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 limit....

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 wate....

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.......

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.......

Materials

List of materials used in this article

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