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

Abstract

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.

Imperfect repeats in the functional amyloid protein FapC reduce the tendency to fragment during fibrillation

“…This repeat‐free construct aggregates in a highly stochastic nature which prevents proper fitting with Amylofit; furthermore, the ensuing fibrils show an unusual mesh‐like structure and are also much less stable against solubilization by formic acid than all the other mutants (Christensen et al, unpublished data). Furthermore, Figure (A) shows a certain level of similarity between the regions flanking the repeats (although much weaker than between the actual repeats), while bioinformatics analyses also indicate a significant amyloidogenicity in these regions, although less so than with the repeats . A likely explanation is that the repeats drive fibrillation so efficiently that they override any alternative aggregative behavior of the linker regions, and it is only the complete removal of the repeats proper that allows the linker regions to “take their place in the sun” and aggregate on their own.…”

“…Furthermore, Figure 1(A) shows a certain level of similarity between the regions flanking the repeats (although much weaker than between the actual repeats), while bioinformatics analyses also indicate a significant amyloidogenicity in these regions, although less so than with the repeats. 30 A likely explanation is that the repeats drive fibrillation so efficiently that they override any alternative aggregative behavior of the linker regions, and it is only the complete removal of the repeats proper that allows the linker regions to "take their place in the sun" and aggregate on their own. Linker regions may also serve an independent purpose by modulating the physical-chemical surface properties of the ensuing fibrils and thus interactions between individual fibrils.…”

N-Acetyl-cysteine and Mechanisms Involved in Resolution of Chronic Wound Biofilm

“…In prokaryotes, disulfide bonds also play an important role in the stability of the extracellular substances. For example, P. aeruginosa secretes an array of proteins, many of which contain disulfide bonds, such as FapC, an amyloid protein abundant in P. aeruginosa EPS, which requires two highly conserved cysteines to form disulfide linkages between two monomers to form mature fibrils [47]. A 29 kDa extracellular lipase secreted by P. aeruginosa contains two cysteine residues to maintain its active conformation [48].…”

Turncoat Polypeptides: We Adapt to Our Environment

“…An amyloidogenic or amyloid‐forming protein (Figure H) is a protein that misfolds upon certain conditions, and the misfolded structure experiences irreversible aggregation forming amyloid fibrils. They are mostly related to neurodegenerative diseases, but some functional amyloids have also been described …”

“…They are mostly relatedt on eurodegenerative diseases,b ut some functional amyloids have also been described. [27][28][29] Hereinafter,w ef ocus on three closely related types of sequences:c hameleon sequences, metamorphic proteins, and switchp eptides. We use the generic term "turncoat" polypeptides to encompass all of them.…”