Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria.

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 10 3 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 Δ lpqY , Δ sugA , Δ sugB , and Δ sugC and fully recovered in the complemented strain of Δ sugC . For the mycolyl transferase antigen 85 complex (Ag85), however, only a slight reduction of uptake was observed in the Ag85 deletion mutant Δ Ag85C , 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.