Abstract
Protein amyloids are ubiquitous in natural environments. They typically originate from microbial secretions or spillages from mammals infected by prions, currently raising concerns about their infectivity and toxicity in contexts such as gut microbiota or soils. Exploiting the self-assembly potential of amyloids for their scavenging, here, we report the insertion of an amyloidogenic sequence stretch from a bacterial prion-like protein (RepA-WH1) in one of the extracellular loops (L5) of the abundant Escherichia coli outer membrane porin OmpF. The expression of this grafted porin enables bacterial cells to trap on their envelopes the same amyloidogenic sequence when provided as an extracellular free peptide. Conversely, when immobilized on a surface as bait, the full-length prion-like protein including the amyloidogenic peptide can catch bacteria displaying the L5-grafted OmpF. Polyphenolic molecules known to inhibit amyloid assembly interfere with peptide recognition by the engineered OmpF, indicating that this is compatible with the kind of homotypic interactions expected for amyloid assembly. Our study suggests that synthetic porins may provide suitable scaffolds for engineering biosensor and clearance devices to tackle the threat posed by pathogenic amyloids.
Highlights
Amyloids are β-sheet-structured protein aggregates that are involved in prevalent neurodegenerative and systemic diseases but that recently have been recognized to confer alternative functional states to some proteins.[1−3] Amyloids have yet a hidden post-life as recalcitrant contaminants in natural environments, such as those associated with microbiota in soils and in the gut
We report the conversion of the OmpF porin in the outer membrane (OM) of E. coli into a scavenger of a model amyloid peptide, gaining function as a synthetic amylo-adhesin
The E. coli OM porin OmpF was the scaffold of choice for inserting an amyloidogenic sequence due to its high copy number, clustered as trimers in the membrane plane, which assures high density and valence of potential binding sites;[26] its non-essentiality, due to its partial functional redundancy with other related porins, such as OmpC, that allows for obtaining null strains;[59] and its known three-dimensional structure which, combined with numerous biophysical studies, has unveiled its function as an electroselective import channel.[27,29]
Summary
Amyloids are β-sheet-structured protein aggregates that are involved in prevalent neurodegenerative and systemic diseases but that recently have been recognized to confer alternative functional states to some proteins.[1−3] Amyloids have yet a hidden post-life as recalcitrant contaminants in natural environments, such as those associated with microbiota in soils and in the gut. To get insights into the biochemical nature of the interaction between the amyloidogenic stretches in OmpF-L5 and the probe RepA-WH1_amyl, binding of this peptide was competed with epigallocatechin-3-gallate (EGCG) or resveratrol, two natural polyphenolic molecules known to inhibit amyloid aggregation, and at the same concentration that was reported to interfere with RepA-WH1 amyloidogenesis both in vitro[66] and in vivo.[69] Epifluorescence microscopy revealed that the polyphenols efficiently abolished labeling of the bacterial cells by the fluorescent peptide (Figure 4), providing a strong indication for the amyloid nature of the homotypic interaction between the grafted loop in OmpF and its targeted sequence.
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