Microbial Pathogens that replicate intracellularly must eventually exit the infected cell. Which, when compared to other aspects of host pathogen interactions is relatively understudied. Here, we describe techniques to analyze pathogen efflux.
We show how relatively simply assays can be used to either evaluate efflux efficiency or characterize post-efflux pathogens The general techniques described here are broadly applicable although specific pathogen host cell models may differ in kinetics, dosages and possibly read-outs. As in setting up any experimental system, it is essential that clear controls are established that will ensure that genuine, biological processes are observed in experimental samples or cohorts. Demonstrating the procedure will be Petoria Gayle, a graduate student from my laboratory.
To begin with the infection experiment, use a pipette to add 20 microliters of the freshly prepared Lm inoculum to wells, with the dish slightly tilted and the pipette tip carefully placed in the overlying media. Avoid touching the walls of the well with the pipette tip. Gently pipette the contents of each well to ensure complete mixing.
Do not shake or significantly tilt the plate to avoid spreading LM over the walls of the well. Return cell culture dish to the 37 degree Celsius, five percent carbon dioxide incubator. Use conditioned media from uninfected cells as diluent to prepare a 10 micrograms per milliliter gentamicin solution.
At 30 minutes post-infection, carefully add 30 microliters of the gentamicin solution into the dish to reach a final concentration of two-point-five micrograms per milliliter. Gently pipette the contents of the well to mix and return the cell culture dish to the incubator. To harvest, slightly tilt the dish and carefully use a pipette to remove the entire media from the well.
Add point-five milliliters of distilled, sterile water to the well. After 30 seconds, transfer the resulting water lysate to a one-point-five milliliter microcentrifuge tube and vortex vigorously for 10 seconds. Add four-point-five microliters and 50 microliters of the water lysate to 450 microliters of distilled, sterile water to prepare the tenfold and one-hundredfold dilutions.
Briefly vortex to ensure thorough mixing. Add 50 microliters of the original tenfold diluted and one-hundredfold diluted samples onto lysogeny broth agar plates, and spread using a plate spreader. To assay for emerging LM, extract 10 microliters of the overlaying media from the dish and divide it into two five-microliter aliquots and micro centrifuge tubes.
Add five-microliters of DMEM/FCS to one aliquot and add five-microliters of DMEM/FCS containing five-microliters per milliliter gentamicin to the second aliquot as control. Wait five minutes at room temperature. Add 90 microliters of distilled sterile water to each aliquot and vortex the two vigorously for 10 seconds.
Then add 50 microliters of the sample on LB agar plates and spread using a plate spreader. Store the plates in 37 degrees celsius incubator for two days before enumerating Lm colonies. Using an automated cell counter, adjust the prepared, raw two-six-four-point-seven cells to the concentration of one times 10 to the sixth cells-per-milliliter.
Add one milliliter of the sample to wells of a six-well tissue culture plate. Infect the cells with a prepared Lm inoculum, corresponding to a multiplicity of infection of 50. At one-point-five hours post-infection, in a one-point-five milliliter microcentrifuge tube filled with 500 microliters of four percent PFA, collect media from the first set of wells and place the tube on ice.
To collect intracellular Lm, add one milliliter of distilled, sterile water to each well. After 60 seconds, transfer the resulting water lysate to a one-point-five milliliter microcentrifuge tube with 500 microliters of four-percent PFA. Vortex the tube vigorously for 10 seconds and place it on ice.
For remaining wells, remove media and replace with one-milliliter of DMEM/FCS with five-micrograms per milliliter gentamicin to kill extracellular Lm.Promptly return the dish to the incubator. After 30 minutes, remove media and replace with DMEM/FCS without gentamicin. After another another two and four hours, collect media in lysates for the second and third time points into the tubes and place them on ice to chill.
Centrifuge tubes at 10, 000 times G for seven minutes. Using a pipette, remove supernatant without disturbing the pellet. Add staining cocktail containing 50 microliters of four-percent PFA and 15 microliters of phalloidin into the tube and mix to suspend each pellet.
Incubate tubes in the dark at four degrees Celsius for 20 minutes. After incubation, add one milliliter of FACS buffer to each tube and pipette up and down to mix. Spin tubes in the centrifuge at 10, 000 times G for seven minutes.
Remove supernatant without disturbing the pellet. For immediate use, suspend each pellet in 400 microliters of FAX buffer. If storing for later analysis, add 200 microliters of FAX buffer and 200 microliters of four percent PFA into the tube and mix to suspend.
Using the infection conditions in the brief treatment with the antibiotic gentamicin to eliminate non-internalized Lm, zero-point-one-five percent of the wild-type Lm is recovered after one-point-five hours of co-incubation with cultured macrophages. In the subsequent co-incubation, there was a fourfold and seven-point-fivefold increase in the recovery of viable Lm, which exclusively represent intracellular proliferation. Comparable profile was observed for the Lm strain possessing a hypervirulent prfA during initial infection but not between three and six HPI.
When the prfA was deleted from the Lm strain, there is a subsequent reduction in the recovery of the strain after prolonged co-incubation. When gentamicin is removed from the wells at six HPI, extracellular Lm can be detected one hour later for both wild-type and hypervirulent prfA strains, but not for the strain with prfA deleted. Media overlaying both uninfected and infected macrophages contained light-scattering, bacterium-sized particulate matter.
However, only media from infected macrophages possessed GFP positive signals within the size gating. A time-dependent increase in the appearance of phalloidin-positive Lm was observed with infected macrophages. When plating out the macrophages in the 48-well tissue culture dish, leave some wells without cells.
These no-cell control wells are then treated exactly the same as the cell-containing wells, in terms of the addition of the pathogen, antibiotics, etc. to ensure that experimental signals are dependent on the presence of host cells. Similar to the experiments shown in figures one and three, pathogen or host factors and or small molecules can be tested as to their impact on pathogen cellular efflux.
