Uncontrolled misfolding of proteins leading to the formation of amyloid deposits is associated with more than 40 types of diseases, such as neurodegenerative diseases and type-2 diabetes. These irreversible amyloid fibrils typically assemble in distinct stages. Transitions among the various intermediate stages are the subject of many studies but are not yet fully elucidated. Here, we combine high-resolution atomic force microscopy and quantitative nanomechanical mapping to determine the self-assembled structures of the decapeptide hIAPP _20–29 , which is considered to be the fibrillating core fragment of the human islet amyloid polypeptide (hIAPP) involved in type-2 diabetes. We successfully follow the evolution of hIAPP _20–29 nanostructures over time, calculate the average thickening speed of small ribbon-like structures, and provide evidence of the coexistence of ribbon and helical fibrils, highlighting a key step within the self-assembly model. In addition, the mutations of individual side chains of wide-type hIAPP _20–29 shift this balance and destabilize the helical fibrils sufficiently relative to the twisted ribbons to lead to their complete elimination. We combine atomic force microscopy structures, mechanical properties, and solid-state NMR structural information to build a molecular model containing β sheets in cross-β motifs as the basis of self-assembled amyloids.

Protein aggregation and amyloid formation have historically been linked with various diseases such as Alzheimer's and Parkinson's disease, but recently functional amyloids have gained a great deal of interest in not causing a disease and having a distinct function in vivo. Functional bacterial amyloids form the structural scaffold in bacterial biofilms and provide a survival strategy for the bacteria along with antibiotic resistance. The formation of functional amyloids happens extracellularly which differs from most disease related amyloids. Studies of functional amyloids have revealed several distinctions compared to disease related amyloids including primary structures designed to optimize amyloid formation while still retaining a controlled assembly of the individual subunits into classical cross-β-sheet structures, along with a unique cross-α-sheet amyloid fold. Studies have revealed that functional amyloids interact with components found in the extracellular matrix space such as lipids from membranes and polymers from the biofilm. Intriguingly, a level of complexity is added as functional amyloids also interact with several disease related amyloids and a causative link has even been established between functional amyloids and neurodegenerative diseases. It is hence becoming increasingly clear that functional amyloids are not inert protein structures found in bacterial biofilms but interact with many different components including human proteins related to pathology. Gaining a clear understanding of the factors governing the interactions will lead to improved strategies to combat biofilm associated infections and the correlated antibiotic resistance. In the current review we summarize the current state of the art knowledge on this exciting and fast growing research field of biofilm forming bacterial functional amyloids, their structural features and interaction partners.

A wide range of bacterial pathogens have been shown to form biofilms, which significantly increase their resistance to environmental stresses, such as antibiotics, and are thus of central importance in the context of bacterial diseases. One of the major structural components of these bacterial biofilms are amyloid fibrils, yet the mechanism of fibril assembly and its importance for biofilm formation are currently not fully understood. By studying fibril formation in vitro, in a model system of two common but unrelated biofilm-forming proteins, FapC from Pseudomonas fluorescens and CsgA from Escherichia coli, we found that the two proteins have a common aggregation mechanism. In both systems, fibril formation proceeds via nucleated growth of linear fibrils exhibiting similar measured rates of elongation, with negligible fibril self-replication. These similarities between two unrelated systems suggest that convergent evolution plays a key role in tuning the assembly kinetics of functional amyloid fibrils and indicates that only a narrow window of mechanisms and assembly rates allows for successful biofilm formation. Thus, the amyloid assembly reaction is likely to represent a means for controlling biofilm formation, both by the organism and by possible inhibitory drugs. IMPORTANCE Biofilms are generated by bacteria, embedded in the formed extracellular matrix. The biofilm9s function is to improve the survival of a bacterial colony through, for example, increased resistance to antibiotics or other environmental stresses. Proteins secreted by the bacteria act as a major structural component of this extracellular matrix, as they self-assemble into highly stable amyloid fibrils, making the biofilm very difficult to degrade by physical and chemical means once formed. By studying the self-assembly mechanism of the fibrils from their monomeric precursors in two unrelated bacteria, our experimental and theoretical approaches shed light on the mechanism of functional amyloid assembly in the context of biofilm formation. Our results suggest that fibril formation may be a rate-limiting step in biofilm formation, which in turn has implications on the protein self-assembly reaction as a target for potential antibiotic drugs.