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

Summary

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Abstract

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

Introduction

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. Drug-sensitive TB is a treatable disease that requires multiple antibiotics for a period of 6 months. Despite being a treatable disease, TB mortality was estimated to be 1.5 million in 2015¹. In the past 10 years, there have been increasing concerns over the prevalence of drug-resistant M. tuberculosis. Multidrug-resistant TB (MDR-TB) is defined as TB that is resistant to at least Isoniazid (INH) and Rifampicin (RMP), and most MDR-TB strains are also resistant to select second-line TB drugs, thus limiting treatment options. The effects of drug resistance create a greater challenge for treating patients co-infected with Human Immunodeficiency Virus (HIV); 400,000 patients worldwide died of HIV-associated TB in 2015¹. Disappointingly, the United States Food and Drug Administration has approved only one new TB drug against MDR-TB, bedaquiline, in the past 40 years². Advances in finding better and shorter TB therapies are urgently needed in order to stay ahead in the fight against TB and MDR-TB.

Traditionally, TB drug screens are performed under in vitro growth conditions in growth medium, whereby compounds are added to a growing culture and their effectiveness in eradicating the microorganisms are determined by counting colony forming units (CFU) on solid medium. As CFU counts are labor intensive, time consuming, and costly, various indirect methods have been developed to alleviate this problem. Such methods include the Alamar Blue viability assay³, the determination of fluorescence⁴ from green fluorescent protein (GFP) or luminescence⁵ from luciferase-expressing bacteria, and the estimation of total adenosine triphosphate (ATP)^6,7.

Typical TB is characterized by an M. tuberculosis infection of the lung, where the bacteria reside and replicate inside the phagosomes of alveolar macrophages⁸. The simple in-broth phenotypic screen may suit extracellular pathogens; however, in the historical perspective, hit compounds against M. tuberculosis identified using this method often fail to live up to expectations during downstream validation steps in infection models. We propose that TB drug is best performed in an intracellular host cell infection model. Nevertheless, intracellular models possess many technological and biological barriers to high-throughput screening (HTS) development. A big hurdle is the complexity of the infection process, exemplified by numerous steps and the elaborate removal of extracellular bacteria by in-between washing. A second major hurdle is the lengthy time requirements, as growth detection, normally done by CFU counting on culture plates, is a process that takes over 3 weeks to complete. One solution to replace CFU counts has been provided by automated fluorescent microscopy in combination with fluorescent bacteria. However, this solution requires an initial equipment investment that is out of reach for many research labs. A simple, low-cost, and disease-relevant HTS method would greatly enhance the drug discovery process.

In this study, we report a new, modular HTS system that is aimed at providing a rapid, and highly scalable, yet economical, assay suitable for determining the activity of compounds against intracellular M. tuberculosis. This system is composed of three modules: (i) intracellular, (ii) cytotoxicity, and (iii) in-broth assays. The combined final result provides a comprehensive description of the compound properties, with additional information as to the potential mode of action. This screening system has been used in several projects with various compound libraries that target mode of action, including the analysis of drug synergy⁹, the stimulation of autophagy¹⁰, and the inhibition of M. tuberculosis-secreted virulence factor (unpublished). Compounds of unknown mode of action have also been studied¹¹. A modified version of this method was also adopted by our industrial partner as the primary screening method to identify new compounds against intracellular M. tuberculosis¹¹.

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Protocol

1. Bacterial Strain and Growth Medium

1. Make albumin dextrose and salt stock solution (ADS) by solubilizing 25 g of bovine serum albumin, 10.0 g of dextrose, and 4.05 g of sodium chloride in 460 mL of deionized water. Filter-sterilize the ADS and store at 4 °C.
2. Make 7H9 broth by adding 4.7 g of 7H9 powder and 2 mL of glycerol to 900 mL of purified water. Autoclave the 7H9 broth at 121 °C for 10 min and allow it to cool to room temperature before proceeding. Make 7H9ADST by adding 100 mL of ADS and 0.5 mL of Tween80 to 900 mL of 7H9 broth. Store at 4 °C.
3. Weigh 50 mg of kanamycin sulfate and dissolve in 1 mL of deionized water; the final concentration is 50 mg/mL. Filter-sterilize and store at -20 °C. Add 0.5 mL of kanamycin stock solution per 1 L of 7H9ADST.
   NOTE: This medium should be made fresh, so scale the volumes appropriately according to the culture size.
4. Grow M. tuberculosis in 7H9ADST supplemented with kanamycin in standing culture. Shake the culture daily and dilute it before the OD600 reaches 1.0 to avoid clumping.
   ​NOTE: The M. tuberculosis strain used for the development of this method was H37Rv transformed with pJAK2.A plasmid¹². pJAK2.A is an integrative plasmid based on the pMV361 vector, which allows high-level expression of the firefly luciferase gene from the hsp60 promoter and can be selected using kanamycin.

2. THP-1 Medium and Maintenance

1. Add 50 mL of heat-inactivated fetal bovine serum (FBS) and 5 mL of 200 mM L-glutamine to 500 mL of RPMI 1640 to make RPMI incomplete medium (approximately 10% FBS and 2 mM glutamine).
2. Maintain an THP-1 cell culture according to standard protocol¹³. Briefly, grow THP-1 cells in RPMI incomplete medium while maintaining a cell density of 0.2 to 1 million per mL of medium between passages.

3. High-throughput Intracellular Screening Using Luciferase-expressing M. tuberculosis H37Rv

1. Measure the optical density of an actively growing bacterial suspension in a spectrophotometer at a wavelength of 600 nm. Calculate the bacterial density using the conversion factor of 0.1 OD₆₀₀ = 3 x 10⁷ bacteria per mL.
2. Pipette out sufficient bacteria for a multiplicity of infection (MOI) of 10:1 into a new centrifuge tube. Pellet at 3,000 x g for 10 min and aspirate the liquid. Add 50 µL of human serum to 450 µL of RPMI1640. Scale the volume to appropriate values for the experiment.
3. To opsonize the bacteria, resuspend the pellet at a density of 1 x 10⁸ bacteria per 500 µL of RPMI1640 containing 10% human serum. Allow the mixture to incubate at 37 °C for 30 min. Determine the THP-1 cell culture density by counting with a hemocytometer and an inverted microscope.
4. Pellet the cells in sterile centrifuge tubes at 100 x g and 37 °C for 10 min. Aspirate the supernatant and resuspend the cells in RPMI incomplete at a density of 1 million cells per mL. Add phorbol-12-myristate-13-acetate (PMA) to a 40 ng/mL final concentration.
   NOTE: This will be referred to as the differentiation mix.
5. Combine opsonized M. tuberculosis with THP-1 differentiation mix at a MOI of 10:1 and aliquot the final mix at 100 µL per well in a 96-well flat-bottom white plate. Regularly stir the mixture to ensure uniformity. Allow the differentiation and infection to proceed overnight at 37 °C in a humidified incubator containing 5% CO₂.
6. Wash the wells twice with 100 µL of RPMI each. Add compounds diluted to the desired concentrations in RPMI incomplete and incubate for 3 days.
7. Aspirate the medium from the wells. Add 50 µL of luciferase assay reagent to each well. Seal the plates with transparent adhesive plate sealers. Allow 5 min of incubation at 22 °C and then obtain a readout in a luminometer at 1 s per well.

4. Cytotoxicity Analysis Using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide (MTT) Assay¹⁴

1. Differentiate THP-1 cells in RPMI incomplete supplemented with 40 ng/mL of PMA in clear 96-well plates. Maintain a cell density of 1 million per mL and aliquot 100 µL per well. Allow differentiation to proceed overnight at 37 °C in a humidified incubator containing 5% CO₂ .
2. Aspirate the medium from the wells and wash them twice with RPMI 1640. Add compounds diluted in RPMI incomplete to the wells. Incubate for 3 days.
3. Dissolve 0.5 g of MTT in 100 mL of phosphate-buffered saline (PBS) to make a stock solution of 5 mg/mL. Sterile filter and store at -20 °C, away from light; it is best to make this solution fresh.
4. 2.5 h before the end of the 3-day incubation period, add 25 µL of MTT solution to each well and complete the incubation period.
5. Prepare 50% N,N-dimethyl formamide (DMF) by mixing 250 mL of DMF with 250 mL of deionized water.
6. Prepare MTT extraction buffer as follows: Weigh 100 g of SDS in a 500-mL bottle and add 300 mL of 50% DMF. Apply low heat to allow the SDS to dissolve. Add 10 mL of pure acetic acid and 12.5 mL of 1 M HCl. Fill up to the 500-mL mark with 50% DMF; the final composition of the extraction buffer is 50% DMF, 20% SDS, 2.5% acetic acid, and 2.5% 1 M hydrochloric acid.
7. At the end of the treatment period, add 100 µL of extraction buffer (warmed to 45 °C to dissolve any crystals) to each well. Allow the mixture to incubate overnight at 37 °C in a humidified incubator containing 5% CO₂. Read the absorbance at 570 nm.
   ​NOTE: The cytotoxicity assay is best performed in parallel with an intracellular screen using same-batch and age of THP-1 cells.

5. In-broth Activity Analysis Using a Resazurin Assay³

1. Grow M. tuberculosis in 7H9ADST to the mid-log phase (~ 0.5 - 0.8 OD₆₀₀). Dilute the culture with the 7H9ADST to 0.01 OD₆₀₀. Dilute the compounds in 7H9ADST to 2x the testing concentrations and aliquot 100 µL of each diluted compound into each well.
2. Transfer 100 µL of the diluted bacterial suspension into each well. Allow the plates to incubate at 37 °C in a humidified incubator for 5 days. Dissolve 10 mg of resazurin in 100 mL of deionized water and sterile filter.
3. Add 30 µL of resazurin solution and monitor the color change after 48 h; bacterial growth is indicated by a color conversion from blue to pink.
   NOTE: A quantitative analysis can also be performed by measuring either the fluorescence at 590 nm with excitation at 530 - 560 nm or the absorbance at 570 nm and 600 nm¹⁵.

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Results

High-throughput intracellular screening using M. tuberculosis expressing the luciferase gene

Figure 2A and Table 1 contain the raw data collected by the luminometer, expressed in relative luminescent units (RLU), showing the effect of an increasing concentration of the TB drug rifampicin on M. tuberculosis inside THP-1 cells. Figure 2A is a scatter plot of the raw...

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Discussion

The goal of this study was to create a simple and cost-effective HTS method using a human intracellular infection model for M. tuberculosis. Tuberculosis is a human disease characterized by the infection of alveolar macrophages by M. tuberculosis. Due to biosafety issues, research involving biological models of both the bacterium and the host cells has been used in the past. However, it has been shown that the usage of surrogate bacteria and non-human models are poor predictors of hit-to-lead success in...

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Materials

Name	Company	Catalog Number	Comments
RPMI 1640	Sigma-Aldrich	R5886
L-glutamine	Sigma-Aldrich	G7513
Fetal bovine serum (FBS)	Thermo Fisher Scientific	12483020
Middlebrook 7H9	Becton, Dickinson and Company	271210
Tween80	Fisher Scientific	T164
Albumin, Bovine pH7	Affymetrix	10857
Dextrose	Fisher Scientific	BP350
Sodium Chloride	Fisher Scientific	BP358
kanamycin sulfate	Fisher Scientific	BP906
PMA	Sigma-Aldrich	P8139
MTT	Sigma-Aldrich	M2128
N,N-Dimethylformamide (DMF)	Fisher Scientific	D131
1 M Hydrocholoric acid (HCl)	Fisher Scientific	351279212
Acetic acid	Fisher Scientific	351269
SDS	Fisher Scientific	BP166
Resazurin	Alfa Aesar	B21187
DMSO	Sigma-Aldrich	D5879
Glycerol	Fisher Scientific	BP229
THP-1	American Type Culture Collection	TIB-202
M. tuberculosis H37Rv
96-well flat bottom white plate	Corning	3917
95-well flat bottom clear plate	Corning	3595
Transparent plate sealer	Thermo Fisher Scientific	AB-0580
Spectrophotometer	Thermo Fisher Scientific	Biomate 3
Microplate spectrophotometer	Biotek	Epoch
luminometer	Applied Biosystems	Tropix TR717