Amyloid Structures as Biofilm Matrix Scaffolds

Agustina Taglialegna,a Iñigo Lasa,a,b Jaione Vallea

Laboratory of Microbial Biofilms, Idab-CSIC-Universidad Pública de Navarra-Gobierno de Navarra, and Departamento Producción Agraria, Pamplona, Spaina;Navarrabiomed-Universidad Pública de Navarra, IdiSNA, Pamplona, Spainb

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

Amyloid structures have been traditionally associated with sev-eral incurable degenerative human diseases, including Alz-

heimer’s disease, Parkinson’s disease, Huntington’s disease, andprion diseases (1). In these cases, amyloid fiber formation occursupon aberrant misfolding of soluble and functional proteins (2).However, numerous pieces of evidence indicate that amyloid fi-bers are not just the result of aberrant protein folding but areproduced on purpose under different nonpathological conditionsto provide organisms with beneficial properties (3). The microbialworld makes use of this type of protein folding for a number ofprocesses relevant for bacterial growth and survival in the envi-ronment, such as morphological differentiation of filamentousbacteria (4, 5), adhesion to host tissues (6), detoxification of toxiccompounds (7–9), electron transport (10), and structural scaf-folds in biofilm matrices (1).

The amyloid structure is an attractive building material be-cause it is generally resistant to harsh denaturing conditions and inmost cases cannot be degraded by proteases (1, 11). In addition,polymerization of amyloid proteins is metabolically cheap be-cause it occurs in the absence of energy. Thus, the amyloid con-formation is suitable as a component of the extracellular matrixwhere the energy sources are limited (12).

The aim of this review is to describe the current knowledge onthose functional bacterial amyloids involved in the building ofbiofilm matrices. We emphasize the differences between amyloidorganelles with machineries dedicated to control the expression,secretion, and polymerization of protein subunits into amyloidfibers and cell surface proteins that build amyloid structures onlyunder specific environmental conditions. We also discuss newcompounds that are able to destabilize bacterial biofilms throughtheir specific action on amyloid structures, recent evidence show-ing the implication of biofilm components, such as extracellularDNA (eDNA), in the self-assembly process of some functionalamyloids, and the striking use of such proteinaceous nanostruc-tures as tools to promote functional biofilms with biotechnologi-cal purposes.

AMYLOID STRUCTURES WITH DEDICATED FIBER ASSEMBLYMACHINERY

The most studied and characterized bacterial amyloid system iscurli fimbriae in Escherichia coli. Curli fimbriae show the proto-typical biophysical properties of amyloid fibers: ordered

�-sheet-rich fibers, resistance to proteases, and ability to bindamyloid-specific dyes (Congo red or thioflavin T). Curli assem-bly requires the products of at least two divergently transcribedoperons, csgBAC and csgDEFG (13). CsgA and CsgB are the pro-tein subunits that make up curli, whereas the rest of the proteinsare involved in the regulation of the production, stabilization, andsecretion of CsgA and CsgB subunits (1, 14–16) (Fig. 1B). CsgAhas the propensity to self-aggregate; however, it requires the CsgBsubunit for nucleation into fibers in vivo (14, 17). CsgA and CsgBare translocated into the periplasm by the Sec translocon systemand exported to the extracellular medium through a pore-likestructure constituted by the CsgG protein formed in the outermembrane (18). Associated with this pore are the CsgE and CsgFproteins, which modulate subunit stability and proper assembly ofcurli fibers (18, 19). The periplasmic protein CsgC may be in-volved in regulating the export of curli subunits through the reg-ulation of the redox state of CsgG (20), and it was shown to inhibitCsgA amyloid formation in vitro (21). CsgD is the master regula-tor that controls at the transcriptional level the expression of thecsgBAC operon and also adrA, which is involved in posttranscrip-tional regulation of cellulose synthesis and encodes another com-mon component of the biofilm matrix (22–25). CsgD activity isalso controlled by a variety of regulatory systems that respond toenvironmental signals, including osmolarity, pH, temperature,and nutrient availability (26, 27). Curli fibers mediate bacterialadhesion to a variety of host proteins or abiotic surfaces, promot-ing biofilm formation and pathogenicity (28–33).

Production of curli fibers has been described in Salmonella,Citrobacter, and Enterobacter (34), but genes encoding productswith homology to proteins coding for the curli system are found inmembers of Proteobacteria, Bacteroidetes, Firmicutes, and Ther-modesulfobacteria (35), suggesting that the formation of curli fi-

Accepted manuscript posted online 16 May 2016

Citation Taglialegna A, Lasa I, Valle J. 2016. Amyloid structures as biofilm matrixscaffolds. J Bacteriol 198:2579 –2588. doi:10.1128/JB.00122-16.

Editor: G. A. O’Toole, Geisel School of Medicine at Dartmouth

Address correspondence to Jaione Valle, jaione.valle@unavarra.es.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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FIG 1 Dedicated or simple amyloid-like fiber formation systems in Gram-positive and Gram-negative bacteria. (A) The Sec export system secretes the productsof 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 arepropitious for biofilm formation, SinI antagonizes SinR. The C terminus of SipW also has a regulatory effect on the operon. In S. aureus � PSM, � PSMs, and�-toxin monomers are transported outside the cell through an ATP-dependent ABC transporter (PmtB and PmtD transmembrane proteins linked to ATPasesPmtA and PmtC). PSMs can be present as soluble monomers or, when required, can form amyloid-like fibers. Expression of � and � psm genes is induced byAgrA, 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 theystart to polymerize into amyloidogenic fibers. Accessory proteins CsgC and CsgE regulate export by CsgG, and CsgF assist the nucleation of CsgA. CsgD is themaster regulator of the csgBAC operon. In Pseudomonas, Fap B, FapC, and FapE are further secreted across the outer membrane through FapF, where FapBnucleates 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 ultimatelyself-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 �-sheet-rich V region and C-terminal domain. (D) The autotransporter Ag43 present in E. coli, thoughnot considered a real amyloid, represent a very simple model of amyloid folding due to the high �-helix with �-strand conformation adopted by the Ag43�subunit at the bacterial envelope.

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bers is a common strategy to provide structural integrity to thebiofilm matrix (15).

Examples of extracellular fibers with structural and functionalsimilarity to curli fimbriae of E. coli have been described in variousspecies of Pseudomonas, including P. aeruginosa, P. fluorescens,and P. putida (36). In this case, the protein components for amy-loid assembly are encoded in the fapABCDEF operon (37). Bio-physical analysis of purified Fap fimbriae showed the presence ofan extensive �-sheet structure, as well as binding to the specificthioflavin T dye (37). Biochemical analysis of the fibril composi-tion identified the FapC protein as the major protein component,although small amounts of FapB and FapE were also present (37).Like for CsgB in the curli system, FapB would act as a fibrillationnucleator of FapC (Fig. 1B). Fap amyloid fibers facilitate attach-ment to abiotic surfaces and also provide strength to the maturebiofilm matrix structure, for instance, in the plant rhizosphere(38). Fap fibrils have been shown to bind extracellular metaboliteswithin the biofilm, such as quorum-sensing molecules and redoxmediators, modulating their release and retention when required.This implies a potential new role for functional amyloids in mo-lecular retention and indirect mediation of cell function (7, 39, 40)Again, genes encoding proteins with homology to the Fap systemsare present within species belonging to the Beta-, Gamma-, andDeltaproteobacteria (38).

Functional fibers with amyloidogenic properties have beenshown to be part of the extracellular matrix of biofilms formed bythe soil bacteria Bacillus subtilis and Bacillus cereus. In this case, thebiofilm matrix contains TasA-made amyloid-like fibers that holdcells together and provide structural support for biofilm mainte-nance (41, 42). TasA is expressed from the tapA-sipW-tasAoperon, together with two proteins, TapA and SipW, on whichformation of the fibrils depends. The sipW gene encodes a type Isignal peptidase that is specifically required for the processing andsecretion of TasA and TapA (41), whereas TapA is an accessoryprotein that promotes the efficient polymerization of TasA sub-units at the cell envelope (9, 43, 44) (Fig. 1A). Like for curli, theformation of TasA amyloid fibers is a highly regulated mechanismthat involves the participation of a master regulator, SinR, whichdirectly represses the expression of the tapA-sipW-tasA operonand the biosynthesis of an exopolysaccharide (EPS) (45). Underconditions favorable for biofilm development, SinI antagonizesthe repressive activity of SinR, leading to matrix formation (41). Afurther level of control of amyloid assembly in B. subtilis might begiven by the bifunctional signal peptidase SipW. Disruption of thepeptidase active site prevented the processing of TasA but did notaffect biofilm formation capacity, while deletion of the carboxy-terminal region of SipW affected the expression of the repressorSinR, thus impairing the induction of biofilm matrix genes (46).

A special example of extracellular fibers formed by the poly-merization of a monomer that it is secreted by a dedicated secre-tion apparatus corresponds to the phenol-soluble modulins(PSMs) in Staphylococcus aureus (47). PSMs can be classified ac-cording to their length as �-PMSs (20 to 25 amino acids), and�-PSMs (43 to 45 amino acids). S. aureus expresses four �-PSMpeptides encoded in the � psm locus, two �-PSM peptides en-coded in the � psm locus, and the �-toxin encoded within RNAIII,the effector molecule of the Agr system (48). These peptides areexported by an ATP-binding cassette (ABC) transporter with twoseparate membrane components (PmtB and PmtD) and two sep-arate ATPases (PmtA and PmtC) (49). Interestingly, PSM expres-

sion is directly regulated by binding of the AgrA response regula-tor to psm loci instead of the effector molecule RNAIII of theaccessory gene regulator system (Agr) (50). at the extracellularmedium, PSMs can be present either as soluble peptides or aspolymerized amyloid structures. When soluble, PSMs form anamphipathic �-helix with surfactant-like properties that promotebiofilm disassembly (47, 51). Under still poorly understood envi-ronmental conditions, the soluble �-helical peptides switch to�-sheet-rich protein aggregates, and in this state, they providefunctional support to the biofilm community (52) (Fig. 1A). Thus,PSMs are bifunctional proteins that may be stored as inert fibrilsin a sessile biofilm until conditions arise that favor their dissocia-tion to promote biofilm disassembly and virulence (47). Recentevidence suggests that PSM aggregation can be accelerated by thepresence of trace amounts of AgrD, the signal peptide of the S.aureus quorum-sensing system, since AgrD would self-aggregateand act as seeds, allowing the incorporation of PSMs into thegrowing aggregate (53).

SURFACE-ASSOCIATED PROTEINS WITH AMYLOIDOGENICPROPERTIES

Amyloid assembly mechanisms like the ones described above rep-resent sophisticated machineries that strictly control the poly-merization of protein subunits into amyloid fibers outside the cell.This is achieved by the expression of accessory genes generallyorganized within an operon, each of which encodes a protein witha specific function in amyloid assembly machinery. However, lesscomplex models of autoaggregative cell surface proteins with am-yloid properties have been also studied in different bacterial gen-era (54–56).

The Bap case is the most recent example of a surface proteinwith amyloid behavior involved in biofilm development. Bap is ahigh-molecular-weight protein present in many staphylococcithat is covalently anchored to the peptidoglycan through a sor-tase-dependent reaction recognizing the LPXTG motif (57, 58).Bap is a multidomain protein structurally organized into four do-mains. Region A contains two short repeats of 32 amino acids.Region B contains two EF-hand motifs that regulate Bap function-ality upon binding to calcium (59). Region C consists of a series ofidentical repeats of 86 amino acids and is followed by a C-terminalregion D containing a serine-aspartate-rich repeated sequence.Because Bap is covalently linked to the cell wall, it was believedthat Bap mediated intercellular adhesion through homophilic in-teractions between opposing proteins in neighboring cells, as hasbeen shown for the SasG protein (60–65). However, recent resultsindicate that Bap behaves in a completely different manner. Aftersecretion, Bap is proteolytically processed, and the N-terminalregion (amino acids 49 to 819) is released (66) (Fig. 1C). Theresulting N-terminal fragments adopt a molten globule-like con-formation that proceeds to a �-sheet-rich form that self-assemblesinto amyloid-like aggregates when the pH becomes acidic and thecalcium concentration is below 1 mM (Fig. 2). At higher calciumconcentrations, binding of the cation to the EF-hand domains ofregion B stabilizes the molten globule-like state, hindering theproper self-assembly of the N-terminal region. These findings de-fine a dual function for Bap, first as a sensor and then as a scaffoldprotein that promotes biofilm development under specific envi-ronmental conditions. This mechanism is substantially differentfrom the curli amyloid assembly machinery for several reasons: (i)Bap secretion does not require a dedicated secretion machinery,

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because it is efficiently exported when an S. aureus Newman bap-negative strain is complemented with the bap gene (67); (ii) Bapdoes not require a chaperon to prevent toxic premature assemblyinside the cytoplasm, since Bap is assembled extracellularly afterprotein processing; and (iii) Bap expression does not require atight transcriptional regulation, since amyloid formation dependsnot only on the presence of enough Bap but also on specific envi-ronmental conditions (66).

Bap is present in coagulase-negative staphylococci, and manyother bacteria use Bap-like proteins to build a biofilm matrix,suggesting that the amyloid-like aggregation described for Bapmay be widespread among pathogenic bacteria (58, 66).

Another example of a surface-associated protein with amy-loidogenic properties is adhesin P1 (also called antigen I/II) fromthe cariogenic pathogen Streptococcus mutans (54). This protein isfound covalently liked to the peptidoglycan and also in the culturemedium (54). The crystal structure of P1 shows that the proteinhas a highly extended fibrillar structure in which an alanine-richrepeat region intertwines through an �-helix with the noncontig-uous type II helix adopted by the proline-rich repeat region (68).P1 also contains two globular domains, the V region and the Cterminus, that assemble into �-sheet-rich fibers characteristics ofthe amyloid fold (54, 68, 69) (Fig. 1C).

The � module of the Ag43 autotransporter of E. coli constitutesanother example of a surface protein with amyloid propensity.Ag43 is composed of two domains: the � domain, which forms anouter membrane integrated pore, and the � domain, which issecreted to the extracellular environment through the folded �domain after processing of the Ag43 preprotein. The � and �domains remain in contact via noncovalent interactions (70).Ag43� promotes bacterial autoaggregation and biofilm forma-tion, and it adopts a parallel right-handed �-helix conformationthat resembles the amyloid structure established for curli (Fig. 1D)(55, 71). However, this structure cannot be considered a typicalamyloid fibril because the protein remains anchored to the cellsurface and is unable to polymerize into fibers. In addition, it wasdescribed that pairs of � domains mediate interaction betweendifferent cells through a “Velcro-like” mechanism in which theformation of � homodimers organized in a head-to-tail confor-mation and stabilized by hydrogen bonds and electrostatic inter-actions results in the aggregation of cells (70).

Another fascinating mechanism of amyloid-mediated cellular

interaction and biofilm development is given by the Als family ofyeast cell adhesins. The Als protein family consists of eight cellsurface multidomain glycoproteins. These proteins contain an N-terminal signal sequence followed by three tandem Ig-like se-quences, a Thr-rich conserved domain (T), a variable number ofglycosylated 36-residue tandem repeats (TR), an extended andunstructured highly N- and O-glycosylated stalk, and finally aC-terminal glycosylphosphatidylinositol (GPI) signal sequencethat covalently links the protein to the wall glucan (72–74). Im-portantly, the T domain contains a single amyloid-forming se-quence present in each Als paralog (72). To start cell adhesion, Alsproteins undergo conformational changes, accompanied by sur-face protein clustering, that spread around the entire surface of thecells (73, 75). These adhesin clusters, called nanodomains, areforce induced (76, 77) and are composed of adhesion moleculesaggregated through side chain amyloid-like interactions on thecell surface (74, 78). This clustering is possible even though Alsadhesins are anchored to the cell wall polysaccharide, since it isfacilitated by the length and conformational flexibility of theirextended molecules (73, 74). The formation of such Als amyloidclusters has implications in the formation of Saccharomyces cerevi-siae and Candida albicans biofilms, as demonstrated by studieswith the Als5p adhesin from C. albicans. A substitution in theamyloid-forming region of the protein prevented formation ofadhesion nanodomains and as a consequence the establishment ofcellular aggregates and biofilm development on a polystyrene sur-face (77).

ROLE OF BIOFILM-ASSOCIATED EXTRACELLULAR DNA INAMYLOID POLYMERIZATION

When studying the role of functional amyloids in the building ofan extracellular bacterial matrix, an often-asked question is whatactually assists or triggers the assembly of monomeric proteinsinto fibers, a reaction that is thermodynamically unfavorable andconstitutes the rate-limiting step of the reaction (79).

The possibilities to overcome this kinetic limitation include thepresence of trace amounts of preformed aggregates of the same ora heterologous protein, binding to other elements present in thecell envelope, or the presence of amyloid prone-to-aggregatestretches in the amino acid sequence of the protein. As describedpreviously, CsgB, FapB, TapA, and AgrD act as fibrillation nucle-ators. Some recent information pointed out that DNA might also

FIG 2 Amyloid-like fibers formed by the Bap protein of S. aureus. Transmission electron micrographs of negatively stained fibers formed by the recombinant Bdomain 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.

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play this role. Direct interaction of DNA with two major bacterialamyloids, curli and PSMs, reveals its role as a nucleator of amyloidpolymerization (80). In the case of curli fibers, Gallo et al. (80)showed that Salmonella curli fibers bind tightly to extracellularDNA (eDNA), protecting it from degradation by DNases. Fur-thermore, addition of three different types of nucleic acids (syn-thetic oligonucleotide, Salmonella genomic DNA, or eukaryoticDNA) indistinctly reduced the lag phase for curli fiber polymer-ization, suggesting a mechanism by which DNA molecules triggerthe assembly of CsgA monomers into highly stable complexes thatwould provide stability to Salmonella enterica serovar Typhimu-rium biofilms (80).

The presence of eDNA seems to also play a key role in theformation of amyloid fibers in S. aureus biofilms. In a recent re-port, Schwartz et al. (81) showed that biofilms formed by S. aureusmutant strains that do not contain eDNA in the extracellular ma-trix failed to assemble PSM amyloid fibrils, even when the mono-meric amyloid-like subunits were present (81). Further analysisrevealed that DNA was able to promote the polymerization ofpurified PSM�1 at concentrations at which this peptide is not ableto self-polymerize, suggesting that DNA increases the peptideconcentration above the threshold needed to start fiber assemblyby attracting the positively charged PSMs (81).

Further characterization of the interactions between amyloidfibers and extracellular DNA is necessary to understand how DNAtriggers amyloid fiber assembly and how the interaction betweenthese two structures protects the DNA from degradation byDNases. By extending our current knowledge on the polymeriza-tion process and the factors that govern it, the design of moleculesor strategies aimed at disrupting amyloid would appear as a won-derful biotechnological tool to fight against biofilms. Examples offirst attempts in this direction are provided below.

FUNCTIONAL AMYLOIDS IN MICROBIOME-HOSTINTERACTIONS

As a result of the lifelong commensal permanence of bacteriawithin the human body, it must be considered that microbiallygenerated amyloids, together with other related microbial secre-tory products, could be potential contributors to the pathology ofprogressive immunological and neurological disorders, some ofthem associated with amyloidogenesis (82). Curli fibers are able tostimulate the innate immune system by recognizing the Toll-likereceptor 2 (TLR2)–TLR1 heterocomplex (83–85). Interestingly, avery recent study highlights the role of curli-DNA complexes inthe stimulation of the innate and adaptive immune systems andsuggests that these complexes can accelerate autoimmunity con-tributing to systemic lupus erythematosus pathogenesis (80). Inan indirect manner, the strong innate immune response activatedby bacterial amyloids could stimulate neuropathogenic signalsthat promote amyloid aggregation and inflammatory degenera-tion characteristic of age-related neurological diseases such as Alz-heimer’s disease. Bacterial amyloid leakage through compromisedgastrointestinal tract or blood-brain barriers could also directlycontribute to progressive neuroamyloidogenesis (82). These dataprovide evidences of how biofilm-forming bacteria can contributeto the progression of some immunological and neurological dis-eases and point to amyloid components as potential moleculartargets for their treatment.

On the other hand, owing to their immunomodulatory activ-ity, bacterial amyloids have been proposed as potential therapeu-

tic candidates to treat intestinal inflammatory disorders, as re-cently shown by Oppong et al. (86). TLR2 activation by curli fibrilsfrom both commensal and pathogenic microbiota resulted in theproduction of an immunomodulatory cytokine of intestinal ho-meostasis that ameliorated inflammation in a mouse model ofcolitis (86). However, further studies are needed to probe the im-munomodulatory function of bacterial amyloids and their use astherapeutic candidates to treat chronic inflammatory disorders.

AMYLOIDS AS TARGETS TO FIGHT AGAINST BIOFILMS

Because biofilms can cause devastating consequences in clinicaland industrial settings, there has been an increasing interest indeveloping biofilm control strategies. The apparent ubiquity ofamyloid fibers in biofilm matrices has led to the screening of mol-ecules that might interrupt their production or assembly.

The first molecule to show inhibitory properties against bio-film amyloid fibrils was the ring-fused 2-pyridone called FN075,which is an analogue of the BipC10 pilicide compound. FN075proved to significantly reduce the polymerization of the main curlisubunit CsgA and the biogenesis of curli and type 1 pili in uro-pathogenic E. coli (87). This double curlicide-pilicide property ofFN075 is important from a therapeutic point of view because E.coli can use both structures, pili and curli, to promote surfaceattachment and subsequent biofilm formation (87). The authorsfurther tested FN075 in a mouse urinary tract infection model toevaluate inhibitory activity in vivo. Pretreatment with FN075 ofuropathogenic E. coli grown under pilus-forming but not curli-forming conditions significantly attenuated virulence, demon-strating the importance of inhibiting pilus formation to decreaseinfection (87). However, direct treatment of the mice with theinhibitor to validate its protective role in vivo remains to be done.

Romero and collaborators (88) used a simple screeningmethod based on the alteration of the B. subtilis wrinkly biofilmphenotype to search for possible antibiofilm compounds. Theysuccessfully identified that AA-861, a benzoquinone derivativewith anti-inflammatory activity, and parthenolide, a sesquiter-pene lactone with anti-inflammatory and anticancer activities, in-hibited TasA polymerization into amyloid-like fibers in vitro (88).Interestingly, AA-861 was more efficient in inhibiting TasA po-lymerization, whereas parthenolide was more potent in destroy-ing preformed biofilms in vivo, indicating that these molecules canwork additively to inhibit biofilm formation (88). Because thesetwo compounds showed antibiofilm activity against E. coli and B.cereus and also impeded the conversion of the yeast New1 proteinto its prion state, they seem to have activity against proteins ofdifferent origins (88).

Within the field of amyloid fibers and the possible ways toavoid their polymerization, is important to emphasize all the ef-forts made in fighting against human amyloidogenic degenerativediseases. Several small polyphenol molecules have been demon-strated to remarkably inhibit the formation of fibrillar assem-blies in vitro and their associated cytotoxicity, their mechanismbeing based primarily on antioxidative properties or structuralconstraints (89). The compound (�)-epi-gallocatechine gal-late (EGCG), for example, inhibits the fibrillogenesis of �-sy-nuclein and amyloid-�, two amyloid peptides crucially involvedin Alzheimer’s and Parkinson’s diseases, respectively, and is a po-tent agent for remodeling of mature amyloid fibrils (90, 91).Moreover, the efficiency of EGCG in inhibiting Streptococcus mu-tans and S. aureus biofilms has been shown (54, 66). This gives us

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the clue that compounds able to inhibit human aberrant amyloidscould be active also against bacterial functional amyloids and viceversa. In this regard, it has been recently reported that theperiplasmic protein CsgC is highly effective in inhibiting hu-man �-synuclein and CsgA amyloid formation, while it doesnot affect polymerization of A�42 (21).

In a recent study, rifapentine, an antibiotic that targets RNApolymerase and is often used to treat tuberculosis, was shown toinhibit curli-dependent biofilm formation on different surfaces ata concentration that did not alter bacterial growth (92). Specifi-cally, this antibiotic prevented curli operon transcription, asshown by reduction of the levels of mRNA of each csg gene in thepresence of increasing concentrations of rifapentine. Moreover,expression of cellulose-related genes (bscA and adrA) that are reg-ulated by CsgD was also decreased in the presence of rifapentine(92).

Finally, the property of protein aggregation and its cytotoxicitycan be used to fight against bacterial pathogens during infection(93). Bednarska and collaborators developed an ingenious strat-egy in this regard: they designed peptides carrying aggregation-prone sequence stretches of bacterial proteins, some of them withan amyloid-like nature, that specifically penetrate bacteria, caus-ing aggregation of polypeptides in the cytosol and ultimately lead-ing to bacterial death. The interesting point is that these peptideshad no detectable effects on host cells (94).

BIOTECHNOLOGICAL APPLICATIONS OF BIOFILM AMYLOIDFIBERS

Amyloid fibrils are attractive natural building blocks for the de-sign of new nanostructures, nanomaterials, and networks of fila-ments which can be functionalized for specific applications innanotechnology and nanomedicine (95). Several applications arebeing considered for using amyloids as nanowires and nanotubesfor electronics, as nanosensors, as amyloid-based gels in cell ad-hesion and wound healing, and as drug delivery systems (96).

Recently, two elegant studies have utilized the properties ofbacterial curli fibers to engineer cellular consortia and obtain ar-tificial biofilms for biotechnological applications. Chen and co-workers (97) developed controllable and autonomous patterningof curli fibrils assembled in E. coli cells. The system used externalchemical inducers to activate synthetic gene circuits that pro-duced engineered CsgA subunits that self-assemble in functional-ized curli fibrils and biofilms. By combining inducer gradients andsubunit engineering, this system gives the possibility to fabricatemultiscale patterning fibers. The proposed system constitutes aversatile scaffold that is able to synthesize fluorescent quantumdots, gold nanowires, nanorods, and nanoparticles, composingswitchable conductive biofilms. The other work describes a bio-film-integrated nanofiber display (BIND) system that consists ofgenetically programming the E. coli biofilm extracellular matrix byfusing functional peptide domains to the CsgA protein. This tech-nique is compatible with a wide range of peptide domains of var-ious lengths and secondary structures, since they maintain theirfunction after secretion and assembly, conferring artificial func-tions to the biofilm as a whole (98) (Fig. 3A). Based on this work,Botyanszki et al. (99) exploited the curli system of E. coli to createa biocatalytic biofilm in which a network of functional nanofibersis capable of covalently and site specifically immobilizing an in-dustrially relevant enzyme, �-amylase.

So far, curli have been the most engineered bacterial amyloid

protein. However, manipulation of others amyloid componentspresent in biofilm matrices of different bacteria might allow thefabrication of functionalized biofilms with different properties.For example, taking advantage of the amyloid assembly propertiesof Bap, this protein might serve as a potential biotechnologicaltool by fusing of its N-terminal domain to specific peptides tags.The key role of pH and Ca2� in the formation of Bap assembliescould be used to control the disruption or formation of pro-grammed S. aureus biofilms (Fig. 3B). Because most biofilms innature are composed of more than one species, it would be inter-esting to study microbial consortia where multiple engineeredamyloidogenic proteins produced by different bacteria couldwork as a complex nanomaterial to fulfill several technologicalapplications.

CONCLUDING REMARKS

Functional amyloids are common structural components ofthe extracellular matrix in bacterial biofilms. In regard to thisfunction, the amyloid conformation provides at least two ma-jor advantages compared to other protein mechanisms. First,the assembly of protein monomers into the amyloid insolubleaggregates implies a switch in protein conformation that usu-ally depends on specific environmental conditions. This simplerequirement broadens the biofilm regulatory network by pro-viding an additional level of control that enables biofilm devel-opment only under particular environmental conditions. Sec-ond, the stability of the amyloid structure and its intrinsicresistance against proteases prevent the destruction of the bio-film matrix scaffold in the presence of extracellular proteases.These two obvious advantages may explain why different bac-teria coincide in producing proteins with the capacity to formamyloid structures to build the biofilm matrix. However, addi-tional, still unknown reasons could justify the use of amyloidfibers as basic building blocks of the bacterial biofilm matrix.Deepening our knowledge of the properties that amyloids pro-vide to the biofilm matrix will be fundamental to understandthe benefits of living in microbial communities embedded in aself-produced extracellular matrix.

Several intriguing issues remain to be addressed in the fu-ture. In the particular case of the Bap surface protein, a detailedunderstanding of the functional similarities with structurallyrelated Bap-like surface proteins present in other bacterial gen-era will help to establish new directions for the elucidation ofamyloid-dependent biofilm development. In this regard, thecapacity of Bap to build functional fibers that reversibly disag-gregate provides one of the first examples of how bacteria canswitch between planktonic and sessile lifestyles depending onthe specific environmental conditions. Also, the discovery ofother bacterial and host molecules capable of interacting withamyloids or seeding their polymerization, as occurs witheDNA, will not only enable the development of new strategiesaimed at preventing amyloid assembly and interaction with thehost by targeting these molecular complexes but will also per-mit the design of new amyloid-based nanostructures and theoptimization of their stability and functionality through cou-pling with these other molecules. Regarding biotechnologicalpurposes of amyloids, there is still much work to do. In the nearfuture, the exploitation of newly discovered functional amyloidsystems with special regulatory and assembly features like theones described in this review may transform deleterious bio-

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films into useful bacterial communities with novel technolog-ical applications to suit specific needs.

FUNDING INFORMATIONThis work, including the efforts of Jaione Valle, was funded by Ministeriode Economía y Competitividad (MINECO) (AGL2011-23954). Thiswork, including the efforts of Lasa Iñigo, was funded by Ministerio deEconomía y Competitividad (MINECO) (BIO2011-30503-C02-02 andBIO2014-53530-R).

The funders had no role in study design, data collection and interpreta-tion, or the decision to submit the work for publication.

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