Fluorescent β-ketoenole AmyGreen dye for visualization of amyloid components of bacterial biofilms

Green-emitting water-soluble amino-ketoenole dye AmyGreen is proposed as an efficient fluorescent stain for visualization of bacterial amyloids in biofilms and the detection of pathological amyloids in vitro. This dye is almost non-fluorescent in solution, displays strong green emission in the presence of amyloid fibril of proteins. AmyGreen is also weakly fluorescent in presence to biomolecules that are components of cells, extracellular matrix or medium: nucleic acids, polysaccharides, lipids, and proteins. Thus, the luminescence turn-on behavior of AmyGreen can be utilized for visualization of amyloid components of bacterial biofilm extracellular matrix. Herein we report the application of AmyGreen for fluorescent staining of a number of amyloid-contained bacteria biofilms produced by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Bordetella avium, and Staphylococcus aureus. The effectiveness of AmyGreen was compared to traditional amyloid sensitive dye Thioflavine T. The main advantage of AmyGreen (concentration 10^-5 M) is a higher sensitivity in the visualization of amyloid biofilm components over Thioflavine T (10^-4 M) as it was revealed when staining E. coli and K. pneumoniae bacterial biofilms. Besides, AmyGreen displays lower cross-selectivity to nucleic acids as demonstrated both in in-solution experiments and upon staining of eukaryotic human mesenchymal stem cells used as amyloid-free negative control over amyloid-rich bacterial biofilms. The results point to a lower risk of false-positive response upon determination of amyloid components of bacterial biofilm using AmyGreen. Co-staining of biofilm by AmyGreen and cellulose sensitive dye Calcofluor White show difference in their staining patterns and localization, indicating separation of polysaccharide-rich and amyloid-rich regions of investigated biofilms. Thus, we suggest the new AmyGreen stain for visualization and differentiation of amyloid fibrils in bacterial biofilms to be used solely and in combination with other stains for confocal and fluorescence microscopy analysis.