Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated β-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer’s disease-associated amyloid-β, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, as previously shown, CsgA fibrils cross-seeded fibrillation of amyloid-β, providing support for the proposed structural resemblance and potential for cross-species amyloid interactions. The presented findings provide structural insights into amyloidogenic regions important for curli formation, suggest a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases.

These authors contributed equally to this work †Correspondence to: mlandau@technion.ac.il Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curlin subunit, CsgA, revealing steric zipper fibrils of tightly mated β-sheets, demonstrating a structural link between curli and human pathological amyloids. We propose that these cross-β segments structure the highly robust curli amyloid core. D-enantiomeric peptides, originally developed to interfere with Alzheimer's disease-associated Amyloid-β, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, CsgA fibrils cross-seeded fibrillation of Amyloid-β, providing further support for the proposed structural resemblance and potential for cross-species amyloid interactions. In this study, we provide structural insights into curli formation, offer a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases. SignificanceAtomic resolution structural insights into the biofilm-associated curli amyloid fibril secreted by Enterobacteriaceae revealed elements of fibrillar architecture conserved between bacterial and human amyloids. This inspired us to repurpose anti-amyloid drugs designed to target human pathological amyloids as a novel class of anti-biofilm agents. Moreover, the results provide a molecular basis for understanding interspecies cross-seeding of amyloids through the generation of prion-like agents by molecular mimicry. This raises concerns regarding human exposure to exogenous sources of amyloids, such as contaminated food and amyloid-secreting microbes. Overall, we provide a novel framework for investigating interspecies amyloid interactions at the molecular level and offer novel insights into mechanisms which may underlie the evolutionary and etiological relationships between the human and microbial amylomes.

Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-β spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked β-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-β structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-β structures are often involved in fungal pathogenesis and potentially in antifungal therapy.

Antibiotic resistance of bacteria is considered one of the most alarming developments in modern medicine. While varied pathways for bacteria acquiring antibiotic resistance have been identified, there still are open questions concerning the mechanisms underlying resistance. Here, we show that alpha phenol-soluble modulins (PSMαs), functional bacterial amyloids secreted by Staphylococcus aureus, catalyze breakup of β-lactams, a prominent class of antibiotic compounds. Specifically, we show that PSMα2 and, particularly, PSMα3 catalyze hydrolysis of the amide-bond four-member ring of nitrocefin, a widely used β-lactam surrogate. Microscopic and spectroscopic analyses of several PSMα3 variants and correlation with their catalytic activities allowed mapping of the catalytic sites on the amyloid fibrils surface, specifically underscoring the key roles of the cross-α fibril organization, and the combined electrostatic and nucleophilic functions of the lysine residue array. This study unveils a previously unknown role of functional bacterial amyloids as catalytic agents for antibiotic compounds, pointing to possible mechanisms for antibiotic resistance of bacteria.

The remarkable variety of microbial species of human pathogens and microbiomes generates significant quantities of secreted amyloids, which are structured protein fibrils that serve diverse functions related to virulence and interactions with the host. Human amyloids are associated largely with fatal neurodegenerative and systemic aggregation diseases, and current research has put forward the hypothesis that the interspecies amyloid interactome has physiological and pathological significance. Moreover, functional and molecular-level connections between antimicrobial activity and amyloid structures suggest a neuroimmune role for amyloids that are otherwise known to be pathological. Compared to the extensive structural information that has been accumulated for human amyloids, high-resolution structures of microbial and antimicrobial amyloids are only emerging. These recent structures reveal both similarities and surprising departures from the typical amyloid motif, in accordance with their diverse activities, and advance the discovery of novel antivirulence and antimicrobial agents. In addition, the structural information has led researchers to postulate that amyloidogenic sequences are natural targets for structural mimicry, for instance in host–microbe interactions. Microbial amyloid research could ultimately be used to fight aggressive infections and possibly processes leading to autoimmune and neurodegenerative diseases.