Table 1.
Strains and Plasmids.
Figure 1.
Luminescence of mycobacteria during growth in bacteriological media.
Luminescent (FFlux and CBRlux) and non-luminescent (pJDC89) BCG carrying the vector backbone display similar growth rates in M-ADC-TW plus hygromycin (80 µg/ml) laboratory medium (A). Luminescence for both firefly (FFlux) and click beetle red (CBRlux) luciferase expressing BCG (BCG16 and BCG26) increases steadily over time until approximately 12 days post-inoculation (B). The correlation between luminescence and optical density of BCG cultures expressing FFlux, CBRlux or the vector (pJDC89) alone (C). Data and error bars represent the means and standard deviations, respectively, of triplicate samples. Error bars are often too small to be visible around the marker for the mean. RLU = relative light units. * indicate data points with P<0.05 for FFlux vs. CBRlux.
Figure 2.
Luminescence from mycobacteria within macrophages.
Luminescence detected from firefly (FFlux) and click beetle red (CBRlux) luciferase expressing BCG (BCG16 and BCG26) or BCG carrying the vector (pJDC89) alone within J774A.1 macrophages. Infections in macrophages were carried out with various multiplicities of infection (MOI) from 0.1 to 100 bacteria per cell for 30 min and washed to remove extracellular mycobacteria. Luciferin was added immediately after infection (day 0) and on days 2 and 7 post-infection ∼15 min before luminescence measurements. Data and error bars represent the means and standard deviations, respectively, of triplicate samples. RLU = relative light units. * indicate data points with P<0.05 for luciferase vs. pJDC89 expressing BCG at the same time point and same MOI.
Figure 3.
Correlation of bacterial numbers with luminescence.
Whole body images of luminescence from BALB/C mice (2/group) inoculated subcutaneously with 102 to 107 colony forming units (cfu) of BCG expressing click beetle red (CBRlux) or firefly (FFlux) luciferase (A) at 24 h post-inoculation. After imaging, mice were sacrificed and cfu determined by plating homogenates of skin region from injection site. Bioluminescence from each inoculation site correlates well with the number of bacteria present for both CBRlux and FFlux (B). Template for injection sites using different numbers of cfu or PBS control (C). Data and error bars represent the means and standard deviations, respectively, of duplicate samples. Data are expressed as total Flux in photons per second (p/sec). * indicate data points with P<0.05 for CBRlux vs. FFlux expressing BCG at the same inoculum.
Figure 4.
Luminescence allows imaging during pulmonary infection.
Whole body dorsal and ventral images of luminescence from click beetle red (CBRlux) and firefly (FFlux) luciferase expressing BCG inoculated intratracheally into BALB/C mice (2/group) at 106 cfu (A). Two vector only (pJDC89) control mice were imaged along with each set of two CBRlux or FFlux mice (vector controls on left). Data are expressed as total Flux in photons per second (p/sec). Luminescence is greater with CBRlux than with FFlux and ventral imaging is more sensitive than dorsal imaging (B). Imaging of mice post-mortem with open chest cavities demonstrates the luminescence signal originates from the lungs (C). Data and error bars represent the means and standard deviations, respectively, of duplicate samples. * indicate data points with P<0.05 between data indicated by horizontal bars.
Figure 5.
Spectral characteristics of firefly and click beetle red luciferase.
Collection of whole body images of BALB/C mice (2/group) infected subcutaneously with 106 cfu of BCG expressing click beetle red (CBRlux) or firefly (FFlux) luciferase at defined wavelengths between 540 nm and 720 nm demonstrates that very little luminescence is obtained from CBRlux at short wavelengths and, similarly, less is obtained for FFlux at longer wavelengths (A). Template map for subcutaneous inoculation of BCG strains and PBS control (B). Quantitation of total Flux (p/sec) at different wavelengths for areas with CBRlux of FFlux indicates that luminescence from each reporter can be spectrally separated (C). Comparing luminescence kinetics obtained with mixed cultures of 104 total cfu CBRlux and FFlux expressing BCG at ratios from 1∶1 to 1∶100 shows that the kinetic curve of a mixed culture resembles that of the luciferase that is at higher numbers in the culture. Furthermore, at the similar numbers (1∶1) the kinetic curve appears to be a mix between the two curves for the luciferases. Since the kinetic curves are directly relates to the numbers of each strain present, it is likely that these luciferases compete similarly for available substrate (D). Luminescence was measured using a plate reader after injecting 50 µl of 2 mM luciferin. Measurements began 10 s after adding luciferin and were taken every 10 s up to 5 min.
Figure 6.
Spectral unmixing of luminescence allows quantitation of mixed bacterial infections.
Whole body images were obtained from BALB/C mice (2/group) subcutaneously infected with a total of 107 cfu BCG mixed at different ratios from 1∶10 to 1∶1 expressing click beetle red (CBRlux) or firefly (FFlux) luciferase at defined wavelengths from 520 nm to 720 nm demonstrates ability to quantitatively separate signal from CBRlux and FFlux in mixed infections, similar to those seen in mixed cultures (A). Normalized quantification of signal at defined wavelengths after spectral unmixing for FFlux or CBRlux (left panel) and map of subcutaneous inoculation sites on mice (right panel) for imaging (B). After unmixing signal at each injection site correlates with the number of FFlux or CBRlux bacteria present (C). The composite image (on the right) shows green for quantitative FFlux signal, red for CBRlux signal and orange at positions of co-localization of both CBRlux and FFlux signals.
Figure 7.
Codon optimized luciferases produce more light than non-optimized luciferases.
BCG (104 cfu) expressing wild type click beetle red (CBRlux) or firefly (FFlux) luciferases or the same luciferases synthetically optimized for mycobacterial codon usage (opt) were examined in liquid culture for light production over time in the presence of 2 mM luciferin (A, B). Results shown are for the individual strains of each type selected for maximal luminescence as described in the methods. BCG containing the vector alone (pJDC89) was used as a control. Both the L5 and hsp60 promoters were examined with the codon optimized luciferases to evaluate which promoter results in the greatest light production. Whole body images of BALB/C mice (2/group) subcutaneously infected with 106 cfu of the same BCG strains show similar results to those found in liquid culture where codon optimized luciferases produce greater luminescence (C). A map of subcutaneous inoculation sites on mice is shown in the right panel. Quantitation of each inoculation site for total Flux photons per second (p/sec) indicates that the codon optimized CBRlux expressed from hsp60 emits the most light (D). Data and error bars represent the means and standard deviations, respectively, of two mice. ** indicates p<0.01 as compared to FFlux in the same construct.
Figure 8.
Codon optimized click beetle red luciferase (CBRlux) allows rapid therapeutic evaluation.
Percent inhibition of light production (A) and bacterial killing (B) for 104 of BCG expressing codon optimized CBRlux (optCBRlux) in the presence of different concentrations of isoniazid (INH) plus rifampin (RIF) during culture in bacterial media after 24 (day 1) or 48 h (day 2) as compared to the absence of antimicrobials. Whole body imaging during pulmonary infection in BALB/C mice (4/group) with 4×106 cfu of BCG expressing optCBRlux after 24 and 48 hr treatment with 10 mg/kg INH+RIF results in a reduction in luminescence (C). Quantitation of the percentage of the initial luminescence as compared to each time point out to six days post-treatment confirms the reduced luminescence in treated animals (D). Similarly, ex-vivo imaging of lungs at 2 days to confirm luminescence observed in whole body images is derived from the lungs and is reduced after antibiotic treatment (E). Untreated and treated panels for lung images are indicated in panel C. Correlation of luminescence with cfu present was confirmed by plating homogenates from the same animals at each time point (F). Data and error bars represent the means and standard deviations, respectively, of four mice. * indicates p<0.05 and ** indicates p<0.01 as compared to treated group at the same time point.