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.