Functional amyloid formation by Streptococcus mutans

Abstract

Dental caries is a common infectious disease associated with acidogenic and aciduric bacteria, including Streptococcus mutans. Organisms that cause cavities form recalcitrant biofilms, generate acids from dietary sugars and tolerate acid end products. It has recently been recognized that micro-organisms can produce functional amyloids that are integral to biofilm development. We now show that the S. mutans cell-surface-localized adhesin P1 (antigen I/II, PAc) is an amyloid-forming protein. This conclusion is based on the defining properties of amyloids, including binding by the amyloidophilic dyes Congo red (CR) and Thioflavin T (ThT), visualization of amyloid fibres by transmission electron microscopy and the green birefringent properties of CR-stained protein aggregates when viewed under cross-polarized light. We provide evidence that amyloid is present in human dental plaque and is produced by both laboratory strains and clinical isolates of S. mutans. We provide further evidence that amyloid formation is not limited to P1, since bacterial colonies without this adhesin demonstrate residual green birefringence. However, S. mutans lacking sortase, the transpeptidase enzyme that mediates the covalent linkage of its substrates to the cell-wall peptidoglycan, including P1 and five other proteins, is not birefringent when stained with CR and does not form biofilms. Biofilm formation is inhibited when S. mutans is cultured in the presence of known inhibitors of amyloid fibrillization, including CR, Thioflavin S and epigallocatechin-3-gallate, which also inhibited ThT uptake by S. mutans extracellular proteins. Taken together, these results indicate that S. mutans is an amyloid-forming organism and suggest that amyloidogenesis contributes to biofilm formation by this oral microbe. INTRODUCTIONAmyloid represents a fibrous cross b-sheet quaternary structure comprised of ordered aggregates of peptides or proteins that demonstrate common biophysical properties (Nilsson, 2004). While amyloid formation has been extensively studied in the context of pathological states, for example Alzheimer's disease, the concept of functional amyloid has only recently emerged (Epstein & Chapman, 2008;Fowler et al., 2007;Gebbink et al., 2005;Maury, 2009a;. In bacteria, amyloid formation may be the rule rather than the exception . There are now numerous examples of surface-localized microbial proteins that aggregate and assemble into functional amyloid fibrils.Amyloid fibres have a tensile strength comparable to steel (Smith et al., 2006). The unique physical and morphological properties of amyloid and the fact that it can be formed in a regulated manner suggest that it probably represents a common quaternary structure that is widespread in biology. Therefore, it does not always represent a misfolded protein structure, but instead represents a lowenergy quaternary structure that can occur in the context of function or disease (Fowler et al., 2007). Amyloid is a non-covalent oligomer of extended intermolecular hydrogen-bonded b-sheets that s...

Staphylococcus epidermidis small basic protein (Sbp) forms amyloid fibrils, consistent with its function as a scaffolding protein in biofilms

“…Functional amyloids have been found in a wide range of organisms, from bacteria to mammals, with functions as diverse as biofilm formation, development of aerial structures, scaffolding, regulation of melanin synthesis, epigenetic control of polyamines and information transfer (21). There is a limited but growing by guest on March 28, 2019 http://www.jbc.org/ Downloaded from number of examples, including the curli homolog in Salmonella (23), FapC in many Pseudomonas species (24), TasA in Bacillus subtilis (25), the phenol soluble modulins (PSMs) (26) and the biofilm associated surface protein (27) in Staphylococcus aureus and the adhesin protein P1 in Streptococcus mutans (28), which are found to be able to form amyloid fibrils and are implicated in biofilm formation (29).…”

Streptococcus mutans: a new Gram-positive paradigm?

“…AgI/II family) that are capable of binding to receptors in the pellicle (Gibbons, 1989). Recently, the adhesin P1 (AgI/II) was shown to be an amyloid-forming protein that contributes to biofilm development by S. mutans (Oli et al, 2012). S. mutans also interacts with specific salivary proteins, such as common salivary protein-1 (CSP-1), which, in turn, helps the bacterium to bind saliva-coated apatitic surfaces (Ambatipudi et al, 2010).…”

The Role and Efficacy of Herbal Antimicrobial Agents in Orthodontic Treatment

“…The development of novel technologies and the rapid advances made in dissecting the genetics and physiology of S. mutans, have resulted in the emergence of this bacterium as a new Gram positive model organism and they have proved that S. mutans was an amyloid-forming organism that contributed to biofilm formations [19]. This study may provide an impetus for understanding the interrelations between the plaque biofilm, tooth tissues and the oral environment, and for the development of procedures to modify the course of caries development [20].…”