Widespread Abundance of Functional Bacterial Amyloid in Mycolata and Other Gram-Positive Bacteria

Abstract

Until recently, extracellular functional bacterial amyloid (FuBA) has been detected and characterized in only a few bacterial species, including Escherichia coli, Salmonella, and the gram-positive organism Streptomyces coelicolor. Here we probed gram-positive bacteria with conformationally specific antibodies and revealed the existence of FuBA in 12 of 14 examined mycolata species, as well as six other distantly related species examined belonging to the phyla Actinobacteria and Firmicutes. Most of the bacteria produced extracellular fimbriae, sometimes copious amounts of them, and in two cases large extracellular fibrils were also produced. In three cases, FuBA was revealed only after extensive removal of extracellular material by saponification, indicating that there is integrated attachment within the cellular envelope. Spores of species in the genera Streptomyces, Bacillus, and Nocardia were all coated with amyloids. FuBA was purified from Gordonia amarae (from the cell envelope) and Geodermatophilus obscurus, and they had the morphology, tinctorial properties, and ␤-rich structure typical of amyloid. The presence of approximately 9-nm-wide amyloids in the cell envelope of G. amarae was visualized by transmission electron microscopy analysis. We conclude that amyloid is widespread among gram-positive bacteria and may in many species constitute a hitherto overlooked integral part of the spore and the cellular envelope.

Microbial Amyloids Induce Interleukin 17A (IL-17A) and IL-22 Responses via Toll-Like Receptor 2 Activation in the Intestinal Mucosa

“…Amyloid fibrils are a PAMP that is commonly present in biofilm material produced by members of the phyla Firmicutes, Bacteroides, and Proteobacteria (41,44). The best-characterized member of this family of surface structures is the curli (55), which are amyloid fibrils (13) produced by Salmonella enterica serovar Typhimurium, Escherichia coli, and other closely related members of the Enterobacteriaceae (65,85).…”

Functional amyloid

“…Some filamentous bacteria, e.g., from the phylum Chloroflexi, also express amyloids as a part of the sheath or close to the septum between the individual cells in the filaments. In the Gram-negative Mycolata family, it was necessary to strip off the lipid top layer by harsh treatment with alkaline alcohol to reveal the underlying amyloid structure, indicating that the amyloid, shown to consist of one to two protein components, was deeply integrated into the cellular envelope where it may perform essential structural functions 24 ( Fig. 2B).…”

“…The β-strands are stacked with near-perfect design, and it has been estimated that there is only one misalignment per 30,000 strands in a typical amyloid structure. 25 Unlike pathological amyloid, which often can be dissolved by denaturants, SDS or even pure water, 26 functional amyloid generally resists boiling SDS (though exceptions do occur), thus providing a convenient selection criterion for its isolation 24,27 and is only dissolved by high concentrations of formic acid. 24,27,28 Furthermore, functional amyloid has typically been evolved to fibrillate, meaning that this process can occur over a broad range of conditions rather than a small "window of opportunity" engendered by the fortuitous accumulation of structural flexibility under specific structure-promoting conditions.…”

“…25 Unlike pathological amyloid, which often can be dissolved by denaturants, SDS or even pure water, 26 functional amyloid generally resists boiling SDS (though exceptions do occur), thus providing a convenient selection criterion for its isolation 24,27 and is only dissolved by high concentrations of formic acid. 24,27,28 Furthermore, functional amyloid has typically been evolved to fibrillate, meaning that this process can occur over a broad range of conditions rather than a small "window of opportunity" engendered by the fortuitous accumulation of structural flexibility under specific structure-promoting conditions. The archetypal CsgA, which is the major component of the E. coli amyloid structure called curli, 28 fibrillates to the same amyloid structure in vitro under a broad range of pH, temperature, concentration and ionic strength, according to fiber diffraction and Fourier Transform Infrared Spectroscopy (Dueholm MS and Otzen DE, unpublished observations).…”

“…2B and discussion above). 24 Subsequently 16S rRNA sequencing identifies the bacterial species. Many amyloid-producing species can be identified at this stage, but so far it has only been possible to purify amyloid to single-(or double-) band purity for a very small number of cases.…”