Abstract
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 biofilm's 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.
Highlights
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 of central importance in the context of bacterial diseases
The protein was found to assemble spontaneously to form long, entangled fibrils as observed by transmission electron microscopy (TEM) (Fig. 2b). These findings are similar to those previously observed for the self-assembly of FapC [5, 30] and are consistent with the view that the amyloid-like fibrils formed by FapC are an integral part of the biofilm extracellular matrix
To assess the changes in secondary structure upon FapC aggregation, we used far-UV circular dichroism (CD) where a transition from a spectrum that has a minimum below 200 nm for the freshly purified monomer to a spectrum with a predominant minimum at approximately 218 nm could be observed after 5 days of incubation at 37°C
Summary
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 of central importance in the context of bacterial diseases. 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. Aggregated proteins, in the form of functional bacterial amyloids (FuBAs), are a key component of the EPS, providing structural stability to the biofilm [1, 10] Examples of such FuBAs include the Salmonella Tafi protein, Xanthomonas axonopodia harpins, Bacillus subtilis TasA protein [1,2,3], Escherichia coli curli system, and Pseudomonas fluorescens Fap proteins [4, 5]. Aggregation is regulated through transport proteins, transcription factors, chaperones, and even specific auxiliary nucleator proteins that promote the targeted aggregation of monomers at the cell surface [11,12,13,14,15]
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