Amyloid adhesins are abundant in natural biofilms

Abstract

Surface-associated amyloid fibrils have been described by bacteria in the family Enterbacteriaceae, but it is unknown to what extent amyloid adhesins are present in natural biofilms. In this study, amyloid adhesins were specifically stained with Thioflavin T and two conformationally specific antibodies targeting amyloid fibrils. These three independent detection methods were each combined with fluorescence in situ hybridization using fluorescently labelled oligonucleotide probes in order to link phenotype with identity. Escherichia coli mutants with and without amyloid adhesins (curli) served as controls. In biofilms from four different natural habitats, bacteria producing extracellular amyloid adhesins were identified within several phyla: Proteobacteria (Alpha-, Beta-, Gamma- and Deltaproteobacteria), Bacteriodetes, Chloroflexi and Actinobacteria, and most likely also in other phyla. Quantification of the microorganisms producing amyloid adhesins showed that they constituted at least 5-40% of all prokaryotes present in the biofilms, depending on the habitat. Particularly in drinking water biofilms, a high number of amyloid-positive bacteria were identified. Production of amyloids was confirmed by environmental isolates belonging to the Gammaproteobacteria, Bacteriodetes, Firmicutes and Actinobacteria. The new approach is a very useful tool for further culture-independent studies in mixed microbial communities, where the abundance and diversity of bacteria expressing amyloid adhesins seems much greater than hitherto anticipated.

Amyloid-Like Adhesins Produced by Floc-Forming and Filamentous Bacteria in Activated Sludge

“…Generally, the function of amyloid fibrils is assumed to be related to enhanced adhesion to surfaces (49) and biofilm formation, but they may also increase resistance to chlorine (55) and resistance to chemical and enzymatic digestion (45). The function in activated sludge flocs is still unknown but may be related to the aggregation of microorganisms internally in microcolonies, whereas it is more uncertain what the function is in the filamentous bacteria, which were also shown to produce amyloids (32). Activated sludge flocs are often described as having a strongly and loosely bound fraction of cells and EPS (23,57), so amyloids might be a good candidate for contributing to the high stability of the strongly bound fraction.…”

“…Recently, we found that proteinaceous amyloid adhesins are an abundant component of the EPS fraction in biofilms from different habitats among these activated sludge flocs (32). Bacterial amyloids are usually thin fibrils, insoluble, and extremely resistant to various denaturants (8).…”

“…They share many similarities to amyloid fibrils, which are known to cause neurodegenerative diseases such as Parkinson's and Alzheimer's disease in humans (6). Although many different bacteria are presumably able to produce amyloid adhesins (32), detailed descriptions of the size, amount, and properties of bacterial amyloids are restricted to pure culture studies of mainly Escherichia coli, Salmonella species, and the gram-positive Streptomyces coelicolor (48). Amyloid adhesins (named curli) produced by E. coli are 4 to 12 nm wide and 0.1 to 10 m long (6,15).…”

“…The bacterial amyloids in activated sludge and other biofilms are expressed by a broad range of phylogenetically distant species in the phyla Proteobacteria, Bacteroidetes, Chloroflexi, Firmicutes, and Actinobacteria (32). Generally, the function of amyloid fibrils is assumed to be related to enhanced adhesion to surfaces (49) and biofilm formation, but they may also increase resistance to chlorine (55) and resistance to chemical and enzymatic digestion (45).…”

“…Detection of sessile bacteria producing amyloids can be performed by staining with thioflavin T (ThT) or by labeling with antibodies targeting a generic conformational epitope on amyloidal proteins (32). Antibodies have been found to be very specific for labeling of amyloid adhesins (32,47), whereas ThT suffers from some nonspecificity, since it can also bind to cellulose and DNA (19,51).…”