Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria

Area of Science:

• Biomedical imaging
• Chemical biology
• Microbiology

Background:

• Mycobacteria pose significant diagnostic challenges.
• Existing imaging methods for mycobacteria often lack specificity or require complex procedures.
• Targeting specific bacterial uptake pathways is a promising strategy for developing novel diagnostic probes.

Purpose of the Study:

• To develop and characterize a novel fluorescence turn-on probe for imaging mycobacteria.
• To investigate the uptake mechanism and specificity of the developed probe.
• To assess the potential of the probe for diagnosing mycobacterial infections in clinical samples.

Main Methods:

• Synthesis of a trehalose-derivatized carbazole (Tre-Cz) fluorescence probe.
• Photoactivation of the azide group in Tre-Cz to generate fluorescence.
• Testing probe specificity against various bacterial species (Gram-positive, Gram-negative, and mycobacteria).
• Evaluating probe performance in sputum samples and with different bacterial states (heat-killed, stationary, treated).
• Investigating the role of the trehalose uptake pathway (LpqY-SugABC) and antigen 85 complex (Ag85) in probe uptake using deletion mutants.

Main Results:

• The developed Tre-Cz probe exhibits fluorescence turn-on upon photoactivation, specific for mycobacteria.
• The probe allows for naked-eye visualization of mycobacteria at concentrations as low as 10^3 CFU/mL using a handheld UV lamp.
• Probe uptake is dependent on metabolically active mycobacteria and utilizes the LpqY-SugABC trehalose transporter pathway.
• Uptake was significantly reduced in LpqY-SugABC deletion mutants, confirming the transporter's role.
• Minimal impact on uptake was observed with Ag85 deletion mutants, suggesting a distinct intracellular incorporation mechanism.

Conclusions:

• Tre-Cz is a highly specific and sensitive fluorescence turn-on probe for imaging metabolically active mycobacteria.
• The probe's unique intracellular incorporation mechanism via the LpqY-SugABC transporter differentiates it from other trehalose-based probes.
• Tre-Cz holds promise for developing advanced diagnostic tools and targeted therapeutic delivery systems for mycobacterial infections.