Si materials and Methods Strains and Growth Conditions



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SI Materials and Methods
Strains and Growth Conditions. Mycobacterium tuberculosis (Mtb) strain CDC1551, Erdman, bacillus Calmette-Guérin (Pasteur, BCG) and derivatives were routinely grown in 7H9 broth (Difco, Detroit, MI) supplemented with 0.5% glycerol, 10% OADC (oleic acid dextrose complex without catalase), and 0.05% Tween-80 (M-OADC-TW broth), or on Middlebrook 7H9 supplemented with 10% OADC and 15 g/L Bacto agar (M-OADC agar) or on 7H11 selective media. Stocks were prepared by growth standing at 37°C until an OD600 of 0.5 and stored in aliquots at −80°C until use.
Design and synthesis of CNIR800

A mixture of QC-1 Maleimide (7.8mmol), 1 (8.2 mg, 7.1 mmol) and 2,6-lutidine (3 mL, 25.9 mmol) in DMF (50 mL) was shaken at room temperature for about 30 min. After the reaction completed (monitored by HPLC), ether was added (by the end of HPLC the reaction time was about 1 h) and the precipitate was collected by centrifugation. The precipitate was then mixed with IR800CW-NHS (7.8 mmol) and PB buffer (100 mM, pH 7.4, 50 mL) and shaken at room temperature for 2 hr. HPLC purification (A: 10% PBS in water, B: CH3CN, 22%-27%), followed by desalting in a C18 cartridge to produce the final desired product (0.56 mmol, yield 8%).


Animal Infections. Mice were allowed to acclimate to the facilities for a week and fed commercial alfalfa-free diet (Harlan Teklad, Indianapolis, IN) with ad libitum access to tap water. Subcutaneous infections were carried out by shaving the back of the mice and injecting bacteria in 50 µl of saline subcutaneously at specific site. Intravenous infection was done by injecting 107 CFU of Mtb strain Erdman in 50 µl of saline through the lateral saphenous vein. Alternatively, mice were infected by intratracheal instillation of ~103-107 CFU of bacteria to the lungs as described previously (5, 6, 30). Bacterial numbers were determined by harvesting the skin at the site of infection or lungs and homogenized in 1 ml of PBS followed by plating 10-fold dilutions on the 7H11 selective media. At least 4 mice were infected for each experimental group, unless stated otherwise

Imaging Mycobacterial Infections

Mice were anesthetized using isofluorane (Henry Schein, Melville, NY) and imaged using the IVIS Spectrum (Perkin Elmer, Waltham, MA). CNIR800 (20 μM, 2.5 μl/g of weight) was administered by intraperitoneal injection. Images were acquired at 745 nm excitation and emission was collected in 20 nm increments from 780 nm to 840 nm. For animal whole body imaging, images were acquired with 9-12 trans-illumination points around the lung. Each acquisition was taken using auto exposure, f-stop of 2-3, and medium binning. Image analysis was performed with Living Image software v4.4 using spectral unmixing algorithms to remove auto-fluorescence for images taken in epi-illumination. In the case of the images taken using the trans-illumination, 3-dimensional fluorescent tomography (FLIT) reconstruction algorithms were used to determine fluorescent source distribution within each animal. Fluorescent signal was quantified by measurement of ROI (regions of interest) in the lungs of animal for each image.



Macrophage Infection Assays. Murine macrophages J774A.1 were seeded at 2.5 x 105 cells/well in 24-well dishes or 1 x 105 cells/well in 8-well chamber slides and incubated overnight at 37°C in DMEM to allow formation of a monolayer. The medium was changed just prior to infection with 0.2 ml of medium that contains 106 CFU of bacteria, a multiplicity of infection (MOI) of approximately 10 (bacteria/cell). The bacteria were incubated with the cells for 30 min at 37°C, washed twice with warm PBS and media replaced with DMEM plus 1% FBS. The cells on the slides were then prepared for microscopy. Viability was >98% and was not impacted by the presence of any substrates for the duration of experiments.

Confocal Fluorescence Microscopy. Murine macrophages J774A.1 were seeded in 8-well chamber slides with 1 x 105 cells per well in 200 µl of DMEM supplemented with 10% FBS at 37°C in 5% CO2 overnight. The medium was removed, and a GFP expressing M. bovis BCG strain was added to each well at an MOI of 10 (bacteria per cell) in 200 µl of medium. The bacteria were co-incubated with the cells for 30 min and washed twice with PBS to remove extracellular bacteria. Fresh medium with 200 µg/ml of amikacin was added and incubated for 2 h at 37°C to kill extracellular bacteria. The cells were then washed twice with PBS and placed in fresh medium supplemented with 1 μM of CNIR800 for 24 h at 37°C in 5% CO2 and washed twice with PBS. The cells were then stained with 10 µg/ml of DAPI in PBS by incubation for 5 min at room temperature, followed by washing twice with PBS and fixing in 4% paraformaldehyde in PBS for 30 min at room temperature. After washing with PBS the slides were dried and mounted for viewing by confocal fluorescent microscopy.
In vitro Detection of Mycobacteria with CNIR800. BCG strain (100-105/well) were spun down and the bacterial pellets were co-incubated with CNIR800 (5 µl of 20 µM, in 1×PBS supplemented with gelatin pH=7) at 37 oC for 24 hours. Fluorescence was measured with multimode spectrophotometer at 750 nm excitation and 785 nm emission. Correlation between fluorescence and colony forming units in each well was determined using Pearson’s correlation test.


SI Figure Legends

Fig. S1. Characterization of CNIR800. (A) HRMS of CNIR800. (B) HPLC spectrum of CNIR800.
Fig. S2. CNIR800 allows detection of M. tuberculosis (Mtb) in live animals after subcutaneous infection. (A) Whole-body images of mice subcutaneously infected with 103 to 106 CFU of Mtb. Images were acquired at 6 h post administration of CNIR800 using IVIS epi-illumination. (B) Comparison of the fluorescence over time post-administration of CNIR800. Statistical significance was determined using a Student’s 2-tail t-test with significance set at *P<0.05, **P<0.01, ***P<0.001 comparing with different concentrations at the same time point. Red asterisks indicate comparison between 106 and 105 CFU and black asterisks indicate comparison between 105 and 104 CFU. (C) Cartoon depicting inoculation sites, number of bacteria infected, and regions of interest used for each animal.
Fig. S3. Fluorescence changes in mice after intratracheal delivery of Mtb with CNIR800 administration. Mice were infected with Mtb intratracheally and administered CNIR800 (20 µM, 2.5 µl/g, I.P) at 24 h post-infection. At the indicated times post-administration of CNIR800, whole body images were acquired followed by ex vivo lung imaging. Lungs were homogenized and plated for CFU (A) Representative whole body images and (B) excised lung images at 6 h post-administration of CNIR800. (C) Quantification of fluorescence in whole body images over time post-administration of CNIR800 as compared to uninfected animals that had been given substrate. *P<0.05, **P<0.001 comparing infected with 1×103 CFU to uninfected animals at the same time point.
Fig. S4. REF imaging using CNIR800 allows tracking high dose of Mtb aerosol infection in mice. Mice were infected with ~103 CFU of Mtb by aerosol and imaged over 60 days post-infection with REF substrate CNIR800 (20 µM, 2.5 µl/g, I.P). (A) Representative whole body images at each time point. (B) Quantification of fluorescence in animal whole body. Data represent the mean and standard deviation of 3D FLIT analyses. (C) Bacterial loads as determined by CFU in lung homogenates. Data represent the mean and standard deviation for each group of four animals.

 

Fig. S5. CNIR800 allows detection of Mtb in the guinea pigs. Guinea pigs were infected with Mtb stain CDC1551 at ~50 CFU by aerosol. At 6 weeks post-infection, animals were imaged using REF substrate CNIR800 (40 µM, 1.25 µl/g, I.P.) followed by ex vivo lung imaging and plating lung homogenates for CFU. (A) Representative whole body images taken using trans-illumination at 6 h post-administration of CNIR800. (B) Representative ex vivo lung images taken using epi-illumination. (C) Quantification of fluorescent signal in whole body that was given substrate. Black bar denotes median. (D) Correlation of fluorescent signal in whole body to CFU in the lungs. Correlation was determined using Pearson’s correlation test.


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