System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

The overall goal of this protocol is to provide multidimensional assessment of how compounds affect M.tuberculosis survival inside human macrophages. This method is centered on a Luciferase-based efficacy assay. When combined with cytotoxicity an MIC assay can provide useful information into modification of HIT compounds in TB drug development.

The main advantage of our method is that it is time and labor saving. It is replacing a CFU colony forming unit assay with a Luciferase based assay. Generally individuals new to this technique will struggle with consistency with manual pipetting instead of automation.

A consistent pipetting technique is crucial. Grow THP1 cells in RPMI in complete medium. While maintaining a cell density of 0.2 to one million cells per milliliter of medium between passages.

Use a spectrophotometer to measure the OD600 of an actively growing bacterial suspension. Then calculate the bacterial density using the conversion factor of 0.1 OD600 equals three times 10 to the seventh bacteria per milliliter. Pipette out sufficient bacteria for an MOI of 10 to one into a new centrifuge tube.

Then pellet the bacteria at 3, 000 times G for 10 minutes. To opsonize the bacteria, add 50 microliters of human serum to 450 microliters of RPMI 1640 and use it to resuspend the bacterial pellet at a density of one times 10 to the eighth bacteria per 500 microliters of medium. Allow the mixture to incubate at 37 degrees Celsius for 30 minutes.

Next, using a hemocytometer and an inverted microscope, determine the THP1 cell culture density. Then, in sterile centrifuge tubes, pellet the cells at 100 times G in 37 degrees Celsius for 10 minutes. Aspirate the supernatant and resuspend the cells in RPMI in complete medium at a density of one million cells per milliliter.

Add PMA to a final concentration of 40 nanograms per milliliter. This is differentiation mix. Combined opsonized M.tuberculosis with THP1 differentiation mix at an MOI of 10 to one.

And aliquot the final mix at 100 microliters per well in a 96 well white flat bottom plate. Then allow the differentiation and infection to incubate at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide overnight. The following day, use 100 microliters of RPMI to wash the wells twice.

Then add compounds diluted to the desired concentrations and RPMI in complete medium. Incubate the plates for three days. When using multichannel pipettes, remember to practice slow and steady plunger action and always land the pipette tips in the same spot inside each well to minimize disturbance to the THP1 cells.

Following the incubation, aspirate the medium from the wells and add 50 microliters of luciferase assay reagent to each well. Then use transparent adhesive plate sealers to seal the plates. Incubate the plates at 22 degrees Celsius for five minutes, then use a luminometer to obtain a readout at one second per well.

To differentiate THP1 cells, after preparing differentiation mix as demonstrated earlier in this video, dispense 100 microliters of the differentiation mix into each well of a clear, 96 well plate of cells. Incubate the cells at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide overnight. The following day, aspirate the medium from the wells and use RPMI 1640 to wash them twice.

Add 100 microliters of compounds diluted in RPMI incomplete to the wells. Then, incubate the cells for three days. Dissolve 0.5 grams of MTT in 100 milliliters of PBS to make a stock solution of five milligrams per milliliter.

Then filter sterilize the solution. Two and a half hours before the end of the three day incubation period, add 25 microliters of MTT solution to each well of cells and complete the incubation period. Prepare 50%NN dimethylformamide or DMF by mixing 250 milliliters of DMF with 250 milliliters of deionized water.

Prepare MTT extraction buffer as follows. Lay 100 grams of SDS in a 500 milliliter bottle and add 300 milliliters of 50%DMF. Apply low heat to allow the SDS to dissolve.

Add 10 milliliters of pure acetic acid and 12.5 milliliters of one molar hydrochloric acid. Then, use 50%DMF to fill up the bottle to the 500 milliliter mark. At the end of the treatment period, add 100 microliters of extraction buffer to each well.

Incubate the mixture at 37 degrees Celsius in a humidified incubator containing 5%carbon dioxide overnight. The following day, read the absorbance at 570 nanometers. To carry out a resazurin assay, grow M.tuberculosis in 7H9 ADST to midlock phase.

Dilute the culture with 7H9 ADST to an OD600 of 0.01. Use 7H9 ADST to dilute the compounds to two times the testing concentrations. And aliquot 100 microliters of each diluted compound into each well of a clear 96 well plate.

Transfer 100 microliters of the diluted bacterial suspension into each well. Incubate the plates at 37 degrees Celsius in a humidified incubator for five days. Dissolve 10 milligrams of resazurin in 100 milliliters of deionized water.

And filter sterilize the solution. Add 30 microliters of resazurin solution to the wells. Then incubate the cells for 48 hours.

Bacterial growth is indicated by a color conversion from blue to pink. Carry out quantitative analysis according to the text protocol. This scatterplot shows the raw luminescence data measured in relative luminescence units for the treatment of various concentrations of rifampicin on M.tuberculosis and THP1 cells.

In this plot, the percent reduction and luminescence in treated wells compared to untreated wells is shown. Rifampicin is capable of reducing 99.9%of the relative light units, or RLUs from intracellular M.tuberculosis at a concentration of 0.1 micrograms per milliliter. Similar to previously published results determined from CFU.

In this figure, the cytotoxicity caused by increasing concentrations of G1-1H used in the MTT assay is illustrated. Concentrations of 10 and three micromolar are clearly below the IC 50 cutoff. This figure shows the effects of various concentrations of the antibiotic apramycin on resazurin conversion by M.tuberculosis.

The in-broth MIC90 was between 2.5 and five micrograms per milliliter. This is slightly higher than the previously published value of 1.5 micrograms per milliliter. But it is still well within the acceptable range.

As demonstrated here, evaporation becomes significant for all experiments with prolonged incubation times. So care must be taken to keep incubators well humidified to avoid inconsistencies. Once mastered, this technique can provide reliable intracellular data in as little as four days.

While attempting this procedure it is important to remember to be gentle with THP1 cells, and be consistent with pipetting if attempting without automation. Following this procedure, other methods like high content screening can be used to determine if hit compounds are bacteriostatic or bactericidal. After its development, this protocol paved the way for researchers in the field of tuberculosis drug discovery to rapidly assess compound efficacy against MTB inside human macrophages.

Don't forget that working with microbacterium tuberculosis is dangerous. Use precautions and proper equipment when handling this microorganism.