Nosocomial infections affect hundreds of millions of patients worldwide each year, and ~60% of these infections are associated with biofilm formation on an implanted medical device. Biofilms are dense communities of microorganisms in which cells associate with surfaces and each other using a self-produced extracellular matrix composed of proteins, polysaccharides, and genetic material. Proteins in the extracellular matrix take on a variety of forms, but here we focus on functional amyloid structures. Amyloids have long been associated with protein misfolding and neurodegenerative diseases, but recent research has demonstrated that numerous bacterial species utilize the amyloid fold to fortify the biofilm matrix and resist disassembly. Consequently, these functional amyloids, in particular the soluble oligomeric intermediates formed during amyloidogenesis, represent targets to destabilize the extracellular matrix and interrupt biofilm formation. Our previous studies suggested that these amyloidogenic intermediates adopt a non-standard structure, termed “α-sheet”, as they aggregate into soluble oligomeric species. This led to the design of complementary α-sheet peptides as anti-α-sheet inhibitors; these designs inhibit amyloidogenesis in three unrelated mammalian disease-associated systems through preferential binding of soluble oligomers. Here we show that these anti-α-sheet peptides inhibit amyloid formation in Staphylococcus aureus biofilms. Furthermore, they inhibit aggregation of pure, synthetic phenol soluble modulin α1, a major component of Staphylococcus aureus functional amyloids. As it aggregates phenol soluble modulin α1 adopts α-helix then α-sheet and finally forms β-sheet fibrils. The binding of the designed peptide inhibitors coincides with the formation of α-sheet.

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.

There has been much interest in synthetic peptides as inhibitors of aggregation associated with amyloid diseases. Of particular interest are compounds that target the cytotoxic soluble oligomers preceding the formation of mature, nontoxic fibrils. This study explores physical and chemical differences between two de novo-designed peptides that share an identical primary structure but differ in backbone chirality at six key positions. We show that the presence of alternating L/D-amino acid motifs dramatically increases aqueous solubility, enforces α-sheet secondary structure, and inhibits aggregation of the β-amyloid peptide implicated in Alzheimer’s disease, in addition to neutralizing its cytotoxicity. In contrast, the all-L-amino acid isomer does not form α-sheet structure and is insoluble and inactive.

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