A High-throughput Compatible Assay to Evaluate Drug Efficacy against Macrophage Passaged Mycobacterium tuberculosis

Summary

New models and assays that would improve the early drug development process for next-generation anti-tuberculosis drugs are highly desirable. Here, we describe a quick, inexpensive, and BSL-2 compatible assay to evaluate drug efficacy against Mycobacterium tuberculosis that can be easily adapted for high-throughput screening.

Abstract

The early drug development process for anti-tuberculosis drugs is hindered by the inefficient translation of compounds with in vitro activity to effectiveness in the clinical setting. This is likely due to a lack of consideration for the physiologically relevant cellular penetration barriers that exist in the infected host. We recently established an alternative infection model that generates large macrophage aggregate structures containing densely packed M. tuberculosis (Mtb) at its core, which was suitable for drug susceptibility testing. This infection model is inexpensive, rapid, and most importantly BSL-2 compatible. Here, we describe the experimental procedures to generate Mtb/macrophage aggregate structures that would produce macrophage-passaged Mtb for drug susceptibility testing. In particular, we demonstrate how this infection system could be directly adapted to the 96-well plate format showing throughput capability for the screening of compound libraries against Mtb. Overall, this assay is a valuable addition to the currently available Mtb drug discovery toolbox due to its simplicity, cost effectiveness, and scalability.

Introduction

Tuberculosis (TB) remains a serious global health threat despite the availability of anti-TB chemotherapy regimens for over 40 years¹. This is due in part to the requirement for long treatment periods of over 6 months using multiple drug combinations, which leads to patient non-compliance². The emergence of drug-resistant TB in recent years has further compounded problems in a field where successful development of clinically approved drugs is virtually non-existent³. Indeed, despite exhaustive anti-TB drug development, only a single drug has been FDA approved for clinical use in the past 40 years<....

Protocol

NOTE: As M. tuberculosis mc²6206 is an avirulent strain^17,18, all work in this protocol can be performed in a Biosafety Level 2 facility (BSL-2).

1. Culture Conditions for Green Fluorescent Protein Expressing M. tuberculosis mc²6206 (Mtb-GFP)

NOTE: The M. tuberculosis H37Rv derived auxotroph strain mc²6206 (ΔpanCD, ΔleuCD) transformed with the gfp expressing plasmid pMN437 is used throughout this protocol¹⁶. It is possible to sub....

Results

To confirm the robustness of adapting this infection model to 96-well plate format, we here examined the drug susceptibility of Mtb derived from our 96-well adapted infection model to rifampicin (RIF) and moxifloxacin (MOXI) according to the template given in Figure 1A. We demonstrate that the generation of Mtb/macrophage aggregate structures key to this assay can be reliably produced in a 96-well plate format (Figure 2), thereby enablin.......

Discussion

Here, we have described in detail an alternative Mtb infection model suitable for drug efficacy testing. This model takes into account two key factors that should be given more consideration during the early TB drug development process: the presence of physiologically relevant barriers to drug penetration and metabolic changes of Mtb during infection. While we have previously shown the benefits of our infection model and proposed the possibility of scaling up the infection for throughput compatibility

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
7H9	BD Difco	271310	Follow manufacturer's recommendations
Middlebrook OADC	BD Biosciences	212351
Tyloxapol	Sigma	T8761	Prepare 20% stock solution in H2O; filter sterilize
D-Pantothenic acid hemicalcium salt	Sigma	P5710	Prepare 24 mg/mL stock solution in H2O; filter sterilize
L-leucine	MP Biomedicals	194694	Prepare 50 mg/mL stock solution in H2O; filter sterilize
Hygromycin B	EMD Millipore	400051	Prepare 200 mg/mL stock solution in H2O
Nalgene Square PETG media bottle	Thermo Fisher	2019-0030
RPMI 1640 media	Hyclone	SH30027.01
Fetal Bovine Serum	Atlanta Biologicals	S12450H
L-glutamine	Corning	MT25005CI
HEPES	Hyclone	SH30237.01
Cytation 3 plate reader	Biotek		Interchangable with any fluorescent plate reader and microscope
Gen5 Software	Biotek		Recording and analysis of rezasurin coversion
Rifampicin	Fisher Scientific	BP2679250	Prepare 10 mg/mL stock solution in H2O
Moxifloxacin Hydrochloride	Acros Organics	457960010	Prepare 10 mg/mL stock solution in H2O
Resazurin Sodium Salt	Sigma	R7017	Prepare 800 μg/mL stock solution in H2O; filter sterilize
Tween-80	Fisher Scientific	T164500	Prepare 20% stock solution in H2O; filter sterilize