Fig 1.
Correlation between luciferase activity and the CFU of rBCG-MDP1-luc.
A bacterial suspension of rBCG-MDP1-luc adjusted to an optical density (OD) = 0.001 at 600 nm in 7H9-ADC media containing 10 μg/ml Km was incubated in a 96-well round bottom plate at 37°C. At each time point, 50 μl of bacterial culture was mixed with the same volume of luciferin-ATP mixture (Promega) and the relative light units (RLU) of luciferase was immediately determined using a luminometer. At the same time, bacteria were inoculated on 7H11-OADC agar containing 10 μg/ml of Km after serial dilution. Three weeks after cultivation, the CFU were counted and compared with the corresponding control. The representative data of two independent experiments are presented as the means ± SD.
Fig 2.
TB drugs abrogate the increase of both luciferase activity and the CFU of rBCG-MDP1-luc.
TB drugs, such as INH, RFP, LVFX, and SM, were added to the bacterial suspension at a concentration of 0.5 μg/ml, 8 μg/ml, 1 μg/ml, and 4 μg/ml, respectively (10 fold concentration of the MIC of each drug). At each time point, 50 μl of bacterial suspension was mixed with the same volume of the luciferin-ATP mixture (Promega) and then the luciferase activity was determined immediately as the RLU. At the same time, each bacterial culture was serially diluted and inoculated on 7H11-OADC agar containing 10 μg/ml of Km. Three weeks after cultivation at 37°C, the CFU were counted and compared with the RLU. The representative data of two independent experiments are presented as the means ± SD.
Fig 3.
Kinetics of luciferase activity reduction after treatment with TB drugs.
Three days after culturing rBCG-MDP1-luc, TB drugs were added at the same concentrations as depicted in Fig 2. At each time point, 50 μl of bacterial suspension were mixed with the same volume of luciferin-ATP mixture (Promega) and the luciferase activity was determined immediately as the RLU. At the same time, the bacterial culture was serially diluted and inoculated on 7H11-OADC agar containing 10 μg/ml of Km. Three weeks after cultivation at 37°C, the CFU were counted and compared with the RLU. The representative data of two independent experiments are presented as the means ±SD.
Fig 4.
Pilot screening of actinomycetes-derived extracts using rBCG-MDP1-luc (1st stage of screening).
rBCG-MDP1-luc was cultured in the presence or absence of 1 μl of each actinomycetes extract dissolved in DMSO in a 96-well round bottom plate. (a) Four days after cultivation, 50 μl of bacterial culture was mixed with the same volume of luciferin-ATP mixture and the luciferase activity was measured. The data were normalized against positive control (DMSO). b) The list of extracts numbers is described and the effective extracts are noted in orange. (c) Nine days after incubation, the plate was scanned to observe the macroscopic growth of rBCG-MDP1-luc. Wells where growth inhibition was observed were marked by red circles. The representative data of three independent experiments are presented (a-c).
Fig 5.
Pilot screening of actinomycetes-derived extracts using rBCG-MDP1-luc (2nd stage of screening).
rBCG-MDP1-luc was cultured in media containing 1 μl of a two-fold serially diluted extract in a 96-well round bottom plate. (a) Four days after cultivation, 50 μl of bacterial culture was mixed with the same volume of luciferin-ATP mixture and the luciferase activity was measured. The data were normalized against DMSO. The representative data of two independent experiments are presented as the means ±SD. (b) Nine days after incubation, the plate was scanned to observe the macroscopic growth of rBCG-MDP1-luc. (a and b) Extract 2038-56a was effective even after a 1:25,600 dilution.
Fig 6.
Two-layer distribution test of active 1904–1 extract (4th stage of screening).
The 1904–1 extract was fractionated into a two-layer distribution using acetic ethyl and water, or n-butyl alcohol and water. Extracts of each layer were dried and reconstituted in methanol for the ethyl layer and n-butyl layer and in 50% methanol for the water layer. rBCG-MDP1-luc was cultured in the presence or absence of 1 μl of a two-fold serially diluted layered extract. Luciferase activity (a) and macroscopic growth (b) were checked after 96 h and 9 days cultivation, respectively. BtOH: n-butyl alcohol layer, BtOH-W: n-butyl alcohol-water layer, Ace: acetic ethyl layer, Ace-W: acetic ethyl-water layer. The data were normalized against DMSO control and presented as mean ± SD. The representative data of two independent experiments are shown (a and b).
Fig 7.
Separation of the 1904–1 extract by HPLC.
The BtOH layer of the 1904–1 extract was separated by HPLC using a C18 column as described in Material and Methods. A brief method is described on the right side of the chromatogram of (a). (a) UV spectroscopic chromatogram at 254 nm. (b) The retention times of the eluents. Each eluent was incubated with rBCG-MDP1-luc and their activities were obtained by luciferase activity measurement (c) and macroscopic assay (d) 4 and 9 days after incubation, respectively. The peak of the active substance is indicated by the arrow in (a) and its retention times are marked by red in (b). The time of elution is delayed from that of the peak in the chromatogram because of a time lag (a and b). The data were normalized against DMSO control (c). All data of three independent experiments are presented (a-d).
Fig 8.
Electrospray mass spectrometry (ESI)/MS data of the active fractions of the 1904–1 extract.
Effective fractions of the 1904–1 extract obtained by HPLC were analyzed by ESI-mass spectrometry using the positive ion mode.
Fig 9.
Comparison of the efficacy of the active substance of the 1904–1 extract with commercially available TB drugs.
rBCG-MDP1-luc was cultured in the presence of various concentrations of LVFX, REF, SM, INH, and the active substance of the 1904–1 extract. (a) Luciferase activity was measured 4 days after cultivation. The data were normalized against medium alone. (b) Nine days after incubation, the plate was scanned to see the macroscopic growth of rBCG-MDP1-luc. The representative data of three independent experiments are presented (a and b).
Fig 10.
Species-specific efficacy of the active substance of the 1904–1 extract.
Efficacy of species-specificities of the active substance of the 1904–1 extract was studied. M. smegmatis was cultured in the media containing serial dilutions of the 1904-1-derived active substance. (a) Four days after, the macroscopic growth was scanned. (b) Similarly, Pseudomonas fluorescens, Staphylococcus epidermidis, and Escherichia coli were cultured in the presence of the 1904-1-derived active substance, tetracycline, or in the absence of antibiotics for 1 day?. The active substance of the 1904–1 extract and tetracycline were two-fold serially diluted from 0.5 μg/ml and 10 μg/ml, respectively. The macroscopic growth of the bacteria was scanned. The representative data of two independent experiments are presented.
Fig 11.
Analysis of the cytotoxicity of the 1904-1-derived active substance toward eukaryotic cells.
The active substance of the 1904–1 extract was dissolved in saline containing 10% cremophor EL (Cremo), 0.01% BSA (BSA), or saline. Ten-fold serial dilutions of the active substance were added to the culture of both A549 cells and bone marrow derived macrophages of mice (BMDM), seeded at 1×104 cells/100 μl media on a 96-well plate. Three days after cultivation, cell viability (respiration) was checked using a cell viability detection kit. Increases in respiration were monitored at 0, 0.5, 2, 4 and 6 (only for BMDM) h after adding the redox-sensitive growth indicator. The representative data of two independent experiments are presented.
Fig 12.
Bactericidal effect of the 1904-1-derived active substance against intracellular Mtb.
In vitro-cultured 5×104 cells/well of mouse bone marrow derived macrophages (BMDM) (a) or THP1 cells (b) were infected with Mtb H37Rv at a MOI of 1:1 for 12 h. After washing the uninfected bacteria, cells were cultured in the presence or absence of the active substance of 1904–1 at several concentrations as indicated in the graphs. Seven days after cultivation, cells were lysed and aliquots of culture were inoculated on 7H11-OADC agar to count the living bacteria. The CFU was counted after 4 weeks. The representative data of two independent experiments are presented as means ± SD.
Table 1.
1H and 13C NMR data for 1904-1(Cyclomarin A).
Fig 13.
Chemical structure of the active substance of 1904–1 determined by NMR.
The active substance of the 1904–1 extract was dissolved in chloroform-d (CDCl3) and NMR spectra were recorded with a Bruker AVANCE 600 spectrometer (600 MHz for 1H, 150 MHz for 13C and 61 MHz for 15N).
Fig 14.
Susceptibility testing of resistant BCG mutants to the active substance of 1904–1.
Three BCG strains resistant to the active substance of the 1904–1 extract were obtained by selection on agar containing the substance and are designated as 4, 9, and 20. The upper graph (a) and lower picture (b) show the results of susceptibility tests of these mutants to the active substance in the media according to luciferase activity (a) and macroscopic examination (b), respectively. Bacteria were cultured in the media containing the indicated concentrations of the substance for 4 days for the luciferase assay and 9 days for macroscopic examination. Parent, parental rBCG-MDP1-luc. No. 4, 9, and 20, resistant strains to active substance of 1904–1. The representative data of two independent experiments are presented.
Fig 15.
Susceptibility test of resistant strain, No. 4 against conventional TB drugs.
Parental rBCG-MDP1-luc and its resistant derivative; No. 4 were cultured in the media containing isoniazid (INH), ethambutol (EMB), streptomycin (SM), rifampicin (RFP), active substance of 1904–1. Four days after culture, luciferase activity was measured. The data were normalized against medium alone. The representative data of two independent experiments are presented.
Table 2.
Genomic analysis of the 3 resistant isolates.