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

A fluorescence turn-on probe, an azide-masked and trehalose-derivatized carbazole (), 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 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 . The uptake decreased in the presence of trehalose in a concentration-dependent manner, indicating that hijacked the trehalose uptake pathway. Mechanistic studies demonstrated that the trehalose transporter LpqY-SugABC was the primary pathway for the uptake of . The uptake decreased in the LpqY-SugABC deletion mutants Δ, Δ, Δ, and Δ and fully recovered in the complemented strain of Δ. For the mycolyl transferase antigen 85 complex (Ag85), however, only a slight reduction of uptake was observed in the Ag85 deletion mutant Δ, and no incorporation of into the outer membrane was observed. The unique intracellular incorporation mechanism of 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.