Bioluminescence for assessing drug potency against nonreplicating Mycobacterium tuberculosis

Abstract

Targeting dormant Mycobacterium tuberculosis represents a challenge to antituberculosis drug discovery programs. We previously reported and validated the use of the streptomycin (STR)-dependent M. tuberculosis 18b strain as a tool for assessing drug potency against nonreplicating bacteria both in vitro and in vivo. In this study, we generated a luminescent 18b strain, named 18b-Lux, by transforming the bacteria with a vector expressing the luxCDABE operon from Photorhabdus luminescens. Luciferase expression was demonstrated under replicating conditions, and, more importantly, luminescence levels significantly above background were detected following STR removal. The sensitivity of STR-starved 18b-Lux to approved and candidate antituberculosis therapeutic agents was evaluated by means of a luciferase assay in a 96-well format. Results mirrored the data obtained with the standard resazurin reduction microplate assay, and the luminescence readout allowed time course assessments of drug efficacy in vitro. Specifically, we proved that bedaquiline, the rifamycins, and sutezolid displayed time-dependent activity against dormant bacteria, while pyrazinamide and SQ109 showed bactericidal effects at the highest concentrations tested. Overall, we established the optimal conditions for an inexpensive, simple, and very sensitive assay with great potential for future applications.

FIG 1

Growth curves for M. tuberculosis 18b-Lux and evaluation of luciferase expression. (A) 18b-Lux was grown at 37°C, with shaking, in glucose- or acetate-containing 7H9 medium with 50 μg/ml STR. (B) SS18b-Lux was incubated under the same conditions as in panel A except for the addition of STR. The OD₆₀₀ and luminescence were recorded at different time points and used to compile the graphs. RLU, relative luciferase units. The experiments were carried out with two biological replicates.

FIG 2

Sensitivity of the luciferase reporter. (A) Limit of detection of luciferase activity in SS18b-Lux. Serial 2-fold dilutions of bacterial cultures with glucose or acetate were used to determine the detection limit for luciferase activity. Dashed line, background luminesce measured for the culture medium (63 ± 1.7 RLU); dotted line, background luminescence measured for SS18b not transformed with the plasmid carrying the luxCDABE operon (70.7 ± 7.4 RLU). The experiments were carried out with four technical replicates. (B) Luminescence produced by SS18b-Lux, at an OD₆₀₀ of 0.1, at different time points in glucose-containing medium. The mean values and variability range in a 96-well plate are presented. The experiments were carried out with two biological replicates. (C) Luminescence produced by SS18b-Lux, at an OD₆₀₀ of 0.1, at different time points in acetate-containing medium. The mean values and variability range in a 96-well plate are presented. RLU, relative luciferase units. The experiments were carried out with two biological replicates.

FIG 3

Sensitivity of 18b-Lux, with and without STR, to INH and moxifloxacin. (A) Potency of INH at different time points in acetate-containing medium in the presence of STR. (B) Potency of INH at different time points after STR removal. (C) Potency of moxifloxacin (MOX) at different time points in acetate-containing medium in the presence of STR. (D) Potency of moxifloxacin at different time points after STR removal. Relative luminescence, luminescence values normalized to the untreated control values. Data represent the means and standard deviations of two independent experiments (biological replicates).

FIG 4

Luciferase assay with SS18b-Lux. Different concentrations of approved and experimental drugs were assessed in the luciferase-based assay with SS18b-Lux. Luminescence was measured at different time points, and data were used to construct killing curves. Relative luminescence, luminescence values normalized to the untreated control values. Data from one representative experiment of three biological replicates are shown. CFM, clofazimine. ●, day 0; ▲, day 1; ■, day 4; ⬥, day 6.

FIG 5

Drug activity evaluated as CFU. SS18b-Lux was incubated for 1 week with the various compounds at the concentrations indicated. An untreated sample (no drug) served as a control. D0, CFU detected at day 0. CFM, clofazimine. Ten-fold serial dilutions were plated on 7H10 plates containing STR, and CFU were enumerated after 4 to 5 weeks of incubation at 37°C. Mean values and standard deviations from two biological replicates are presented. *, P < 0.05 (Bonferroni's multiple-comparison test); ns, not significant.