Biopolym. Cell. 2021; 37(6):447-458.
Molecular and Cell Biotechnologies
Benzoxazole styrylcyanine dye as a fluorescent probe for functional amyloid visualization in Staphylococcus aureus ATCC25923 biofilm
1Chernii S. V., 1Moshynets O. V., 1Aristova D. I., 1Kryvorotenko D. V., 1Losytskyy M. Yu., 1, 2Iungin O. S., 1Yarmoluk S. M., 1Volynets G. P.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03143
  2. Kyiv National University of Technologies and Design
    2, Nemirovich-Danchenko Str., Kyiv, Ukraine, 01011


Aim. Synthesis and characterization of styrylcyanine dye as a potential fluorescent probe for the detection in vitro of pathological amyloid fibrils and functional amyloid in S. aureus biofilm. Methods. Chemical synthesis, fluorescence spectroscopy, irradiation with a visible light source, confocal laser scanning microscopy, fluorescence microscopy. Results. Styrylcyanine dye is low fluorescent when free, but in the presence of amyloid fibrils in vitro shows an increase in the emission intensity up to 214 times depending on the amyloidogenic protein structure; the most pronounced enhancement was observed for fibrils of beta-lactoglobulin. Photostability of the dye in the free state is low, but binding to amyloid fibrils results in a strong increase of dye photostability. Upon staining S. aureus biofilm, the dye stains extracellular components of the biofilm matrix and does not penetrate the cell. Conclusion. This dye is suggested to visualize the functional amyloids of S. aureus biofilm with a red emission.
Keywords: fluorescence microscopy, styryl dyes, S. aureus, functional amyloids, bacterial biofilm, laser scanning confocal microscopy


[1] Koza A, Kusmierska A, McLauglin K, Moshynets O, Spiers AJ. Adaptive radiation of P. fluorescens SBW25 in experimental microcosms provides an understanding of the evolutionary ecology and molecular biology of A-L interface biofilm-formation. FEMS Microbiol Lett. 2017;364(12).
[2] Moshynets OV, Spiers AJ. Viewing biofilms within the larger context of bacterial aggregations. In "Biofilms". InTech Press, 2016; 3-22.
[3] Robertson M, Hapca SM, Moshynets O, Spiers AJ. Air-liquid interface biofilm formation by psychrotrophic pseudomonads recovered from spoilt meat. Antonie Van Leeuwenhoek. 2013; 103(1):251-9.
[4] De Rycker M, Horn D, Aldridge B, Amewu RK, Barry CE, Buckner FS, Cook S, Ferguson MA, Gobeau N, Herrmann J, Herrling P, Hope W, Keiser J, Lafuente MJ, Leeson PD, Leroy D, Manjunatha UH, McCarthy J, Mizrahi V, Moshynets O, Niles J, Overington JP, Pottage J, Rao SP, Read KD, Ribeiro I, Silver LL, Southern J, Spangenberg T, Sundar S, Taylor C, Voorhis WV, White NJ, Wyllie S, Wyatt PG, Gilbert IH. Setting our sights on infectious diseases. ACS Infectious Disease. 2020; 6(1):3-13
[5] Moshynets O, Bardeau JF, Tarasyuk O, Makhno S, Cherniavska T, Dzhuzha O, Potters G, Rogalsky S. Antibiofilm activity of polyamide 11 modified with thermally stable polymeric biocide polyhexamethylene gua-nidine 2-naphtalenesulfonate.Int J Mol. Sci. 2019; 20(2):348.
[6] Flemming H-C, Wingender J, Szewzyk U, Steinberg P, Rice SA, Kjelleberg S. Biofilms: an emergent form of bacterial life. Nat Rev Microbiol. 2016;14(9): 563-75.
[7] Matz C, Kjelleberg S. Off the hook-how bacteria survive protozoan grazing. Trends Microbiol. 2005;13(7):302-7.
[8] Lee KWK, Periasamy S, Mukherjee M, Xie C, Kjelleberg S, Rice SA. Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm. ISME J. 2014;8(4):894-907.
[9] Burmølle M, Ren D, Bjarnsholt T, Sørensen SJ. Interactions in multiespecies biofilms: do they actually matter? Trends Microbiol. 2014; 22(2):84-91.
[10] Young KD. The selective value of bacterial shape. Microbiol Mol Biol Rev. 2006;70(3):660-703.
[11] Martin M, Hölscher T, Dragos A, Cooper VS, Kovács AT. Laboratory evolution of microbial interactions in bacterial biofilms. J Bacteriol. 2016; 198(19):2564-71.
[12] Moshynets OV, Foster D, Karakhim SA, McLaughlin K, Rogalsky SP, Rymar SY, Volynets GP, Spiers AJ. Examining c-di-GMP and possible QS regulation in Pseudomonas fluorescens SBW25: links between intra and inter-cellular regulation benefits community cooperative activities such as biofilm formation. Ukr Biochem J. 2018; 90 (3): 17-31.
[13] Hobley L, Harkins C, MacPhee CE, Stanley-Wall NR. Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes. FEMS Microbiol Rev. 2015; 39(5):649-69.
[14] Turnbull L, Toyofuku M, Hynen AL, Kurosawa M, Pessi G, Petty NK, Osvath SR, Cárcamo-Oyarce G, Gloag ES, Shimoni R, Omasits U, Ito S, Yap X, Monahan LG, Cavaliere R, Ahrens CH, Charles IG, Nomura N, Eberl L, Whitchurch CB. Explosive cell lysis as a mechanism for the biogenesis of bacterial membrane vesicles and biofilms. Nat Commun. 2016; 7:11220
[15] McLaughlin K, Folorunso AO, Deeni YY, Foster D, Gorbatiuk O, Hapca SM, Immoor C, Koza A, Mohammed IU, Moshynets O, Rogalsky S, Zawadzki K, Spiers AJ. Biofilm formation and cellulose expression by Bordetella avium 197N, the causative agent of bordetellosis in birds and an opportunistic respiratory pathogen in humans. Res Microbiol. 2017; 168(5): 419-30.
[16] Dueholm MS, Otzen D, Nielsen PH. Evolutionary insight into the functional amyloids of the pseudomonads. PLoS One. 2013;8(10):e76630.
[17] Serra D, Richter A, Klauck G, Mika F, Hengge R. Cellulose as an architectural element in spatially structured Escherichia coli biofilms. J Bacteriol. 2013; 4(2):e00103-13.
[18] Archer GL. Staphylococcus aureus: a well-armed pathogen. Clin Infect Dis. 1998;26(5):1179-81.
[19] Pérez-Montarelo D, Viedma E, Murcia M, Muñoz-Gallego I, Larrosa N, Brañas P, Fernández-Hidalgo N, Gavaldà J, Almirante B, Chaves F. Pathogenic characteristics of Staphylococcus aureus endovascular infection isolates from different clonal complexes. Front Microbiol. 2017;8:917.
[20] Cucarella C, Solano C, Valle J, Amorena B, Lasa I, Penadés JR. Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol. 2001;183(9):2888-96.
[21] Van Gerven N, Van der Verren SE, Reiter DM, Remaut H. The role of functional amyloids in bacterial virulence. J Mol Biol. 2018;430(20):3657-84.
[22] Volynets G, Vyshniakova H, Nitulescu G, Nitulescu GM, Ungurianu A, Margina D, Moshynets O, Bdzhola V, Koleiev I, Iungin O, Tarnavskiy S, Yarmoluk S. Identification of novel antistaphylococcal hit compounds targeting Sortase A. Molecules. 2021;26(23):7095.
[23] Kim JY, Sahu S, Yau YH, Wang X, Shochat SG, Nielsen PH, Dueholm MS, Otzen DE, Lee J, Delos Santos MM, Yam JK, Kang NY, Park SJ, Kwon H, Seviour T, Yang L, Givskov M, Chang YT. Detection of pathogenic biofilms with bacterial amyloid targeting fluorescent probe, CDy11. J Am Chem Soc. 2016;138(1):402-7.
[24] Otzen D. Functional amyloid: turning swords into plowshares. Prion. 2010;4(4):256-64.
[25] Oli MW, Otoo HN, Crowley PJ, Heim KP, Nascimento MM, Ramsook CB, Lipke PN, Brady LJ. Functional amyloid formation by Streptococcus mutans. Microbiology. 2012;158(Pt 12):2903-16.
[26] Shewmaker F, McGlinchey RP, Wickner RB. Structural insights into functional and pathological amyloid. J Biol Chem. 2011;286(19):16533-40.
[27] Sugimoto S, Arita-Morioka K, Mizunoe Y, Yamanaka K, Ogura T. Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels. Nucleic Acids Res. 2015;43(14):e92.
[28] Kovalska V, Chernii S, Losytskyy M, Dovbii Y, Tretyakova I, Czerwieniec R, Chernii V, Yarmoluk S, Volkov S. β-ketoenole dyes: synthesis and study as fluorescent sensors for protein amyloid aggregates. Dyes Pigments. 2016;132:274-81.
[29] Kovalska V, Chernii S, Losytskyy M, Tretyakova I, Dovbii Y, Gorski A, Chernii V, Czerwieniec R, Yarmoluk S. Design of functionalized β-ketoenole derivatives as efficient fluorescent dyes for detection of amyloid fibrils. New J Chem. 2018; 42(16):13308-18.
[30] Moshynets O, Chernii S, Chernii V, Losytskyy M, Karakhim S, Czerwieniec R, Pekhnyo V, Yarmoluk S, Kovalska V. Fluorescent β-ketoenole AmyGreen dye for visualization of amyloid components of bacterial biofilms. Methods Appl Fluoresc. 2020;8(3):035006.
[31] Mazaheri M, Moosavi-Movahedi AA, Saboury AA, Khodagholi F, Shaerzadeh F, Sheibani N. Curcumin protects β-Lactoglobulin fibril formation and fibril-induced neurotoxicity in PC12 Cells. PLoS One. 2015;10(7):e0133206.
[32] Kovalska V, Chernii S, Cherepanov V, Losytskyy M, Chernii V, Varzatskii O, Naumovets A, YarmolukS. The impact of binding of macrocyclic metal complexes on amyloid fibrillization of insulin and lysozyme. J Mol Recognit. 2017; 30(8):e2622.
[33] Sambrook J, Fritsch E, Maniatis T. Molecular Cloning: A Laboratory Manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. 1989.
[34] Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-82.