Selective inhibition of the amyloid matrix of Escherichia coli biofilms by a bifunctional microbial metabolite

The propensity of bacteria to grow collectively in communities known as biofilms and their ability to overcome clinical treatments in this condition has become a major medical problem, emphasizing the need for anti-biofilm strategies. Antagonistic microbial interactions have extensively served as searching platforms for antibiotics, but their potential as sources for anti-biofilm compounds has barely been exploited. By screening for microorganisms that in agar-set pairwise interactions could antagonize Escherichia colis ability to form macrocolony biofilms, we found that the soil bacterium Bacillus subtilis strongly inhibits the synthesis of amyloid fibers -known as curli-, which are the primary extracellular matrix (ECM) components of E. coli biofilms. We identified bacillaene, a B. subtilis hybrid non-ribosomal peptide/polyketide metabolite, previously described as a bacteriostatic antibiotic, as the effector molecule. We found that bacillaene combines both antibiotic and anti-curli functions in a concentration-dependent order that potentiates the ecological competitiveness of B. subtilis, highlighting bacillaene as a metabolite naturally optimized for microbial inhibition. Our studies revealed that bacillaene inhibits curli by directly impeding the assembly of the CsgB and CsgA curli subunits into amyloid fibers. Moreover, we found that curli inhibition occurs despite E. coli attempts to reinforce its protective ECM by inducing curli genes via a RpoS-mediated competition sensing response trigged by the threatening presence of B. subtilis. Overall, our findings illustrate the relevance of exploring microbial interactions not only for finding compounds with novel and unique activities, but for uncovering additional functions of compounds previously categorized as antibiotics. IMPORTANCEWhile traditionally serving as sources for novel antibiotics, microbial interactions have a great potential -yet to be more intensely exploited- as sources for compounds with anti-biofilm activities among other functions. Exploring such potential, we uncovered an anti-curli amyloid activity of bacillaene, a B. subtilis secondary metabolite, that prevents E. coli biofilm morphogenesis. We demonstrated that bacillaene inhibits curli by interfering with the assembly of curli subunits into amyloid fibers and that such inhibition occurs despite E. coli fights to reinforce its protective amyloid matrix. Moreover, we showed that bacillaene combines this anti-curli activity with a previously assigned antibiotic activity in a concentration-dependent order that potentiates the inhibitory effect against curli-based E. coli biofilms. The finding of additional activities of compounds previously characterized as antibiotics, as here demonstrated for bacillaene, is relevant to understand both the actual roles of secondary metabolites in modulating microbial interactions in natural niches and the potential implications of the combined activities in therapeutic applications to treat bacterial infections.