A red fluorescent small-molecule for visualization of higher-order cyclic dimeric guanosine monophosphate (c-di-GMP) structure in live bacterial cells and real-time monitoring of biofilm formation on biotic and abiotic surfaces

Cyclic dimeric guanosine monophosphate (c-di-GMP) is an important second messenger in bacteria. It regulates a wide range of bacterial functions and behaviors including biofilm formation that causes chronic infections and antibiotic resistance. C-di-GMP being as a signal transducer in bacteria is known to exist in monomer and dimer form. Recent studies also discover that c-di-GMP can form higher-order oligomers, such as tetramer and octamer, which may have physiological roles in bacterial cells. Moreover, the tetrameric c-di-GMP structure was reported to link two subunits of a transcription factor (BldD), which controls the progression of multicellular differentiation in sporulating actinomycete bacteria and then mediates the dimerization process. Current understanding on higher-order oligomers of c-di-GMP is relatively limited compared to its monomer or dimer structure. To probe and visualize the higher-order structure of c-di-GMP and its associated biofunctions in live bacterial cells with fluorescence techniques for mechanistic study and cellular investigation is important. Nonetheless, the sensitive and selective fluorescent probe with a rapid signal response for higher-order oligomers of c-di-GMP is currently lacking. In the present study, a series of fluorescent probes that preferentially interacted with tetrameric c-di-GMP and generated red fluorescence signal promptly were synthesized and investigated. The interaction mechanism was studied with 1H NMR and molecular docking. In addition, the ligand was demonstrated as an excellent molecular fluorescent probe for bioimaging of tetrameric c-di-GMP structure and monitoring of biofilm formation on both biotic and abiotic surfaces with pathogenic bacteria including Pseudomonas aeruginosa PAO1 and Bacillus subtilis 168.