Bioorthogonal probes for L-form conversion visualization and insights into antimicrobial resistance

Area of Science:

• Microbiology
• Chemical Biology
• Antimicrobial Resistance Research

Background:

• Cell wall-deficient bacteria (CWDB) contribute significantly to antimicrobial resistance (AMR) by forming persistent infections.
• Bacterial transition to L-form states allows evasion of conventional antibiotics, necessitating new detection methods.

Purpose of the Study:

• To develop molecular tools for real-time visualization of CWDB formation and L-form conversion.
• To investigate the impact of antibiotics on L-form conversion and its link to AMR.

Main Methods:

• Development of small-sized, peptidoglycan-specific fluorogenic probes using a two-step bioorthogonal strategy.
• Utilizing a novel d-alanine derivative (TCO-d-Ala) and a tetrazine-based probe (Tz-FL-S) for rapid fluorescence signal generation.
• Assessing probe performance in labeling Gram-positive and Gram-negative bacteria and monitoring N-form to L-form transition.

Main Results:

• The fluorogenic probe platform demonstrated rapid reaction kinetics and a significant increase in fluorescence intensity (4.9-fold).
• Effective labeling of bacterial peptidoglycan was achieved with high signal-to-noise ratios (15 to 305).
• Thirteen out of fourteen tested antibiotics were found to induce CWDB formation, highlighting a broad impact of antibiotics on bacterial adaptation.

Conclusions:

• The developed probes enable real-time visualization of CWDB formation, offering a valuable tool for studying bacterial adaptations.
• This research provides insights into the relationship between L-form conversion and AMR, crucial for combating drug-resistant infections.
• The findings pave the way for novel therapeutic strategies targeting persistent bacterial infections and AMR mechanisms.