Amyloid Structures as Biofilm Matrix Scaffolds

Abstract

Recent insights into bacterial biofilm matrix structures have induced a paradigm shift toward the recognition of amyloid fibers as common building block structures that confer stability to the exopolysaccharide matrix. Here we describe the functional amyloid systems related to biofilm matrix formation in both Gram-negative and Gram-positive bacteria and recent knowledge regarding the interaction of amyloids with other biofilm matrix components such as extracellular DNA (eDNA) and the host immune system. In addition, we summarize the efforts to identify compounds that target amyloid fibers for therapeutic purposes and recent developments that take advantage of the amyloid structure to engineer amyloid fibers of bacterial biofilm matrices for biotechnological applications.

FIG 1

Dedicated or simple amyloid-like fiber formation systems in Gram-positive and Gram-negative bacteria. (A) The Sec export system secretes the products of the tapA-sipW-tasA operon through the inner membrane (IM) of B. subtilis. SipW acts as a signal peptidase cleaving the signal peptide from TasA and TapA. TapA allows the anchorage of fibers to the cell wall (CW), and TasA is the major component of the fibers. SinR represses the operon, but when conditions are propitious for biofilm formation, SinI antagonizes SinR. The C terminus of SipW also has a regulatory effect on the operon. In S. aureus α PSM, β PSMs, and δ-toxin monomers are transported outside the cell through an ATP-dependent ABC transporter (PmtB and PmtD transmembrane proteins linked to ATPases PmtA and PmtC). PSMs can be present as soluble monomers or, when required, can form amyloid-like fibers. Expression of α and β psm genes is induced by AgrA, the response regulator of the Agr system. (B) All Fap and Csg proteins (except for CsgD) are expulsed via Sec across the inner membrane. In the case of curli, the major fiber subunit CsgA and the nucleator CsgB are secreted across the outer membrane (OM) through the CsgG channel, and once on the cell surface they start to polymerize into amyloidogenic fibers. Accessory proteins CsgC and CsgE regulate export by CsgG, and CsgF assist the nucleation of CsgA. CsgD is the master regulator of the csgBAC operon. In Pseudomonas, Fap B, FapC, and FapE are further secreted across the outer membrane through FapF, where FapB nucleates fiber assembly of FapC and, in minor proportion, FapE. FapA likely controls FapB and FapC secretion, and FapD would act as a protease of Fap proteins. (C) Once it is anchored to the cell wall, Bap of S. aureus is processed by proteases present in the medium, liberating the N-terminal fragments that will ultimately self-assemble into amyloid-like fibrillar structures upon acidification of the medium and in the absence of calcium. In S. mutans, nonattached P1 adhesin (AgI/II) would presumably form amyloidogenic fibers through its β-sheet-rich V region and C-terminal domain. (D) The autotransporter Ag43 present in E. coli, though not considered a real amyloid, represent a very simple model of amyloid folding due to the high β-helix with β-strand conformation adopted by the Ag43α subunit at the bacterial envelope.

FIG 2

Amyloid-like fibers formed by the Bap protein of S. aureus. Transmission electron micrographs of negatively stained fibers formed by the recombinant B domain of Bap incubated at pH 4.5 (A) and S. aureus V329 cells grown overnight in LB-glucose (B) at 37°C and 200 rpm are shown.

FIG 3

Development of functionalized amyloid-dependent biofilms in Gram-negative and Gram-positive bacteria. (A) In E. coli, the major curli protein CsgA is engineered by fusing variable tags at its C terminus. Once in the extracellular medium, the fusion protein self-assembles into functional amyloid nanofibers. It is possible to obtain multifunctional biofilms by expressing engineered CsgA proteins under the control of different inducible promoters. The addition of measured concentrations of inducer molecules allows the expression and production of curli fibers containing precise multiple designed functions as a result of random extracellular self-assembly of CsgA monomers. (B) In the case of S. aureus, variable tags fused to the C-terminal part of the B domain of Bap could allow the formation of engineered Bap fibers. Since the B domain of Bap (amino acids 362 to 819) is sufficient to bestow multicellular behavior, it could be possible to express engineered Bap B-domain proteins under the control of an inducible promoter. The property of Bap to reversibly form amyloids according to pH and Ca²⁺ levels in the medium could be used as an external way to control the formation or disruption of functionalized biofilms.