Sequence-targeted Peptides Divert Functional Bacterial Amyloid Towards Destabilized Aggregates and Reduce Biofilm Formation.

Functional bacterial amyloid provides structural stability in biofilm, making it a promising target for anti-biofilm therapeutics. Fibrils formed by CsgA, the major amyloid component in E. coli are extremely robust and can withstand very harsh conditions. Like other functional amyloids, CsgA contains relatively short aggregation-prone regions (APR) which drive amyloid formation. Here, we demonstrate the use of aggregation-modulating peptides to knock down CsgA protein into aggregates with low stability and altered morphology. Remarkably, these CsgA-peptides also modulate fibrillation of the unrelated functional amyloid protein FapC from Pseudomonas, possibly through recognition of FapC segments with structural and sequence similarity with CsgA. The peptides also reduce the level of biofilm formation in E. coli and P. aeruginosa, demonstrating the potential for selective amyloid targeting to combat bacterial biofilm.

Protein Engineering Reveals Mechanisms of Functional Amyloid Formation in Pseudomonas aeruginosa Biofilms.

Amyloids are typically associated with neurodegenerative diseases, but recent research demonstrates that several bacteria utilize functional amyloid fibrils to fortify the biofilm extracellular matrix and thereby resist antibiotic treatments. In Pseudomonas aeruginosa, these fibrils are composed predominantly of FapC, a protein with high-sequence conservation among the genera. Previous studies established FapC as the major amyloid subunit, but its mechanism of fibril formation in P. aeruginosa remained largely unexplored. Here, we examine the FapC sequence in greater detail through a combination of bioinformatics and protein engineering, and we identify specific motifs that are implicated in amyloid formation. Sequence regions of high evolutionary conservation tend to coincide with regions of high amyloid propensity, and mutation of amyloidogenic motifs to a designed, non-amyloidogenic motif suppresses fibril formation in a pH-dependent manner. We establish the particular significance of the third repeat motif in promoting fibril formation and also demonstrate emergence of soluble oligomer species early in the aggregation pathway. The insights reported here expand our understanding of the mechanism of amyloid polymerization in P. aeruginosa, laying the foundation for development of new amyloid inhibitors to combat recalcitrant biofilm infections.