The overall goal of this procedure is to harvest and quantify fluorescently tractable bacteria from an intermediary host for subsequent infection of mammalian host cells. Here Legionella mala expressing GFP are used to infect the natural host protozoan acan amoeba, castellani monolayers. The el mala are then harvested by selective lysis and the concentration of bacteria is then calculated using a formula based on fluorescence emission dilution of protozoan.
Primed bacteria are then used to infect mammalian cell monolayers. Finally, the number of infectious bacteria in the mammalian cells are determined using a combination of fluorescence microscopy, flow cytometry and plating lysates to recover colony forming units. The resulting data demonstrate a pathogenic advantage gained for the bacteria that were cultivated in protozoan cells in the priming stage when compared to the same strain that was cultivated in artificial medium.
We developed this technique by observing that the expression of particular virulence factors in bacteria could not be induced through in vitro cultivation alone. Typical infection models of cultured monolayers of cells use in vitro cultured bacteria for legionella and several other bacteria. Proteasomes provide a natural reservoir for proliferation where the bacteria gains a pathogenic advantage.
The technique that we'll describe allows for quantification of proteasome prime bacteria in order to accurately measure the contribution of a virulence factor that is only produced in the context of intracellular growth. Demonstrating the procedure will be bendon. A research assistant Sam Renan, also a research assistant and amri lama, a post-doctoral fellow, Begin this procedure by using electroporation to transform all L pneumophila strains used in the assay with the plasmid PAM 2 39, which encodes IPTG inducible GFP incubate the bacteria for three hours on a shaker at 37 degrees, using a spreader streak bacteria from each transformation onto a single plate and incubate for 72 hours at 37 degrees Celsius.
After three days, transfer a single colony of the bacterial strain into three milliliters of medium containing one millimolar IPTG, and cultivate overnight to stationary phase on an orbital shaker at 37 degrees Celsius. The next day, transfer 10 microliters of the culture onto a glass slide and apply a cover slip. Then view the slide under a microscope to confirm GFP expression.
Using a 60 x objective under the appropriate excitation and emission filters, numerous bacterial rods should be seen that a pure fluorescent green when viewed using the GFP channel of the microscope, if all bacteria are fluorescing, prepare to infect the amobi by diluting a 100 microliter aliquot of each in vitro culture to one to 10 in sterile H2O. Make a blank using 100 microliters A YE medium containing one millimolar IPTG 6.25 micrograms per milliliter at chloramphenicol and 900 microliters water. Next, take OD 600 measurements of the dilution using a spectrophotometer, using the formula provided in the accompanying document.
Calculate the volume necessary to infect a well at a multiplicity of infection or MOI of 20 for each in vitro culture. 24 hours before starting the bacterial liquid cultures replace the medium in the acan MEbA castellani ABI cultures. The next day collect and count the cells using a hemo cytometer.
To do this, dislodge the cells by tapping the flask and transfer a sample to a 50 milliliter conical tube. Next, mix an aliquot with the dye trian blue and load it on the hemo cytometer slide. After counting the cells under a light microscope, dilute the amobi with fresh medium to a final concentration of one times 10 to the sixth cells per milliliter using a repeat pipetter seed.
12 Well soul culture plates with one milliliter aliquots of the ABI incubated room temperature overnight. The most difficult aspect of this procedure is the robust infection during the priming step. To help accomplish this, we use a manual pipetter when washing the amoeba to prevent loss.
Amoeba are not particularly adherent and can be easily lost or removed during wash or media replacement steps. After the incubation, use a 10 milliliter serological pipette and a manual pipette aid to wash the wells of the 12 well cell culture plates three times with one milliliter of sterile PBS. After aspirating the PBS add one milliliter of infection medium to each.
Well then incubate the plates at room temperature for one hour. Next, infect each of the wells at an MOI of 20 by adding the bacterial suspension to the infection medium in each well centrifuge the plate at 400 times G for five minutes. After the spin, float the plate in a 37 degrees Celsius water bath for five minutes.
Transfer the plate to a 37 degrees Celsius 5%carbon dioxide incubator for 18 hours. Following the incubation is a fluorescence microscope to confirm infection following a successful priming step. 90%of the host cells should contain large VAEs populated with GFP expressing bacteria which appear as green crescent as shown in these merged phase contrast and fluorescence images.
At this time, most a castin eye cells have achieved maximum bacterial loads, yet lysis of the population is minimal. Under light microscopy, the amobi should appear rounded and detached. The dot a mutant strain which cannot support ICM mediated translocation of effectors should not grow in a cast eye.
Therefore, minimal fluorescence should be detected. The amobi will appear flat and the monolayer should be intact. Human acute monocytic leukemia or THP one cells, which serve as the target cells should have been prepared on a 12 well plate as described in the accompanying document.
Wash the THP one cells three times with PBS. Then add one milliliter of fresh RPMI 1640 with 10%FBS to each Well incubate TP one cells for one hour at 37 degrees Celsius, 5%carbon dioxide. To prepare the bacterial lysate, aspirate the medium from prime to Tobi.
Then to lice them, add 500 microliters of ice, cold, sterile, ultra filtered water and set on ice for 10 minutes. Next, pull the lysates according to strain. Type in 15 milliliter conical tubes to determine total fluorescence.
Measure emission at 512 nanometers for each of the pooled lysates. Using a 96 well plate in a fluorescence plate reader, calculate an OD 600 measurement for the pooled lysates according to the formula. In the accompanying document, use the lysates of uninfected amobi subject to the same experimental conditions as the infected amoeba to determine lysate background.
Then for each lysate pool, calculate the volume necessary to infect a well of target cells at an MOI of 20. Keep the lysates on ice to limit any changes in bacterial gene expression. Before infection, within 30 minutes of lysis, infect three wells of TP one cells per condition at the calculated MOI.
Using the pooled lysates allow set of wells to remain uninfected, serving as a negative control. For flow cytometric analysis. Centrifuge the plate at 400 times G for five minutes.
After the spin, float the plate in a 37 degrees Celsius water bath for five minutes. Transfer the plate to a 37 degree Celsius 5%carbon dioxide incubator for one hour, one hour post-infection. Aspirate the medium and wash the wells three times.
With PBS, add one milliliter of fresh RPMI 1640 containing 10%FBS to the wells and return the plate to the incubator. Incubate the TP one cells for 14 to 16 hours to determine levels of infection in both the priming stage and target cell stage infections. Image the infected wells using a fluorescence microscope at 10 x or 20 x magnification.
Then to quantify target cell infection by flow cytometry trypsin eyes, the infected THP one cells and gently wash them from the wells by mixing in PBS. With a pipette pull the cells in 1.5 milliliter micro centrifuge tubes, and then centrifuge at 1, 800 times G for two minutes. After the spin resuspend the pellets in one milliliter of PBS spin again at 1, 800 times G.If after resus suspending the pellets in PBS, the resulting suspensions are highly turbid.
They must be diluted one to three in additional PBS to prevent clogging of the lines in the flow cytometer using uninfected target cells, plot the forward side scatter on a flow cytometer once set, collect 20, 000 total events for each target cell infection sample using 488 nanometer laser excitation and the FL one channel. After transferring the data to a separate computer for analysis gait, post capture cell populations to exclude uninfected cells and plot fluorescence intensity in a histogram as shown here, examine the efficiency of infection by CFU plating of the cells harvested for flow cytometry. Prepare serial dilution of the samples in ultra filtered water at 10 to the minus one 10 to the minus two and 10 to the minus three plate 20 microliters of each dilution onto one third of A-C-Y-E-A plate with 6.25 micrograms per milliliter of chloramphenicol.
Incubate the plates at 37 degrees Celsius for 72 hours following the incubation. Count the colonies using a toothpick and cell counter to obtain a population of protozoan primed bacteria. Wild type L phis strains induced to express GFP.
Were used to infect a castellani monolayers for 18 hours at an MOI of 20. The GFP expressing bacteria were then used to reinfect amobi in a target cell stage infection to compare protozoan primed bacteria with in vitro cultured bacteria. A castella monolayers were incubated.
18 hours at an MOI of 10 as can be seen in these micrographs protozoan primed bacteria have a significant pathogenic advantage over in vitro cultured bacteria at the same MOI to quantify the infection of target cell macrophages using protosome and primed bacteria. PMA differentiated THP one macrophages were infected with wild type L pneumophila harvested from the proteasome and priming stage infection for 14 hours. Flow cytometry was then performed to quantify infection as seen here.
The right shift in fluorescence is a measure of the total infected cells in the population. Increasing intensity of the fluorescent signal can be correlated with the number of bacteria per macrophage fluorescence of uninfected TP one macrophages is shown in red and fluorescence of infected cells is shown in green. Once mastered, this technique can be completed in about two weeks total if everything is performed accurately.
Remember, while doing this procedure is important not to disturb the proteasome and monolayer. Using a manual pipette during your wash steps will help increase the yield for your primed infections. Don't forget that working with legionella can theba can be hazardous.
So take precautions such as preventing the production of aerosols while performing this procedure.
