The overall goal of this procedure is to identify small molecules which modulate the intracellular levels of the second messenger cyclic-di-GMP in the opportunistic pathogen, Pseudomonas aeruginosa by means of a high-throughput bio-reporter screen. This method can help uncover small molecules that interfere with Pseudomonas aeruginosa bioinformation, and other particles by means of cyclic-di-GMP modulation. The main advantage of this technique is that it is very robust and versatile, allowing the screening of upwards of 3, 500 compounds within 48 hours.

The implications of this technique extend towards developing novel therapies which potentially sensitized to Pseudomonas aeruginosa, to the immune system, or to existing antibiotics while exerting a lesser, selective pressure on the bacteria. Innoculate five millileters of lysogeny broth, or LB medium, with a single colony of P.aeruginosa, seven degrees Celsius, with shaking at 200 rpm. Then, transfer two milliliters of the overnight culture into 200 milliliters of fresh LB medium in a one liter flask, and incubate at 37 degrees celsius with shaking at 200 rpm.

Monitor the optical density at 600 nanometers or OD 600, every 30 minutes by taking a 1 milliliter sample from the flask and examining using a spectrophotometer. When the OD 600 reaches between 0.3 and 0.5, place 40 millileters of the culture into a 50 mililiter conical centrifuge tube. Pellet the cells by centrifuging at 8000 rpm for 10 minutes at four degrees Celsius.

Discard the supernatant and resuspend the cells in 40 milliliters of ice-cold sterile 300 millimolar sucrose solution. After centrifuging the cells a second time, resuspend in 20 milliliters of ice-cold sterile 300 millimolar sucrose solution Centrifuge the cells again and resuspend in 400 microliters of the 300 millimolar sucrose solution. Chill the cells on ice for 30 minutes, after which they are ready for electroporation.

Next, add one microliter of a 0.2 microgram per microliter solution of plasmid encoding the CdrA promoter to the gene encoding the green fluorescent protein to 40 microliters of the prepared P.Aeruginosa electro-competent cells in a 1.5 milliliter micro-centrifuge tube that has been pre-chilled on ice. Mix the suspension and transfer it into a pre-chilled 2 millimeter electrode gap electroporation cuvette. Remove moisture on the outside of the cuvette with tissue paper and place the cuvette into the sample chamber of the electroporator.

Pulse the solution with a voltage of 2.5 kilovolts, capacitants of 25 microfarad and a resistance of 200 ohms. Remove the cuvette and add one milliliter of LB medium. Then, transfer the cells to a sterile 1.5 milliliter micro centrifuge tube and incubate for two hours at 37 degrees Celsius with shaking at 200 rpm.

Following incubation, spread 10 microliter, 50 microliter, and 100 microliter aliquots of the culture onto sterile LB agar plates supplimented with Ampicillin and incubate the plates at 37 degrees Celsius overnight. Confirm the GFP expression by examining the plate under a fluorescence microscope with a standard GFP channel. Pick a single colony and inoculate in five milliliters of LB.After overnight incubation, mix 0.5 milliliters of the overnight culture with 0.5 milliliters of 50%glycerol in a 2 milliliter screw-top tube and store at minus 80 degrees Celsius.

Two days before the screen, plate the prepared P.aeruginosa strain from the minus 80 degrees Celsius stock onto an LB agar plate by gently spreading the bacteria over the plate using a sterile inoculation loop. Incubate the plate overnight at 37 degrees Celsius. The evening before the screening, inoculate a single colony of P.aeruginosa from the plate into 10 milliliters of LB medium in a tube.

Incubate the pre-culture overnight at 37 degrees Celsius with shaking at 200 rpm. On the day of the screen, prepare a subculture from the overnight pre-culture by diluting it first with fresh LB medium to an OD 600 of 1.0, and then further diluting with 5%LB to an OD 600 of 04. Add a sterile magnetic stir bar into the container and stir the culture at minimal speed on a magnetic stirrer for 30 minutes at room temperature.

This allows the bacteria to acclimatize to the media before they are dispensed into 384-well plates. To prepare the positive control, add 20 microliters of Tobramycin Sulfate to 10 milliliters of the prepared subculture and mix gently. Pipette 40 microliters of the positive control culture into wells A23 through P23.

To prepare the negative control, add 30 microliters of Dimethyl Sulfoxide to 10 milliliters of the prepared subculture and mix gently. Pipette 40 microliters of the negative control culture into wells A24 through P24. For each of the plates damped with the small molecules, inoculate a volume of 40 microliters of the diluted overnight cultures into wells A1 through P22.

Seal the plates with an air-permeable cover seal and incubate for six hours at 37 degrees Celsius. Approximately 30 minutes before spectrophotometric measurement pre-warm the plate reader to 37 degrees Celsius to avoid condensation. Gently remove the air-permeable cover seal from the plate before reading.

Then measure the optical density at a wavelength of 600 nanometers with a setting of 10 flashes per well and a settling time of 0.2 seconds. Prior to measuring fluorescence, make an automatic gain and focal adjustment based on the negative control well A24 and set the gain target value at 75%Select focus adjustment and channel A on the right of the window. Measure the fluorescence from the GFP reporter at an excitation maximum of 485 nanometers and an emission maximum of 520 nanometers with a setting of 10 flashes per well and a settling time of 0.2 seconds.

Collect data from all plates examined. Data readings are automatically saved as default by the plate reader control software. To analyze the data, view the readings by clicking on the Mars icon within the control software to open the statistical analysis package.

Retrieve data by double-clicking on the plate number of interest to access the data for analysis. Evaluate the uniformity and reproducibility of the assay using a robust Z prime analysis. Then, calculate percent inhibition of growth and assess the percent inhibition of the intracellular level of c-di-GMP as detailed in the text protocol.

This figure describes representative raw data for OD 600 and GFP from the high-throughput screen. A color gradient heat map with hot to cool colors indicating low to high values has been applied to the well values. The generated data is analyzed for percent inhibition and is represented here for both of the OD 600 and GFP readouts.

Small molecules which potentially inhibit growth and intra-cellular c-di-GMP will tend to be in green, while small molecules which potentially promote growth and intra-cellular c-di-GMP levels will tend to be in red. Scatter plots are utilized to compare the percent inhibition obtained from each tested small molecule. Each small molecule is represented by a small dot and the percent inhibition distribution for each of the OD 600 and GFP readouts is shown.

A plus or minus 50%cutoff is selected for hit identification. Potential hits are highlighted in red. Dose-response assays are carried out on each interesting compound.

Here, two identified compounds are tested in a 10 point dose-response assay with a top concentration of two millimolar and a two-fold dilution series. Fitting a four-parameter logistic function to the data, yielded half-maximal inhibitory concentration values for each compound of 158 micromolar and 193 micromolar. Once mastered, this technique can be used to screen upwards of 3, 500 compounds within 48 hours.

After watching this video, you should have a good understanding of how to robustly screen Pseudomonas aeruginosa for compounds interfering with cyclic-di-GMP signaling. While attempting this procedure, it's important to remember to keep the screening conditions comparable when screening over several days. Following a single-point screen running a dirsk response screen using the same protocol can validate the hits.

This technique paves the way for researchers to identify potential small molecules that interfere with Pseudomonas aeruginosa bi fiful information and antibiotic resistance. Importantly, this technique can be adapted to be used for any bacteria of interest and can be modified to examine other outputs or inputs such as an impact on bacterial viability. Finally, don't forget that working with Pseudomonas aeruginosa can be hazardous.

And precautions such as wearing personal protective equipment should always be taken when performing this procedure.

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