Alkyne-Substituted Fimbrolide Analogues as Novel Bacterial Quorum-Sensing Inhibitors

Gram-negative bacteria such as Pseudomonas aeruginosa use furanosyl diesters as autoinducers for quorum sensing (QS), a major regulatory and cell-to-cell communication system for social adaptation, virulence factor production, biofilm formation, and antibiotic resistance. A range of natural and synthetic brominated furanones, i.e. fimbrolide derivatives, have been found to act as inhibitors of QS-dependent bacterial phenotypes, complementing the bactericidal ability of traditional antibiotics. In this work, several novel acetylene analogues of fimbrolides were synthesised in moderate to high yields via Sonogashira coupling reactions of brominated furanones 4-bromo-5-(bromomethylene)furan-2(5H)-one 4 and 5-(dibromomethylene)-3-ethylfuran-2(5H)-one 5. The Sonogashira reaction of acetylenes on 4-bromo-5-(bromomethylene)furan-2(5H)-one 4 was favoured at the C5 methylene bromide over the C4 bromide substituent. On biological testing, the most potent compounds 13 and 14 showed 82 and 98 % bacterial quorum-sensing inhibitory (QSI) activity against Pseudomonas aeruginosa reporter strain respectively.

Furanone at Subinhibitory Concentrations Enhances Staphylococcal Biofilm Formation by luxS Repression

ABSTRACT Brominated furanones from marine algae inhibit multicellular behaviors of gram-negative bacteria such as biofilm formation and quorum sensing (QS) without affecting their growth. The interaction of furanone with QS in gram-positive bacteria is unknown. Staphylococci have two QS systems, agr and luxS, which lower biofilm formation by two different pathways, RNAIII upregulation and bacterial detachment, and polysaccharide intercellular adhesin (PIA) reduction, respectively. We synthesized natural furanone compound 2 [(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone] from Delisea pulchra and three analogues to investigate their effect on biofilm formation in gram-positive bacteria. Compound 2, but not the analogues, enhanced the biofilms of Staphylococcus epidermidis 1457 and 047 and of S. aureus Newman at concentrations between 1.25 and 20 μM. We show the growth inhibition of S. epidermidis and S. aureus by free furanone and demonstrate bactericidal activity. An induction of biofilm occurred at concentrations of 10 to 20% of the MIC and correlated with an increase in PIA. The biofilm effect was agr independent. It was due to interference with luxS, as shown by reduced luxS expression in the presence of compound 2 and independence of the strong biofilm formation in a luxS mutant upon furanone addition. Poly(l-lysine)-grafted/poly(ethylene glycol)-grafted furanone was ineffective on biofilm and not bactericidal, indicating the necessity for free furanone. Free furanone was similarly toxic for murine fibroblasts as for staphylococci, excluding a therapeutic application of this compound. In summary, we observed a biofilm enhancement by furanone in staphylococci at subinhibitory concentrations, which was manifested by an increase in PIA and dependent on luxS.

Brominated Furanones Inhibit Biofilm Formation by Salmonella enterica Serovar Typhimurium

ABSTRACT Salmonella enterica serovar Typhimurium is a main cause of bacterial food-borne diseases. As Salmonella can form biofilms in which it is better protected against antimicrobial agents on a wide diversity of surfaces, it is of interest to explore ways to inhibit biofilm formation. Brominated furanones, originally extracted from the marine alga Delisea pulchra, are known to interfere with biofilm formation in several pathogens. In this study, we have synthesized a small focused library of brominated furanones and tested their activity against S. enterica serovar Typhimurium biofilm formation. We show that several furanones inhibit Salmonella biofilm formation at non-growth-inhibiting concentrations. The most interesting compounds are (Z)-4-bromo-5-(bromomethylene)-3-alkyl-2(5H)-furanones with chain lengths of two to six carbon atoms. A microarray study was performed to analyze the gene expression profiles of Salmonella in the presence of (Z)-4-bromo-5-(bromomethylene)-3-ethyl-2(5H)-furanone. The induced genes include genes that are involved in metabolism, stress response, and drug sensitivity. Most of the repressed genes are involved in metabolism, the type III secretion system, and flagellar biosynthesis. Follow-up experiments confirmed that this furanone interferes with the synthesis of flagella by Salmonella. No evidence was found that furanones act on the currently known quorum-sensing systems in Salmonella. Interestingly, pretreatment with furanones rendered Salmonella biofilms more susceptible to antibiotic treatment. Conclusively, this work demonstrates that particular brominated furanones have potential in the prevention of biofilm formation by Salmonella serovar Typhimurium.