Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare

Abstract

The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of <1% (i.e., >99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time. MATERIALS AND METHODSMycobacterial strains. The strains used in this study were the M. bovis BCG substrain Connaught (ATCC 35745) and M. intracellulare ATCC 35761. The integrating shuttle vector pMV361 was constructed and electroporated into rBCG and M. intracellulare as described previously (10).Culture and growth conditions. Stock strains of mycobacteria were maintained in 7H9 broth with 15% glycerol at Ϫ80ЊC. Subcultures of the microorganisms were made in Middlebrook 7H9 broth (Difco, Detroit, Mich.) containing 10% ADC (albumin-dextrose-catalase) enrichment (BBL, Cockeysville, Md.), 0.05% Tween 80 (BBL), and 20 g of kanamycin (Sigma, St. Louis, Mo.)/ml. Cultures of rBCG and recombinant M. intracellulare were incubated in an ambient atmosphere for 48 and 24 h, respectively. Following incubation, the culture suspension was sonicated for 10 s with a VibraCell Sonicator (Sonics & Materials, Inc., Danbury, Conn.). To prepare the inoculum, the sonicated culture was diluted in Middlebrook 7H9 broth without kanamycin to an optical density at 540 nm of 0.05. This procedure yielded a suspension containing approximately 10 7 CFU/ml, as confirmed by a plate count on 7H11 agar (Remel, Lenexa, Kans.). This diluted suspension was used to inoculate test trays as described below, resulting in a final inoculum of 5 ϫ 10 5 CFU/ml in the reaction tube.

Current perspectives in drug discovery against tuberculosis from natural products

“…The study of Yajko et al [88] was important, since it showed for the first time that MICs of essential anti-TB drugs (isoniazid, rifampicin, streptomycin and ethambutol) could be determined following incubation of MTB isolates for only one to 2 weeks in the presence of the test drugs. This colorimetric method (Alamar blue) was also proposed by Collins and Franzblau [92] for use in a microplate format, microplate Alamar blue assay (MABA), for high-throughput screening of compounds against MTB and Mycobacterium avium, and by Shawar et al [93], for rapid screening of natural products for activity against MTB. The results of the MABA can be read visually [94] and do not require any instruments.…”

“…This method can be applied in a multi-well format with more convenient high throughput detection. Luciferase proteins from the firefly [113][114][115] and from Vibrio harveyi [116] utilize luciferin and n-decylaldehyde substrates, respectively, with n-decylaldehyde yielding a higher signal in mycobacteria. Luciferase enzymes are not ideal for kinetic measurements since they require the addition of a substrate, but they are potentially useful for susceptibility testing in MTB-infected macrophages [113] and in mice [116,117] since the luminescence measurements, performed in a luminometer, have a much higher signal-to-background ratio than is obtained from fluorescence assays.…”

Indian medicinal plants as a source of antimycobacterial agents

“…Fluorescent proteins such as red fluorescent protein (RFP) (Cho et al, 2002) and green fluorescent protein (GFP) (Collins et al, 1998;Changsen et al, 2003) have no exogenous substrate requirement, simplifying quantitation and enabling the easy determination of growth/inhibition kinetics. Luciferase proteins from the firefly (Cooksey et al, 1993;Arain et al, 1996;Shawar et al, 1997) and from Vibrio harveyi (Snewin et al, 1999) utilize luciferin and n-decyl aldehyde substrates, respectively, with the latter yielding a higher signal in mycobacteria. These luminescence measurements, performed in a luminometer, have much higher signal-to-background ratios than is obtained from fluorescence assays thereby making them potentially useful for also measuring inhibitor activity in Mycobacterium tuberculosis-infected macrophages (Arain et al, 1996) and in mice (Snewin et al, 1999).…”

Antimycobacterial activity of two natural alkaloids, vasicine acetate and 2-acetyl benzylamine, isolated from Indian shrub Adhatoda vasica Ness. leaves

“…Vasicine acetate was more active than 2-acetyl benzylamine at all concentrations. Shawar et al (1997) stated that luciferase assay had several advantages owing to its feasibility, amenability and high speed. Cladwell et al (2000) reported that 3-epioleanolic acid and oleanolic acid isolated from Junellia tridens had shown antimycobacterial activity with MIC values ranging from 16 to 128 μg/ml.…”