Identification of E. coli amyloid assembly inhibitors, modes of action, and influence on biofilm formation

Biofilms are multicellular communities of cells surrounded in a self-produced extracellular matrix (ECM), a complex mixture of polymeric substances. This matrix is associated with several beneficial functions for bacteria, depending upon the type of biofilm. These can include resistance to antibiotics and host defenses. Biofilm formation in the human host can contribute to the persistence and recurrence of infection. Uropathogenic E. coli is the most common causative agent of urinary tract infection and can assemble unique biofilms through the production of functional amyloid fibers termed curli and a modified form of cellulose -- phosphoethanolamine (pEtN) cellulose. Curli play a vital role in bacterial adhesion to biotic and abiotic surfaces as well as promoting multicellular biofilm formation. Curli fibers are assembled from CsgA protein subunits that non-covalently polymerize into the fibril form through the nucleation-precipitation pathway. Herein, we describe the characterization of the natural product NDGA and the salicylanilide closantel as inhibitors of curli formation. We demonstrate the effects of inhibitors on bacterial curli production, bacterial adhesion, bacterial biofilm formation as well as protein polymerization in vitro. Molecular analyses are accompanied by the evaluation of curli gene transcription to consider compound effects on gene expression. NDGA exhibits curlicide activity, preventing CsgA polymerization, and does not bind to intact curli. These results are consistent with the inhibition of early steps in curli biogenesis, e.g. amyloid nucleation, transport through CsgG, or CsgA-CsgF interactions. Closantel exhibits curlicide activity at very low concentrations and also binds to intact curli fibers. Thus, closantel is able to bind CsgA in its polymerized amyloid form and may also recognize early amyloidogenic conformations of CsgA or additionally recognize the soluble and unstructured form of CsgA preceding nucleation and polymerization. This comprehensive and mechanistic approach in the full cellular context of bacteria provides an avenue to identify and develop curli-specific amyloid inhibitors and may hold promise in identifying the best inhibitors of amyloidogenesis and assembly of amyloid oligomers and fibers associated with human diseases. Finally, we expanded our understanding of the functions and contributions of both curli and cellulose in uropathogenic E. coli biofilm formation through comparisons of curli and pEtN cellulose gene mutants in the uropathogenic E. coli clinical isolate UTI89. We examined biofilm formation at the agar-air and air-liquid interfaces. We also examined mixed cultures of mutants lacking either curli or modified cellulose and discovered that they could recapitulate the formation of a biofilm similar to wild-type UTI89. This work breaks new ground in understanding the contributions of complex and insoluble biopolymers to the assembly of architectures that extend beyond the surface of single bacteria and that are built for community behaviors

Keywords: Chemistry.
Advisor: Cegelski Lynette
Chen James Kenneth
Kool Eric T.