to remove the cells from the dish and transfer them to a pre-chilled 1.5 milliliter tube. Then use a one milliliter syringe to pass the cell suspension thru a 26-gauge needle at least 15 times. Next, place the cell suspension on the magnetic rack for five minutes.

Particles attached to the magnetic beads are Phagosomes containing coded S.Typhimurium. Transfer then the suspension which contains the rest of the cellular components into a new 1.5 milliliter tube labeled as Cytosol. Remove the tube containing isolated Phagosomes from the rack and re-suspend in one milliliter sterile PBS.

Place the tube back on the magnetic rack for five minutes and then remove the PBS. After repeating this washing with PBS once, remove the PBS and finally, re-suspend S.Typhimurium containing Phagosomes in the required buffer. To assess effectiveness of S.Typhimurium Biotinylation by confocal microscopy, BMDMs were infected with mCherry S.Typhimurium labeled with Biotin.

After fixation and incubation with Cy5-Streptavidin, successful Biotinylation was confirmed by Cy5-Streptavidin fluorescent signal. This signal was observed exclusively in Biotinylated mCherry S.Typhimurium whereas there was no signal in not Biotinylated mCherry S.Typhimurium. Immunoblock analysis of isolated Phagosomes showed significant enrichment of mCherry expressing S.Typhimurium in Phagosomes in comparison to the cytosolic fraction.

Immunoblock analysis using endosome and lysosome markers in isolated Phagosomes showed enrichment of the tested markers in Phagosomes isolated four hours after infection compared to 30 minutes after infection. When this protocol was applied to Staphylococcus aureus, successful Biotinylation of GFP expressing S.aureus was shown. Once mastered, this technique can be done in approximately one hour and a half depending on the incubation time chosen for infection by the investigator.

To ensure the success of the isolation of bacteria containing Phagosomes, please make sure that the buffers contain the indicated concentrations and that the plates are rocked to ensure the homogeneous coverage of the cells. Also, be gentle when scraping the cells and when passing the cell suspension through the needle. Following this procedure, other techniques such as proteomics or metabolomics can be performed with isolated Phagosomes to study alteration caused by pathogens under pathogenic factors involved.