Selective inhibition of the amyloid matrix ofEscherichia colibiofilms by a bifunctional microbial metabolite
Authors: Cordisco Estefanía , Zanor María Inés , Moreno Diego M. , Serra Diego Omar 
Published: July 2023

ABSTRACTThe 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 antagonizeEscherichia coli’sability to form macrocolony biofilms, we found that the soil bacteriumBacillus subtilisstrongly inhibits the synthesis of amyloid fibers –known as curli-, which are the primary extracellular matrix (ECM) components ofE. colibiofilms. We identified bacillaene, aB. subtilishybrid 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 ofB. 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 despiteE. coliattempts to reinforce its protective ECM by inducing curli genes via a RpoS-mediated competition sensing response trigged by the threatening presence ofB. 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, aB. subtilissecondary metabolite, that preventsE. colibiofilm morphogenesis. We demonstrated that bacillaene inhibits curli by interfering with the assembly of curli subunits into amyloid fibers and that such inhibition occurs despiteE. colifights 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-basedE. colibiofilms. 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.