Research ArticleTuberculosis

Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe

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Science Translational Medicine  28 Feb 2018:
Vol. 10, Issue 430, eaam6310
DOI: 10.1126/scitranslmed.aam6310
  • Fig. 1 Strategy for the detection of Actinobacteria by metabolic labeling of the mycomembrane with fluorescent trehalose analogs.

    (A) Schematic illustration depicting how fluorescent trehalose analogs are converted to trehalose mycolates by the antigen 85 (Ag85) protein complex and incorporated into the mycomembrane of bacteria. (B) Bacterial phylogenetic tree (left) highlighting Actinobacteria that have Ag85 protein homologs (red box). These bacteria (examples listed on right) can potentially incorporate trehalose analogs into the mycomembrane. (C) Representative list of Actinomycetales found (green) or not found (black) in the tblastn search results of unique organisms that may label with trehalose dyes, using the amino acid sequence from the reference proteins fbpA, fbpB, and fbpC. The complete tblastn search results are listed in table S1.

  • Fig. 2 An environment-sensitive fluorogenic trehalose derivative for the detection of mycobacteria.

    (A) Schematic illustration depicting the mechanism by which solvatochromic trehalose analogs are converted to trehalose mycolates via the Ag85 complex. Their incorporation into the low dieletric environment of the mycomembrane leads to fluorescence. (B) Structures of 4-N,N-dimethylamino-1,8-naphthalimide–conjugated trehalose (DMN-Tre or 1) and control compounds DMN-glucose (DMN-Glc or 2) and 6-fluorescein-trehalose (6-FlTre or 3). (C) Fluorescence emission spectra of DMN-Tre in mixtures of dioxane/H2O. Inset shows enlargement of spectra in ≤99% dioxane samples.

  • Fig. 3 Specific, no-wash detection of mycobacteria and corynebacteria using DMN-Tre.

    (A) No-wash imaging of Mycobacterium smegmatis (Msmeg), Corynebacterium glutamicum (Cg), and Mycobacterium marinum (Mm) labeled with 100 μM DMN-Tre or 6-FITre for 1, 2, and 6 hours, respectively. (B) Fluorescent labeling of Msmeg and Cg by DMN-Tre as a function of time. Cells were directly imaged without washing. (C) No-wash imaging of Cg, Bacillus subtilis (Bs), Escherichia coli (Ec), Listeria monocytogenes (Lm), and Staphylococcus aureus (Sa) cells incubated with 100 μM DMN-Tre for 2 hours. Background fluorescence with non–acid-fast bacteria is minimal under no-wash imaging conditions. (D) Colabeling of mCherry-expressing Msmeg combined with Bs, Ec, Lm, and Sa labeled with 100 μM DMN-Tre and Hoechst (20 μg/ml) for 1 hour under no-wash conditions. Images were collected in the differential interference contrast (DIC), 4′,6-diamidino-2-phenylindole (DAPI) (Hoechst fluorescence), fluorescein isothiocyanate (FITC)/green fluorescent protein (GFP) (DMN fluorescence), or red fluorescent protein (RFP) (mCherry fluorescence) channels of a Nikon A1R confocal microscope. “Fl merge” represents merged images from the DAPI, GFP, and RFP fluorescence channels. “Merge” represents merged images from all channels. Scale bars, 5 μm.

  • Fig. 4 Labeling of Msmeg with DMN-Tre is fast and specific and depends on Ag85A function.

    (A) Flow cytometry mean fluorescence intensity (MFI) analysis of Msmeg labeling time course. Cells were incubated with 100 μM DMN-Tre for 20, 60, 90, or 240 min. a.u., arbitrary units. (B) Quantitative microscopy analysis of mCherry Msmeg cells after incubation with 100 μM DMN-Tre for 30 min, displaying the ratio of total area of DMN fluorescence over total area of mCherry fluorescence. (C to E) Flow cytometry MFI analysis of Msmeg cells (C) incubated for 30 min with 100 μM DMN-Tre or DMN-Glc and competition of DMN-Tre labeling (D) with free trehalose using the same labeling conditions as in (C); (E) Msmeg cells preincubated with ebselen, an Ag85 inhibitor, for 3 hours and labeled with DMN-Tre as in (C). Data are means ± SEM from at least two independent experiments. Data were analyzed by Student’s t test in (B) and one-way analysis of variance (ANOVA) test in (A) and (C) to (E) (***P < 0.001 and ****P < 0.0001; ns, not significant).

  • Fig. 5 DMN-Tre labeling is selective for live mycobacteria.

    (A and B) No-wash imaging (A) and flow cytometry MFI analysis (B) of live and heat-killed (95°C for 30 min) mCherry Msmeg cells in the presence of 100 μM DMN-Tre for 30 min. (C) Flow cytometry MFI analysis of Msmeg cells in exponential (log) or stationary growth phase incubated with 100 μM DMN-Tre for 1 hour or overnight (~16 hours). (D) Flow cytometry analysis of live or heat-killed cells resuspended with or without culture filtrates from live cells, followed by incubation with DMN-Tre as in (A). Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (B) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by two-way ANOVA test (****P < 0.0001).

  • Fig. 6 DMN-Tre labeling is inhibited by tuberculosis drugs.

    (A and B) Flow cytometry MFI analysis (A) and no-wash imaging (B) of control and drug-treated (37°C for 3 hours) Msmeg cells labeled with 100 μM DMN-Tre for 30 min. Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (20 μg/ml), and isoniazid (20 μg/ml) in 7H9 medium. (C) Flow cytometry MFI analysis of wild-type (WT) Msmeg or KatG mutant cells treated with isoniazid (INH) at the indicated final concentrations (0, 3, 6, or 10 μg/ml of isoniazid) for 3 hours, followed by incubation with DMN-Tre for 30 min. Images were collected in the DIC, FITC/GFP (for DMN fluorescence), or RFP (for mCherry fluorescence) channels of a Nikon A1R confocal microscope. Scale bars, 5 μm. In (A) and (C), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (****P < 0.0001).

  • Fig. 7 DMN-Tre labeling of Mycobacterium tuberculosis is inhibited by tuberculosis drug cocktail, unlike auramine staining.

    (A) Microscopy analysis of Mycobacterium tuberculosis (Mtb) cells incubated with 100 μM DMN-Tre for 2 hours. (B) Flow cytometry MFI analysis of Mtb cells incubated with DMN-Tre for the indicated times: 0.5, 1, 2, and 3 hours or overnight (~16 hours). Error bars denote three biological replicates. (C and D) Microscopy analysis of control and drug-treated Mtb cells labeled with 100 μM DMN-Tre overnight (C) or stained with the auramine-based Fluorescent Stain kit for mycobacteria (05151, Sigma-Aldrich) (D). Drug cocktail contents: ethambutol (1 μg/ml), rifampicin (0.2 μg/ml), SQ109 (10 μg/ml), and isoniazid (10 μg/ml) in 7H9 medium. Images were collected in the DIC or FITC/GFP (for DMN and Auramine fluorescence) channels of a Nikon A1R confocal microscope. Cells stained with auramine were given an orange pseudocolor. Scale bars, 5 μm. In (B), data are means ± SEM from at least two independent experiments. Data were analyzed by one-way ANOVA test (*P < 0.05, ***P < 0.001, and ****P < 0.0001).

  • Fig. 8 DMN-Tre detects Mtb in sputum samples from patients with tuberculosis, similar to the auramine stain.

    (A) Illustration of sputum sample labeling protocol: 16 sputum samples were collected from diagnosed treatment-naïve patients with tuberculosis and were decontaminated with N-acetyl-l-cysteine (NalC)/NaOH mixture following recommended standards. Decontaminated sputum samples were split in two equal aliquots, each incubated with 1 mM DMN-Tre or smeared with Auramine O stain, followed by imaging. (B) Microscopy analysis of four decontaminated sputum samples incubated with 1 mM DMN-Tre in 7H9 liquid medium for 2 hours in a 37°C atmospheric incubator. (C) Microscopy analysis of decontaminated sputum samples either treated with 1 mM DMN-Tre for 30 min at 37°C (top) or directly fixed onto microscope slide for Auramine O staining following standard kit protocol (bottom, orange pseudocolor). (D) Bar graph depiction of the total Mtb cell number detected over eight fields of view per sample with either DMN-Tre or Auramine O in the 16 sputum samples treated as in (C). Images were collected in the DIC and FITC (for DMN and auramine fluorescence) channels of a Zeiss Observer Z1-inverted fluorescent microscope. Scale bars, 5 μm.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/430/eaam6310/DC1

    Materials and Methods

    Fig. S1. Synthesis of DMN-Tre and DMN-Glc.

    Fig. S2. DMN-Tre labeling is concentration-dependent.

    Fig. S3. Optimization of DMN-Tre detection.

    Fig. S4. DMN-Tre labeling is specific to bacteria bearing mycomembranes.

    Fig. S5. DMN-Tre labeling does not primarily rely on the intracellular trehalose metabolic pathway.

    Fig. S6. Msmeg ΔMSMEG_6396-6399 gene deletion mutant exhibits reduced labeling with DMN-Tre.

    Fig. S7. DMN-Tre is incorporated into trehalose monomycolate in the mycobacterial outer membrane.

    Fig. S8. Loss of lipid tails from DMN-Tre–corynemonomycolates is detected by tandem mass spectrometry.

    Fig. S9. Drug cocktail kills Msmeg cells in vitro.

    Fig. S10. DMN-Tre time course labeling of H37Rv axenic cultures.

    Fig. S11. Drug cocktail kills Mtb cells and inhibits DMN-Tre incorporation into the mycomembrane.

    Fig. S12. Rifampin-treated Mtb cells do not label with DMN-Tre, even in the presence of culture filtrates from live Mtb cells.

    Fig. S13. DMN-Tre detects Mtb cells in sputum samples comparably to auramine staining.

    Table S1. List of strains with Ag85 homology.

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

    Reference (46)

  • Supplementary Material for:

    Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe

    Mireille Kamariza, Peyton Shieh, Christopher S. Ealand, Julian S. Peters, Brian Chu, Frances P. Rodriguez-Rivera, Mohammed R. Babu Sait, William V. Treuren, Neil Martinson, Rainer Kalscheuer, Bavesh D. Kana, Carolyn R. Bertozzi*

    *Corresponding author. Email: bertozzi{at}stanford.edu

    Published 28 February 2018, Sci. Transl. Med. 10, eaam6310 (2018)
    DOI: 10.1126/scitranslmed.aam6310

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Synthesis of DMN-Tre and DMN-Glc.
    • Fig. S2. DMN-Tre labeling is concentration-dependent.
    • Fig. S3. Optimization of DMN-Tre detection.
    • Fig. S4. DMN-Tre labeling is specific to bacteria bearing mycomembranes.
    • Fig. S5. DMN-Tre labeling does not primarily rely on the intracellular trehalose metabolic pathway.
    • Fig. S6. Msmeg ΔMSMEG:6396-6399 gene deletion mutant exhibits reduced labeling with DMN-Tre.
    • Fig. S7. DMN-Tre is incorporated into trehalose monomycolate in the mycobacterial outer membrane.
    • Fig. S8. Loss of lipid tails from DMN-Tre–corynemonomycolates is detected by tandem mass spectrometry.
    • Fig. S9. Drug cocktail kills Msmeg cells in vitro.
    • Fig. S10. DMN-Tre time course labeling of H37Rv axenic cultures.
    • Fig. S11. Drug cocktail kills Mtb cells and inhibits DMN-Tre incorporation into the mycomembrane.
    • Fig. S12. Rifampin-treated Mtb cells do not label with DMN-Tre, even in the presence of culture filtrates from live Mtb cells.
    • Fig. S13. DMN-Tre detects Mtb cells in sputum samples comparably to auramine staining.
    • Table S1. List of strains with Ag85 homology.
    • Reference (46)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Appendix S1 (.pdf format). 1H and 13C nuclear magnetic resonance spectra.

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