A Far‐Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug‐Susceptibility Testing

Banahene, Nicholas; Gepford, Dana M.; Biegas, Kyle J.; Swanson, Daniel H.; Hsu, Yen-Pang; Murphy, Brennan; Taylor, Zachary E.; Lepori, Irene; Siegrist, M. Sloan; Obregón-Henao, Andrés; VanNieuwenhze, Michael S.; Swarts, Benjamin M.

doi:10.1002/anie.202213563

Cited by 12 publications

(12 citation statements)

References 36 publications

(2 reference statements)

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“…Several fluorescence turn-on probes based on the action of trehalose have been reported, including solvatochromic dyes, − quencher-fluorophore, molecular rotor turn-on fluorophore, FRET, or through enzyme action . The solvatochromic dye-derivatized trehalose probe developed by Bertozzi and co-workers was incorporated into the mycomembrane through the action of Ag85 .…”

Section: Introductionmentioning

confidence: 99%

Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria

Liyanage

Raviranga

Ryan

et al. 2023

JACS Au

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A fluorescence turn-on probe, an azide-masked and trehalose-derivatized carbazole (Tre-Cz), was developed to image mycobacteria. The fluorescence turn-on is achieved by photoactivation of the azide, which generates a fluorescent product through an efficient intramolecular C–H insertion reaction. The probe is highly specific for mycobacteria and could image mycobacteria in the presence of other Gram-positive and Gram-negative bacteria. Both the photoactivation and detection can be accomplished using a handheld UV lamp, giving a limit of detection of 103 CFU/mL, which can be visualized by the naked eye. The probe was also able to image mycobacteria spiked in sputum samples, although the detection sensitivity was lower. Studies using heat-killed, stationary-phase, and isoniazid-treated mycobacteria showed that metabolically active bacteria are required for the uptake of Tre-Cz. The uptake decreased in the presence of trehalose in a concentration-dependent manner, indicating that Tre-Cz hijacked the trehalose uptake pathway. Mechanistic studies demonstrated that the trehalose transporter LpqY-SugABC was the primary pathway for the uptake of Tre-Cz. The uptake decreased in the LpqY-SugABC deletion mutants ΔlpqY, ΔsugA, ΔsugB, and ΔsugC and fully recovered in the complemented strain of ΔsugC. For the mycolyl transferase antigen 85 complex (Ag85), however, only a slight reduction of uptake was observed in the Ag85 deletion mutant ΔAg85C, and no incorporation of Tre-Cz into the outer membrane was observed. The unique intracellular incorporation mechanism of Tre-Cz through the LpqY-SugABC transporter, which differs from other trehalose-based fluorescence probes, unlocks potential opportunities to bring molecular cargoes to mycobacteria for both fundamental studies and theranostic applications.

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Section: Introductionmentioning

confidence: 99%

Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria

Liyanage

Raviranga

Ryan

et al. 2023

JACS Au

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“…We could also observe that the staining was not homogenously distributed at the surface of the cells. While similar morphological differences have been reported for other receptor systems, [59,65] additional experiments such as DNA‐PAINT or super‐resolution confocal microscopy will be required to identify the origin of this non‐homogenous protein expression.…”

Section: Discussionmentioning

confidence: 59%

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization

et al. 2023

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Chemical cell surface modification is a fast-growing field of research, due to its enormous potential in tissue engineering, cell-based immunotherapy, and regenerative medicine. However, engineering of bacterial tissues by chemical cell surface modification has been vastly underexplored and the identification of suitable molecular handles is in dire need. We present here, an orthogonal nucleic acid-protein conjugation strategy to promote artificial bacterial aggregation. This system gathers the high selectivity and stability of linkage to a protein Tag expressed at the cell surface and the modularity and reversibility of aggregation due to oligonucleotide hybridization. For the first time, XNA (xeno nucleic acids in the form of 1,5anhydrohexitol nucleic acids) were immobilized via covalent, SNAP-tag-mediated interactions on cell surfaces to induce bacterial aggregation.

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“…The first near-infrared “off-to-on” fluorescent substrate for β-lactamase (BlaC) was developed by Cirillo et al . Then, Rao et al reported a series of fluorescent probes based on cephalosporin lactams that can be hydrolyzed by BlaC and release hydrolyzed β-lactam to generate fluorescence. − Fluorescent probes conjugated with trehalose − or trehalose mono- or dimycolate motifs , have been reported to be incorporated into the mycolic acid membrane of Mtb via the mycomembrane metabolic pathway and successfully detect live Mtb . However, in addition to short emission wavelength, low signal-to-noise ratio, and autofluorescence, these probes exhibit the inability to label nonreplicating bacteria, which may limit further clinical applications.…”

Section: Introductionmentioning

confidence: 99%

From Bench to Clinic: A Nitroreductase Rv3368c-Responsive Cyanine-Based Probe for the Specific Detection of Live Mycobacterium tuberculosis

Hong,

Geng,

Tian

et al. 2024

Anal. Chem.

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Tuberculosis (TB), characterized by high mortality and low diagnosis, is caused by a single pathogen, Mycobacterium tuberculosis (Mtb). Imaging tools that can be used to track Mtb without pre-labeling and to diagnose live Mtb in clinical samples can shorten the gap between bench and clinic, fuel the development of novel anti-TB drugs, strengthen TB prevention, and improve patient treatment. In this study, we report an unprecedented novel nitroreductase-responsive cyanine-based fluorescent probe (Cy3-NO 2 -tre) that rapidly and specifically labels Mtb and detects it in clinical samples. Cy3-NO 2 -tre generated fluorescence after activation by a specific nitroreductase, Rv3368c, which is conserved in the Mycobacteriaceae. Cy3-NO 2 -tre effectively imaged mycobacteria within infected host cells, tracked the infection process, and visualized Mycobacterium smegmatis being endocytosed by macrophages. Cy3-NO 2 -tre also detected Mtb in the sputum of patients with TB and exhibited excellent photostability. Furthermore, the Cy3-NO 2 -tre/auramine O percentage change within 7 ± 2 days post drug treatment in the sputum of inpatients was closely correlated with the reexamination results of the chest computed tomography, strongly demonstrating the clinical application of Cy3-NO 2 -tre as a prognostic indicator in monitoring the therapeutic efficacy of anti-TB drugs in the early patient care stage.

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A Far‐Red Molecular Rotor Fluorogenic Trehalose Probe for Live Mycobacteria Detection and Drug‐Susceptibility Testing

Cited by 12 publications

References 36 publications

Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria

Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria

Controlled E. coli Aggregation Mediated by DNA and XNA Hybridization

From Bench to Clinic: A Nitroreductase Rv3368c-Responsive Cyanine-Based Probe for the Specific Detection of Live Mycobacterium tuberculosis

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