Research ArticleTuberculosis

Rapid and specific labeling of single live Mycobacterium tuberculosis with a dual-targeting fluorogenic probe

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Science Translational Medicine  15 Aug 2018:
Vol. 10, Issue 454, eaar4470
DOI: 10.1126/scitranslmed.aar4470
  • Fig. 1 Strategy for specific labeling of Mtb by targeting BlaC and DprE1.

    (A) Structure of dual-targeting fluorogenic probes (CDG-DNBs) and their reaction with BlaC and DprE1: blue, DprE1-targeting group; gray, caged fluorophore; orange, BlaC-sensing group; red, the covalent bond formed between DprE1 and uncaged fluorophore. (B) Mechanism of Mtb imaging by CDG-DNBs: (I) CDG-DNBs specifically label Mtb complex expressing BlaC and DprE1 but not other bacterial species not expressing BlaC or DprE1. Gray bulb with a light string and an anchor, corresponding to CDG-DNBs structure in (A), indicates the lactam (orange) caged fluorophore (gray bulb) with a trapping unit (blue). (II) Cartoon illustration of Mtb cell wall and membrane layer with glycolipids and mycolic acids (Mycomembrane), lipoarabinomannan (LAM), arabinogalactan (Arabinan), peptidoglycan (PG), and cytoplasmic membrane (CM). CDG-DNBs traverse the mycolate arabinogalactan layer through porins and are activated by BlaC and DprE1. (III) Proposed dual processing of CDG-DNBs by BlaC and DprE1 in periplasm (dashed circle in II). Upon BlaC (yellow pacman) activation, the lactam is hydrolyzed to uncage the fluorophore, which restores fluorescing (the string is pulled away to switch the light bulb green). The uncaged flurophore is retained in Mtb through DprE1 modification (the green bulb is anchored on DprE1).

  • Fig. 2 TG-DNBs as the optimal candidates for labeling DprE1-expressing bacteria.

    (A) Structures of TG, DprE1 inhibitor DNB1, and designed fluorophore-DNB analogs (TG-DNB1 and TG-DNB2). Red rectangle in TG-DNB2 indicates the structural similarity to DNB1. (B) Fluorescent labeling of DprE1SM by fluorescein-DNB (FI-DNB), rhodol-DNB (Rd-DNB), and TG-DNB analogs (10 μM). The whole lysate of freshly cultured M. smegmatis provided the natural substrate DPR and the cofactor FAD that are essential for the generation of DNB-DprE1 covalent product. (C) Fluorescence labeling of DprE1SM by TG-DNB2 (10 μM). 5*: DprE1SM was preheated at 90°C for 1 hour to denature the protein. 6**: Sample buffer was adjusted to 7 M urea and 20 mM DTT after incubation. These samples were further incubated for 1 hour at 37°C. 7***: DprE1SM was incubated with 50 μM DNB1 before addition of TG-DNB2. (D) Overlaid confocal images (bright-field and green fluorescence; 63×/oil; excitation, 490 nm; emission, 520 nm) of freshly cultured E. coli, S. pneumoniae, S. aureus, M. abscessus, C. diphtheriae, and M. smegmatis stained with 10 μM TG, TG-DNB1, or TG-DNB2 at room temperature for 1 hour. (E) Confocal images of individual M. smegmatis bacilli treated with TG-DNB2 showing polarized localization of green fluorescence. Scale bars, 2 μm. (F) Histogram of fluorescence-activated flow cytometry data from bacteria in (D). 1: TG-DNB1–treated group; 2: TG-DNB2–treated group. The relative fluorescence (F/FPBS) was calculated by normalizing the mean fluorescence intensity (MFI) against the autofluorescence intensity of PBS-treated bacteria (table S2).

  • Fig. 3 Characterization of the dual-targeting probe CDG-DNB2.

    (A) Structure of CDG-DNB2. (B) Overlaid confocal images (bright-field and green fluorescence; 63×/oil; excitation, 490 nm; emission, 520 nm) of freshly cultured E. coli expressing TEM-1 β-lactamase, S. pneumoniae, S. aureus, M. abscessus, C. diphtheriae, and M. smegmatis stained with 10 μM CDG-DNB2 in PBS at room temperature for 1 hour. (C) Histogram of normalized fluorescence from fluorescence-activated flow cytometry data with bacteria in (B). (D) Normalized fluorescence from M. smegmatis treated with CDG-DNB2 with and without β-lactamase inhibitor clavulanic acid (10 mM) and DNB1 (50 μM), analyzed by flow cytometry. M. smegmatis treated with CDG-DNB2 exhibited an average 90-fold increase of MFI over PBS, which was arbitrarily set to 1 to normalize the other groups with inhibitors. (E) Overlaid confocal images of freshly cultured BCG and H37Rv Mtb stained with CDG-DNB2. (F) Real-time confocal imaging of CDG-DNB2–treated BCG aggregates showing a time-dependent enhancement of green fluorescence.

  • Fig. 4 Specific labeling of live Mtb by CDG-DNB3.

    (A) Structure of CDG-DNB3. (B) Histogram of fluorescence-activated flow cytometry analysis with freshly cultured E. coli (TOP10) expressing TEM-1 β-lactamase, E. coli (TOP10) expressing BlaC, M. smegmatis, and BCG stained with 10 μM CDG-DNB2 or CDG-DNB3 in PBS at room temperature for 1 hour. 1: CDG-DNB2–treated group; 2: CDG-DNB3–treated group. (C) Microscopic imaging of freshly cultured (top row) and 121°C autoclaved (bottom row) BCG stained by Ziehl-Neelsen reagents, auramine O (green; excitation, 490 nm; emission, 520 nm), PI (red; excitation, 535 nm; emission, 617 nm), or CDG-DNB3 (10 μM, 1 hour). (D) Infection of macrophages by CDG-DNB3–labeled individual BCG bacilli: (I) bright-field image of BCG-infected macrophages (bacilli are indicated by arrows); (II) overlay of bright-field and fluorescence images showing the localization of CDG-DNB3–stained bacilli (green; excitation, 490 nm; emission, 520 nm); (III) overlay of fluorescence images showing the bacilli (green) and 4′,6-diamidino-2-phenylindole (DAPI)–stained nucleus (blue; excitation, 358 nm; emission, 461 nm); (IV) overlay of fluorescence image showing the bacilli, nucleus, and the macrophage plasma membrane (magenta; excitation, 649 nm; emission, 666 nm); (V to VII) orthogonal views (XY, XZ, and YZ ) of the bacilli indicated by arrows in (IV). ImageJ was used for image processing and stack projection.

  • Fig. 5 Rapid and specific quantification of live BCG with a microfluidic chip.

    (A) Overview of workflow. BCG was stained with CDG-DNB3 in PBS at room temperature (RT) for 1 hour, and then 10 μl of incubated mixture was applied onto the loading/sample collector in the chip. (B) Schematic of image capture using a microfluidic device. The capillary pump with a vent drove the flow of the sample from the loading/sample collector, whereas the collector and delay valves filtrated and retarded the mixture to allow single BCG imaging in detection chamber under fluorescence illumination. Cartoon shows a microscopic view of the channels with flowing BCG. LED, light-emitting diode. (C) Representative frame of image captured by the camera over the detection chamber (green fluorescence; excitation, 490 nm; emission, 520 nm) with fluorescence intensity peaks quantified in arbitrary units (a.u.) of up to 250 and 50 per scale (right). Yellow lines define the detection window used by the software to analyze the fluorescent signal. Each peak (red triangle) indicates one fluorescently labeled bacterium.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/454/eaar4470/DC1

    Materials and Methods

    Fig. S1. Synthesis of FI-DNB1, FI-DNB2, and FI-MNB.

    Fig. S2. Synthesis of Rd-DNB and Rd-MNB.

    Fig. S3. Synthesis of TG-DNB1.

    Fig. S4. Synthesis of TG-DNB2.

    Fig. S5. Synthesis of CDG-DNB1.

    Fig. S6. Synthesis of CDG-DNB2.

    Fig. S7. Synthesis of CDG-DNB3.

    Fig. S8. SDS-PAGE analysis of purified M. smegmatis DprE1.

    Fig. S9. DprE1 DNA sequence alignment.

    Fig. S10. Microscopic imaging and flow cytometry analysis of bacteria labeled by TG and TG-DNB analogs.

    Fig. S11. Characterization of TG-DNB2 in M. smegmatis.

    Fig. S12. Growth curve of M. smegmatis and BCG.

    Fig. S13. Inhibition study of TG-DNB2 with DNB1.

    Fig. S14. Characterization of CDG-1 and CDG-DNB analogs in bacteria.

    Fig. S15. Western blots detecting the expression of TEM-1 Bla and BlaC in transformed E. coli.

    Fig. S16. Labeling of BCG and H37Rv by CDG-DNB2.

    Fig. S17. Inhibition study with BlaC KO, BlaC compensated, and control vector transformed H37Rv and DNB1.

    Fig. S18. Labeling of Mtb in processed sputum by CDG-DNB2.

    Fig. S19. Evaluation of selectivity of CDG-DNB2/3 for BlaC.

    Fig. S20. Fluorescence intensity of CDG-DNB3 and TG-DNB2 with or without clavulanic acid, DNB1, or DprE1.

    Fig. S21. Characterization of CDG-DNB3 in M. smegmatis, BCG, and TEM-1 Bla or BlaC transformed E. coli.

    Fig. S22. Specific labeling of viable BCG by CDG-DNB3.

    Fig. S23. Labeling of Mtb in processed sputum by CDG-DNB3.

    Fig. S24. Overall layout and the functional regions of the microfluidic bacteria-counting device.

    Table S1. List of NTMs stained with CDG-DNB3.

    Table S2. List of individual subject-level data shown on bar graphs in all figures.

    Movie S1. Real-time fluorescence imaging of CDG-DNB2–treated BCG aggregates.

    Movie S2. Real-time bright-field imaging of CDG-DNB2–treated BCG aggregates in movie S1.

    Movie S3. Infection of macrophages by CDG-DNB3–labeled individual BCG bacilli.

    Movie S4. Automated counting of CDG-DNB3–labeled BCG bacilli with microfluidic chip.

    Appendix S1. 1H and 13C nuclear magnetic resonance and mass spectroscopy spectra.

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Synthesis of FI-DNB1, FI-DNB2, and FI-MNB.
    • Fig. S2. Synthesis of Rd-DNB and Rd-MNB.
    • Fig. S3. Synthesis of TG-DNB1.
    • Fig. S4. Synthesis of TG-DNB2.
    • Fig. S5. Synthesis of CDG-DNB1.
    • Fig. S6. Synthesis of CDG-DNB2.
    • Fig. S7. Synthesis of CDG-DNB3.
    • Fig. S8. SDS-PAGE analysis of purified M. smegmatis DprE1.
    • Fig. S9. DprE1 DNA sequence alignment.
    • Fig. S10. Microscopic imaging and flow cytometry analysis of bacteria labeled by TG and TG-DNB analogs.
    • Fig. S11. Characterization of TG-DNB2 in M. smegmatis.
    • Fig. S12. Growth curve of M. smegmatis and BCG.
    • Fig. S13. Inhibition study of TG-DNB2 with DNB1.
    • Fig. S14. Characterization of CDG-1 and CDG-DNB analogs in bacteria.
    • Fig. S15. Western blots detecting the expression of TEM-1 Bla and BlaC in transformed E. coli.
    • Fig. S16. Labeling of BCG and H37Rv by CDG-DNB2.
    • Fig. S17. Inhibition study with BlaC KO, BlaC compensated, and control vector transformed H37Rv and DNB1.
    • Fig. S18. Labeling of Mtb in processed sputum by CDG-DNB2.
    • Fig. S19. Evaluation of selectivity of CDG-DNB2/3 for BlaC.
    • Fig. S20. Fluorescence intensity of CDG-DNB3 and TG-DNB2 with or without clavulanic acid, DNB1, or DprE1.
    • Fig. S21. Characterization of CDG-DNB3 in M. smegmatis, BCG, and TEM-1 Bla or BlaC transformed E. coli.
    • Fig. S22. Specific labeling of viable BCG by CDG-DNB3.
    • Fig. S23. Labeling of Mtb in processed sputum by CDG-DNB3.
    • Fig. S24. Overall layout and the functional regions of the microfluidic bacteria-counting device.
    • Table S1. List of NTMs stained with CDG-DNB3.
    • Legend for Movies S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). List of individual subject-level data shown on bar graphs in all figures.
    • Movie S1 (.avi format). Real-time fluorescence imaging of CDG-DNB2–treated BCG aggregates.
    • Movie S2 (.avi format). Real-time bright-field imaging of CDG-DNB2–treated BCG aggregates in movie S1.
    • Movie S3 (.avi format). Infection of macrophages by CDG-DNB3–labeled individual BCG bacilli.
    • Movie S4 (.mp4 format). Automated counting of CDG-DNB3–labeled BCG bacilli with microfluidic chip.
    • Appendix S1 (PDF format). 1H and 13C nuclear magnetic resonance and mass spectroscopy spectra.

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