Table 1.
Plasmids and primers.
Fig 1.
In vitro comparison of the brightness among different fluorescent protein-expressing mycobacterial strains.
A. Comparison of the brightness among different fluorescent protein expressing mycobacterial strains in culture medium. Different fluorescent protein expressing M. bovis BCG strains were cultured in MOAD medium to OD600 0.5~1, and then the fluorescent signal of them were detected with a multimode reader at the optimal wavelength condition for each FP. For each FP expressing strain, fluorescence signal was calculated by subtracting the background fluorescence of the strain carrying the backbone vector for the FP. The Y-axis value was calculated mean of fluorescence of triplicates divided by their OD at 600 nm. The error bars are standard deviations. One-way ANOVA test was conducted for assessing overall differences among groups, and Turkey’s multiple comparison tests were applied to assess differences between two groups. * P<0.05; ** P<0.01; and *** P<0.001. B. Comparison of the brightness among M. bovis BCG-L5-tdTomato, M. bovis BCG-Hsp60-tdTomato, M. bovis BCG-L5-mCherry, and M. bovis BCG-Hsp60-mCherry. Five concentrations for each strain of bacteria were loaded into a 96-well plate. The fluorescence of samples were compared by a multimode reader with 530 nm excitation and 590 nm emission wavelengths for tdTomato; 570 nm excitation and 620 nm emission wavelengths for mCherry. Y-axis is in log scale. Data represent one of at least three independent replicate experiments. The error bars are standard deviations.
Fig 2.
Comparison of strains expressing fluorescent proteins under L5 and Hsp60 promoter expression in infected cells.
J774A.1 cells were infected with various multiplicities of infection (MOI) of the BCG strains expressing L5-tdTomato or Hsp60-tdTomato (A); L5-mCherry or Hsp60-mCherry (B). Fluorescence was measured with a multimode reader with excitation wavelength 530 nm and emission wavelength 590 nm for tdTomato and excitation wavelength 570 nm and emission wavelength 620 nm for mCherry. Value in Y-axis represents mean of fluorescence of triplicates divided by their OD at 600 nm. The error bars are standard deviations. Dotted lines represent background fluorescence from strains carrying backbone vectors. Data represent one of at least three independent replicate experiments.
Fig 3.
Comparison of brightness among BCG strains expressing tdTomato, or mCherry in subcutaneously infected mice.
A. Images of subcutaneous infected mice with M. bovis BCG strains expressing L5-tdTomato, Hsp60-tdTomato, L5-mCherry, or Hsp60-mCherry. 108, 107, and 106 CFU of bacteria were injected subcutaneously into BALB/c mice. Images were acquired by epi-illumination at day 1 post infection. B. The locations of the injections were shown on the map. C. Quantitative analysis of the images. Four mice were infected with technical replicates of the same culture. Values in the Y-axis represent mean of fluorescence for each dose of bacteria. The error bars are standard deviations. Data represent one of two independent replicate experiments. One-way ANOVA test was conducted for assessing overall differences among groups, and Turkey’s multiple comparison tests were applied to assess differences between two groups. * P<0.05; ** P<0.01; and *** P<0.001.
Fig 4.
Evaluation of plasmid stability and the absence of effects on bacterial growth.
A. Growth curves for M. bovis BCG strains carrying L5-tdTomato, Hsp60-tdTomato or the vector plasmids alone (no fluorescent protein expressed). B. Optical density (OD) of cultures at 600 nm in 7H9 media with (kan 25) or without (no kan) 25 μg/ml kanamycin over 18 days of culture. C. Fluorescence changes of the strain in media with or without kanamycin over 18 days of culture. D. Correlation between fluorescence and OD at 600 nm for the strain grown in medium without kanamycin. E. Correlation between fluorescence and OD at 600 nm for the strain grown in medium with kanamycin. For correlation analyses, samples for OD600 within the range of 0.05–1 were selected. Data represent one of at least three independent replicate experiments. Values in the Y-axis represent mean of fluorescence or OD values. The error bars are standard deviations.
Fig 5.
Imaging BALB/c mice infected intratrachealy with M. bovis BCG expressing L5-tdTomato.
A. IVIS images of mice infected intratrachealy with the L5-tdTomato expressing strain. Images were acquired by trans-illumination at day 1 post infection. B. Images of the lungs harvested from the infected mice at day 1 post infection. Images were acquired by epi-illumination. Colony forming units (CFU) from plating dilutions on agar plates are indicated in the images. C. Quantitative analysis of images of the infected mice and the correlation between fluorescence ratios and CFUs. Four mice were infected for each dose of bacteria from technical replicates of the same culture. Data represents one of two independent replicate experiments. Value in Y-axis represents mean of fluorescence for each dose of bacteria. The error bars are standard deviations. D. Fluorescent microscopy of lung cryosections from BCG-L5-tdTomato intratracheally infected mice. Upper left: DAPI; upper right: tdTomato; lower left: merge DAPI and tdTomato; and lower right: merge DAPI, tdTomato and phase). White bars represent 20 micrometers.
Fig 6.
In vitro evaluation of anti-TB therapeutic efficacy using the M. bovis BCG L5-tdTomato strain.
Fluorescence of treated or untreated M. bovis BCG L5-tdTomato strain was measured with a multimode reader. The initial CFU for the treated or untreated was 106. The treated group was given 64 ng/μl rifampicin and isoniazid. At each time point, bacteria were loaded into a 96-well plates as quadruplicate samples for each group. The Y-axes represent mean fluorescence for the quadruplicates. Data represents one of two independent replicate experiments. Error bars are standard deviations. Repeated Measures Two-way ANOVA was performed to examine overall difference between the two groups (treated vs. untreated) through multiple time points. Comparison of treated with untreated group was matched at each time point. P<0.01.
Fig 7.
In vivo evaluation of anti-TB therapeutic efficacy using the M. bovis BCG L5-tdTomato strain.
A. Images of infected mice treated or untreated with anti-TB therapy. Mice were infected intratracheally with M. bovis BCG with L5-tdTomato and treated or untreated with anti-TB therapy (10 μg/g rifampicin and isoniazid; intraperitoneal injection.). Images were acquired by trans-illumination at day 0, 2, and 6 post infection. B. Quantitative analysis of in vivo images. C. Images of lungs harvested from the mice sacrificed at day 0, 2, and 6 post infection after in vivo imaging. Images were acquired by epi-illumination. D. Quantitative analysis of images of harvested lungs. E. Colony forming units (CFU) in mouse lungs recovered from agar plating. Y-axis is in log scale. There were four infected mice and one uninfected mouse per group per time point. Data represents two independent replicate experiments. Two-way ANOVA was performed to examine overall difference between the two groups (treated vs. untreated) through multiple time points. Bonferroni post-hoc tests have been performed between treated and untreated groups at each time point. * P<0.05; ** P<0.01; and ***P<0.01
Fig 8.
Comparison between L5-tdTomato and Hsp60-tdTomato M. bovis BCG strains in protein levels, transcript numbers and gene copies of tdTomato.
A. Comparison of the fluorescence from supernatant of bacterial lysates between M. bovis BCG L5-tdTomato and Hsp60-tdTomato strains. M. bovis BCG L5-tdTomato and Hsp60-tdTomato strains were grown until OD600 = 0.75. 106 bacteria/ml were ultrasonically lysed in 1x PBS with 0.1% Triton X-100. After centrifugation, the supernatant from the samples was compared using a multimode reader with 530 nm excitation and 590 nm emission wavelengths. B. Western Blotting to compare tdTomato protein levels expressed from an L5 promoter with that expressed from the Hsp60 promoter. The strain carrying the Hsp60 vector was used as a negative control. Bacterial housekeeping gene GroEL2 was used as the loading control. C. Quantification of relative band intensities in D by densitometry. D. Real time RT-PCR to compare transcripts of tdTomato expressed from the L5 promoter with transcripts levels expressed from the Hsp60 promoter. E. Real time q-PCR to compare copy numbers of tdTomato expressing plasmid with the L5 promoter and that carrying the Hsp60 promoter. Data represent one of three replicate experiments. Student’s t-test was performed to examine difference between the L5-tdTomato and Hsp60-tdTomato strains in fluorescence of extracted protein, transcript numbers and gene copies. * represents P<0.05; and ** represents P<0.01.