Amyloid Structures as Biofilm Matrix Scaffolds.

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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 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.

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myloid structures have been traditionally associated with several incurable degenerative human diseases, including Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and prion diseases (1). In these cases, amyloid fiber formation occurs upon aberrant misfolding of soluble and functional proteins (2). However, numerous pieces of evidence indicate that amyloid fibers are not just the result of aberrant protein folding but are produced on purpose under different nonpathological conditions to provide organisms with beneficial properties (3). The microbial world makes use of this type of protein folding for a number of processes relevant for bacterial growth and survival in the environment, such as morphological differentiation of filamentous bacteria (4, 5), adhesion to host tissues (6), detoxification of toxic compounds (7–9), electron transport (10), and structural scaffolds in biofilm matrices (1). The amyloid structure is an attractive building material because it is generally resistant to harsh denaturing conditions and in most cases cannot be degraded by proteases (1, 11). In addition, polymerization of amyloid proteins is metabolically cheap because it occurs in the absence of energy. Thus, the amyloid conformation is suitable as a component of the extracellular matrix where the energy sources are limited (12). The aim of this review is to describe the current knowledge on those functional bacterial amyloids involved in the building of biofilm matrices. We emphasize the differences between amyloid organelles with machineries dedicated to control the expression, secretion, and polymerization of protein subunits into amyloid fibers and cell surface proteins that build amyloid structures only under specific environmental conditions. We also discuss new compounds that are able to destabilize bacterial biofilms through their specific action on amyloid structures, recent evidence showing the implication of biofilm components, such as extracellular DNA (eDNA), in the self-assembly process of some functional amyloids, and the striking use of such proteinaceous nanostructures as tools to promote functional biofilms with biotechnological purposes. AMYLOID STRUCTURES WITH DEDICATED FIBER ASSEMBLY MACHINERY

The most studied and characterized bacterial amyloid system is curli fimbriae in Escherichia coli. Curli fimbriae show the prototypical biophysical properties of amyloid fibers: ordered

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␤-sheet-rich fibers, resistance to proteases, and ability to bind amyloid-specific dyes (Congo red or thioflavin T). Curli assembly requires the products of at least two divergently transcribed operons, csgBAC and csgDEFG (13). CsgA and CsgB are the protein subunits that make up curli, whereas the rest of the proteins are involved in the regulation of the production, stabilization, and secretion of CsgA and CsgB subunits (1, 14–16) (Fig. 1B). CsgA has the propensity to self-aggregate; however, it requires the CsgB subunit for nucleation into fibers in vivo (14, 17). CsgA and CsgB are translocated into the periplasm by the Sec translocon system and exported to the extracellular medium through a pore-like structure constituted by the CsgG protein formed in the outer membrane (18). Associated with this pore are the CsgE and CsgF proteins, which modulate subunit stability and proper assembly of curli fibers (18, 19). The periplasmic protein CsgC may be involved in regulating the export of curli subunits through the regulation of the redox state of CsgG (20), and it was shown to inhibit CsgA amyloid formation in vitro (21). CsgD is the master regulator that controls at the transcriptional level the expression of the csgBAC operon and also adrA, which is involved in posttranscriptional regulation of cellulose synthesis and encodes another common component of the biofilm matrix (22–25). CsgD activity is also controlled by a variety of regulatory systems that respond to environmental signals, including osmolarity, pH, temperature, and nutrient availability (26, 27). Curli fibers mediate bacterial adhesion to a variety of host proteins or abiotic surfaces, promoting biofilm formation and pathogenicity (28–33). Production of curli fibers has been described in Salmonella, Citrobacter, and Enterobacter (34), but genes encoding products with homology to proteins coding for the curli system are found in members of Proteobacteria, Bacteroidetes, Firmicutes, and Thermodesulfobacteria (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 matrix scaffolds. 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, [email protected]. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Journal of Bacteriology

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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 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 ␣ PSM, ␤ PSMs, and ␦-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 ␣ and ␤ 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 ␤-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 ␤-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 the biofilm matrix (15). Examples of extracellular fibers with structural and functional similarity to curli fimbriae of E. coli have been described in various species of Pseudomonas, including P. aeruginosa, P. fluorescens, and P. putida (36). In this case, the protein components for amyloid assembly are encoded in the fapABCDEF operon (37). Biophysical analysis of purified Fap fimbriae showed the presence of an extensive ␤-sheet structure, as well as binding to the specific thioflavin T dye (37). Biochemical analysis of the fibril composition 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 fibrillation nucleator of FapC (Fig. 1B). Fap amyloid fibers facilitate attachment to abiotic surfaces and also provide strength to the mature biofilm matrix structure, for instance, in the plant rhizosphere (38). Fap fibrils have been shown to bind extracellular metabolites within the biofilm, such as quorum-sensing molecules and redox mediators, modulating their release and retention when required. This implies a potential new role for functional amyloids in molecular retention and indirect mediation of cell function (7, 39, 40) Again, genes encoding proteins with homology to the Fap systems are present within species belonging to the Beta-, Gamma-, and Deltaproteobacteria (38). Functional fibers with amyloidogenic properties have been shown to be part of the extracellular matrix of biofilms formed by the soil bacteria Bacillus subtilis and Bacillus cereus. In this case, the biofilm matrix contains TasA-made amyloid-like fibers that hold cells together and provide structural support for biofilm maintenance (41, 42). TasA is expressed from the tapA-sipW-tasA operon, together with two proteins, TapA and SipW, on which formation of the fibrils depends. The sipW gene encodes a type I signal peptidase that is specifically required for the processing and secretion of TasA and TapA (41), whereas TapA is an accessory protein that promotes the efficient polymerization of TasA subunits at the cell envelope (9, 43, 44) (Fig. 1A). Like for curli, the formation of TasA amyloid fibers is a highly regulated mechanism that involves the participation of a master regulator, SinR, which directly represses the expression of the tapA-sipW-tasA operon and the biosynthesis of an exopolysaccharide (EPS) (45). Under conditions favorable for biofilm development, SinI antagonizes the repressive activity of SinR, leading to matrix formation (41). A further level of control of amyloid assembly in B. subtilis might be given by the bifunctional signal peptidase SipW. Disruption of the peptidase active site prevented the processing of TasA but did not affect biofilm formation capacity, while deletion of the carboxyterminal region of SipW affected the expression of the repressor SinR, thus impairing the induction of biofilm matrix genes (46). A special example of extracellular fibers formed by the polymerization of a monomer that it is secreted by a dedicated secretion apparatus corresponds to the phenol-soluble modulins (PSMs) in Staphylococcus aureus (47). PSMs can be classified according to their length as ␣-PMSs (20 to 25 amino acids), and ␤-PSMs (43 to 45 amino acids). S. aureus expresses four ␣-PSM peptides encoded in the ␣ psm locus, two ␤-PSM peptides encoded in the ␤ psm locus, and the ␦-toxin encoded within RNAIII, the effector molecule of the Agr system (48). These peptides are exported by an ATP-binding cassette (ABC) transporter with two separate membrane components (PmtB and PmtD) and two separate ATPases (PmtA and PmtC) (49). Interestingly, PSM expres-


sion is directly regulated by binding of the AgrA response regulator to psm loci instead of the effector molecule RNAIII of the accessory gene regulator system (Agr) (50). at the extracellular medium, PSMs can be present either as soluble peptides or as polymerized amyloid structures. When soluble, PSMs form an amphipathic ␣-helix with surfactant-like properties that promote biofilm disassembly (47, 51). Under still poorly understood environmental conditions, the soluble ␣-helical peptides switch to ␤-sheet-rich protein aggregates, and in this state, they provide functional support to the biofilm community (52) (Fig. 1A). Thus, PSMs are bifunctional proteins that may be stored as inert fibrils in a sessile biofilm until conditions arise that favor their dissociation to promote biofilm disassembly and virulence (47). Recent evidence suggests that PSM aggregation can be accelerated by the presence of trace amounts of AgrD, the signal peptide of the S. aureus quorum-sensing system, since AgrD would self-aggregate and act as seeds, allowing the incorporation of PSMs into the growing aggregate (53). SURFACE-ASSOCIATED PROTEINS WITH AMYLOIDOGENIC PROPERTIES

Amyloid assembly mechanisms like the ones described above represent sophisticated machineries that strictly control the polymerization of protein subunits into amyloid fibers outside the cell. This is achieved by the expression of accessory genes generally organized within an operon, each of which encodes a protein with a specific function in amyloid assembly machinery. However, less complex models of autoaggregative cell surface proteins with amyloid properties have been also studied in different bacterial genera (54–56). The Bap case is the most recent example of a surface protein with amyloid behavior involved in biofilm development. Bap is a high-molecular-weight protein present in many staphylococci that is covalently anchored to the peptidoglycan through a sortase-dependent reaction recognizing the LPXTG motif (57, 58). Bap is a multidomain protein structurally organized into four domains. Region A contains two short repeats of 32 amino acids. Region B contains two EF-hand motifs that regulate Bap functionality upon binding to calcium (59). Region C consists of a series of identical repeats of 86 amino acids and is followed by a C-terminal region D containing a serine-aspartate-rich repeated sequence. Because Bap is covalently linked to the cell wall, it was believed that Bap mediated intercellular adhesion through homophilic interactions between opposing proteins in neighboring cells, as has been shown for the SasG protein (60–65). However, recent results indicate that Bap behaves in a completely different manner. After secretion, Bap is proteolytically processed, and the N-terminal region (amino acids 49 to 819) is released (66) (Fig. 1C). The resulting N-terminal fragments adopt a molten globule-like conformation that proceeds to a ␤-sheet-rich form that self-assembles into amyloid-like aggregates when the pH becomes acidic and the calcium concentration is below 1 mM (Fig. 2). At higher calcium concentrations, binding of the cation to the EF-hand domains of region B stabilizes the molten globule-like state, hindering the proper self-assembly of the N-terminal region. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein that promotes biofilm development under specific environmental conditions. This mechanism is substantially different from the curli amyloid assembly machinery for several reasons: (i) Bap secretion does not require a dedicated secretion machinery,


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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.

because it is efficiently exported when an S. aureus Newman bapnegative strain is complemented with the bap gene (67); (ii) Bap does not require a chaperon to prevent toxic premature assembly inside the cytoplasm, since Bap is assembled extracellularly after protein processing; and (iii) Bap expression does not require a tight transcriptional regulation, since amyloid formation depends not only on the presence of enough Bap but also on specific environmental conditions (66). Bap is present in coagulase-negative staphylococci, and many other bacteria use Bap-like proteins to build a biofilm matrix, suggesting that the amyloid-like aggregation described for Bap may be widespread among pathogenic bacteria (58, 66). Another example of a surface-associated protein with amyloidogenic properties is adhesin P1 (also called antigen I/II) from the cariogenic pathogen Streptococcus mutans (54). This protein is found covalently liked to the peptidoglycan and also in the culture medium (54). The crystal structure of P1 shows that the protein has a highly extended fibrillar structure in which an alanine-rich repeat region intertwines through an ␣-helix with the noncontiguous type II helix adopted by the proline-rich repeat region (68). P1 also contains two globular domains, the V region and the C terminus, that assemble into ␤-sheet-rich fibers characteristics of the amyloid fold (54, 68, 69) (Fig. 1C). The ␣ module of the Ag43 autotransporter of E. coli constitutes another example of a surface protein with amyloid propensity. Ag43 is composed of two domains: the ␤ domain, which forms an outer membrane integrated pore, and the ␣ domain, which is secreted 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 formation, and it adopts a parallel right-handed ␤-helix conformation that resembles the amyloid structure established for curli (Fig. 1D) (55, 71). However, this structure cannot be considered a typical amyloid fibril because the protein remains anchored to the cell surface and is unable to polymerize into fibers. In addition, it was described that pairs of ␣ domains mediate interaction between different cells through a “Velcro-like” mechanism in which the formation of ␣ homodimers organized in a head-to-tail conformation and stabilized by hydrogen bonds and electrostatic interactions results in the aggregation of cells (70). Another fascinating mechanism of amyloid-mediated cellular

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interaction and biofilm development is given by the Als family of yeast cell adhesins. The Als protein family consists of eight cell surface multidomain glycoproteins. These proteins contain an Nterminal signal sequence followed by three tandem Ig-like sequences, a Thr-rich conserved domain (T), a variable number of glycosylated 36-residue tandem repeats (TR), an extended and unstructured highly N- and O-glycosylated stalk, and finally a C-terminal glycosylphosphatidylinositol (GPI) signal sequence that covalently links the protein to the wall glucan (72–74). Importantly, the T domain contains a single amyloid-forming sequence present in each Als paralog (72). To start cell adhesion, Als proteins undergo conformational changes, accompanied by surface protein clustering, that spread around the entire surface of the cells (73, 75). These adhesin clusters, called nanodomains, are force induced (76, 77) and are composed of adhesion molecules aggregated through side chain amyloid-like interactions on the cell surface (74, 78). This clustering is possible even though Als adhesins are anchored to the cell wall polysaccharide, since it is facilitated by the length and conformational flexibility of their extended molecules (73, 74). The formation of such Als amyloid clusters has implications in the formation of Saccharomyces cerevisiae and Candida albicans biofilms, as demonstrated by studies with the Als5p adhesin from C. albicans. A substitution in the amyloid-forming region of the protein prevented formation of adhesion nanodomains and as a consequence the establishment of cellular aggregates and biofilm development on a polystyrene surface (77). ROLE OF BIOFILM-ASSOCIATED EXTRACELLULAR DNA IN AMYLOID POLYMERIZATION

When studying the role of functional amyloids in the building of an extracellular bacterial matrix, an often-asked question is what actually assists or triggers the assembly of monomeric proteins into fibers, a reaction that is thermodynamically unfavorable and constitutes the rate-limiting step of the reaction (79). The possibilities to overcome this kinetic limitation include the presence of trace amounts of preformed aggregates of the same or a heterologous protein, binding to other elements present in the cell envelope, or the presence of amyloid prone-to-aggregate stretches in the amino acid sequence of the protein. As described previously, CsgB, FapB, TapA, and AgrD act as fibrillation nucleators. Some recent information pointed out that DNA might also



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play this role. Direct interaction of DNA with two major bacterial amyloids, curli and PSMs, reveals its role as a nucleator of amyloid polymerization (80). In the case of curli fibers, Gallo et al. (80) showed that Salmonella curli fibers bind tightly to extracellular DNA (eDNA), protecting it from degradation by DNases. Furthermore, addition of three different types of nucleic acids (synthetic oligonucleotide, Salmonella genomic DNA, or eukaryotic DNA) indistinctly reduced the lag phase for curli fiber polymerization, suggesting a mechanism by which DNA molecules trigger the assembly of CsgA monomers into highly stable complexes that would provide stability to Salmonella enterica serovar Typhimurium biofilms (80). The presence of eDNA seems to also play a key role in the formation of amyloid fibers in S. aureus biofilms. In a recent report, Schwartz et al. (81) showed that biofilms formed by S. aureus mutant strains that do not contain eDNA in the extracellular matrix failed to assemble PSM amyloid fibrils, even when the monomeric amyloid-like subunits were present (81). Further analysis revealed that DNA was able to promote the polymerization of purified PSM␣1 at concentrations at which this peptide is not able to self-polymerize, suggesting that DNA increases the peptide concentration above the threshold needed to start fiber assembly by attracting the positively charged PSMs (81). Further characterization of the interactions between amyloid fibers and extracellular DNA is necessary to understand how DNA triggers amyloid fiber assembly and how the interaction between these two structures protects the DNA from degradation by DNases. By extending our current knowledge on the polymerization process and the factors that govern it, the design of molecules or strategies aimed at disrupting amyloid would appear as a wonderful biotechnological tool to fight against biofilms. Examples of first attempts in this direction are provided below. FUNCTIONAL AMYLOIDS IN MICROBIOME-HOST INTERACTIONS

As a result of the lifelong commensal permanence of bacteria within the human body, it must be considered that microbially generated amyloids, together with other related microbial secretory products, could be potential contributors to the pathology of progressive immunological and neurological disorders, some of them associated with amyloidogenesis (82). Curli fibers are able to stimulate the innate immune system by recognizing the Toll-like receptor 2 (TLR2)–TLR1 heterocomplex (83–85). Interestingly, a very recent study highlights the role of curli-DNA complexes in the stimulation of the innate and adaptive immune systems and suggests that these complexes can accelerate autoimmunity contributing to systemic lupus erythematosus pathogenesis (80). In an indirect manner, the strong innate immune response activated by bacterial amyloids could stimulate neuropathogenic signals that promote amyloid aggregation and inflammatory degeneration characteristic of age-related neurological diseases such as Alzheimer’s disease. Bacterial amyloid leakage through compromised gastrointestinal tract or blood-brain barriers could also directly contribute to progressive neuroamyloidogenesis (82). These data provide evidences of how biofilm-forming bacteria can contribute to the progression of some immunological and neurological diseases and point to amyloid components as potential molecular targets for their treatment. On the other hand, owing to their immunomodulatory activity, bacterial amyloids have been proposed as potential therapeu-


tic candidates to treat intestinal inflammatory disorders, as recently shown by Oppong et al. (86). TLR2 activation by curli fibrils from both commensal and pathogenic microbiota resulted in the production of an immunomodulatory cytokine of intestinal homeostasis that ameliorated inflammation in a mouse model of colitis (86). However, further studies are needed to probe the immunomodulatory function of bacterial amyloids and their use as therapeutic candidates to treat chronic inflammatory disorders. AMYLOIDS AS TARGETS TO FIGHT AGAINST BIOFILMS

Because biofilms can cause devastating consequences in clinical and industrial settings, there has been an increasing interest in developing biofilm control strategies. The apparent ubiquity of amyloid fibers in biofilm matrices has led to the screening of molecules that might interrupt their production or assembly. The first molecule to show inhibitory properties against biofilm amyloid fibrils was the ring-fused 2-pyridone called FN075, which is an analogue of the BipC10 pilicide compound. FN075 proved to significantly reduce the polymerization of the main curli subunit CsgA and the biogenesis of curli and type 1 pili in uropathogenic E. coli (87). This double curlicide-pilicide property of FN075 is important from a therapeutic point of view because E. coli can use both structures, pili and curli, to promote surface attachment and subsequent biofilm formation (87). The authors further tested FN075 in a mouse urinary tract infection model to evaluate inhibitory activity in vivo. Pretreatment with FN075 of uropathogenic E. coli grown under pilus-forming but not curliforming conditions significantly attenuated virulence, demonstrating the importance of inhibiting pilus formation to decrease infection (87). However, direct treatment of the mice with the inhibitor to validate its protective role in vivo remains to be done. Romero and collaborators (88) used a simple screening method based on the alteration of the B. subtilis wrinkly biofilm phenotype to search for possible antibiofilm compounds. They successfully identified that AA-861, a benzoquinone derivative with anti-inflammatory activity, and parthenolide, a sesquiterpene lactone with anti-inflammatory and anticancer activities, inhibited TasA polymerization into amyloid-like fibers in vitro (88). Interestingly, AA-861 was more efficient in inhibiting TasA polymerization, whereas parthenolide was more potent in destroying preformed biofilms in vivo, indicating that these molecules can work additively to inhibit biofilm formation (88). Because these two compounds showed antibiofilm activity against E. coli and B. cereus and also impeded the conversion of the yeast New1 protein to its prion state, they seem to have activity against proteins of different origins (88). Within the field of amyloid fibers and the possible ways to avoid their polymerization, is important to emphasize all the efforts made in fighting against human amyloidogenic degenerative diseases. Several small polyphenol molecules have been demonstrated to remarkably inhibit the formation of fibrillar assemblies in vitro and their associated cytotoxicity, their mechanism being based primarily on antioxidative properties or structural constraints (89). The compound (⫺)-epi-gallocatechine gallate (EGCG), for example, inhibits the fibrillogenesis of ␣-synuclein and amyloid-␤, two amyloid peptides crucially involved in Alzheimer’s and Parkinson’s diseases, respectively, and is a potent agent for remodeling of mature amyloid fibrils (90, 91). Moreover, the efficiency of EGCG in inhibiting Streptococcus mutans 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 amyloids could be active also against bacterial functional amyloids and vice versa. In this regard, it has been recently reported that the periplasmic protein CsgC is highly effective in inhibiting human ␣-synuclein and CsgA amyloid formation, while it does not affect polymerization of A␤42 (21). In a recent study, rifapentine, an antibiotic that targets RNA polymerase and is often used to treat tuberculosis, was shown to inhibit curli-dependent biofilm formation on different surfaces at a concentration that did not alter bacterial growth (92). Specifically, this antibiotic prevented curli operon transcription, as shown by reduction of the levels of mRNA of each csg gene in the presence of increasing concentrations of rifapentine. Moreover, expression of cellulose-related genes (bscA and adrA) that are regulated by CsgD was also decreased in the presence of rifapentine (92). Finally, the property of protein aggregation and its cytotoxicity can be used to fight against bacterial pathogens during infection (93). Bednarska and collaborators developed an ingenious strategy in this regard: they designed peptides carrying aggregationprone sequence stretches of bacterial proteins, some of them with an amyloid-like nature, that specifically penetrate bacteria, causing aggregation of polypeptides in the cytosol and ultimately leading to bacterial death. The interesting point is that these peptides had no detectable effects on host cells (94). BIOTECHNOLOGICAL APPLICATIONS OF BIOFILM AMYLOID FIBERS

Amyloid fibrils are attractive natural building blocks for the design of new nanostructures, nanomaterials, and networks of filaments which can be functionalized for specific applications in nanotechnology and nanomedicine (95). Several applications are being considered for using amyloids as nanowires and nanotubes for electronics, as nanosensors, as amyloid-based gels in cell adhesion and wound healing, and as drug delivery systems (96). Recently, two elegant studies have utilized the properties of bacterial curli fibers to engineer cellular consortia and obtain artificial biofilms for biotechnological applications. Chen and coworkers (97) developed controllable and autonomous patterning of curli fibrils assembled in E. coli cells. The system used external chemical inducers to activate synthetic gene circuits that produced engineered CsgA subunits that self-assemble in functionalized curli fibrils and biofilms. By combining inducer gradients and subunit engineering, this system gives the possibility to fabricate multiscale patterning fibers. The proposed system constitutes a versatile scaffold that is able to synthesize fluorescent quantum dots, gold nanowires, nanorods, and nanoparticles, composing switchable conductive biofilms. The other work describes a biofilm-integrated nanofiber display (BIND) system that consists of genetically programming the E. coli biofilm extracellular matrix by fusing functional peptide domains to the CsgA protein. This technique is compatible with a wide range of peptide domains of various lengths and secondary structures, since they maintain their function after secretion and assembly, conferring artificial functions 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 create a biocatalytic biofilm in which a network of functional nanofibers is capable of covalently and site specifically immobilizing an industrially relevant enzyme, ␣-amylase. So far, curli have been the most engineered bacterial amyloid

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protein. However, manipulation of others amyloid components present in biofilm matrices of different bacteria might allow the fabrication of functionalized biofilms with different properties. For example, taking advantage of the amyloid assembly properties of Bap, this protein might serve as a potential biotechnological tool by fusing of its N-terminal domain to specific peptides tags. The key role of pH and Ca2⫹ in the formation of Bap assemblies could be used to control the disruption or formation of programmed S. aureus biofilms (Fig. 3B). Because most biofilms in nature are composed of more than one species, it would be interesting to study microbial consortia where multiple engineered amyloidogenic proteins produced by different bacteria could work as a complex nanomaterial to fulfill several technological applications. CONCLUDING REMARKS

Functional amyloids are common structural components of the extracellular matrix in bacterial biofilms. In regard to this function, the amyloid conformation provides at least two major advantages compared to other protein mechanisms. First, the assembly of protein monomers into the amyloid insoluble aggregates implies a switch in protein conformation that usually depends on specific environmental conditions. This simple requirement broadens the biofilm regulatory network by providing an additional level of control that enables biofilm development only under particular environmental conditions. Second, the stability of the amyloid structure and its intrinsic resistance against proteases prevent the destruction of the biofilm matrix scaffold in the presence of extracellular proteases. These two obvious advantages may explain why different bacteria coincide in producing proteins with the capacity to form amyloid structures to build the biofilm matrix. However, additional, still unknown reasons could justify the use of amyloid fibers as basic building blocks of the bacterial biofilm matrix. Deepening our knowledge of the properties that amyloids provide to the biofilm matrix will be fundamental to understand the benefits of living in microbial communities embedded in a self-produced extracellular matrix. Several intriguing issues remain to be addressed in the future. In the particular case of the Bap surface protein, a detailed understanding of the functional similarities with structurally related Bap-like surface proteins present in other bacterial genera will help to establish new directions for the elucidation of amyloid-dependent biofilm development. In this regard, the capacity of Bap to build functional fibers that reversibly disaggregate provides one of the first examples of how bacteria can switch between planktonic and sessile lifestyles depending on the specific environmental conditions. Also, the discovery of other bacterial and host molecules capable of interacting with amyloids or seeding their polymerization, as occurs with eDNA, will not only enable the development of new strategies aimed at preventing amyloid assembly and interaction with the host by targeting these molecular complexes but will also permit the design of new amyloid-based nanostructures and the optimization of their stability and functionality through coupling with these other molecules. Regarding biotechnological purposes of amyloids, there is still much work to do. In the near future, the exploitation of newly discovered functional amyloid systems with special regulatory and assembly features like the ones described in this review may transform deleterious bio-



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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.

films into useful bacterial communities with novel technological applications to suit specific needs. 5.

FUNDING INFORMATION This work, including the efforts of Jaione Valle, was funded by Ministerio de Economía y Competitividad (MINECO) (AGL2011-23954). This work, including the efforts of Lasa Iñigo, was funded by Ministerio de Economía y Competitividad (MINECO) (BIO2011-30503-C02-02 and BIO2014-53530-R). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

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REFERENCES 1. Blanco LP, Evans ML, Smith DR, Badtke MP, Chapman MR. 2012. Diversity, biogenesis and function of microbial amyloids. Trends Microbiol 20:66 –73. http://dx.doi.org/10.1016/j.tim.2011.11.005. 2. Schwartz K, Boles BR. 2013. Microbial amyloids-functions and interactions within the host. Curr Opin Microbiol 16:93–99. http://dx.doi.org/10 .1016/j.mib.2012.12.001. 3. Otzen D. 2010. Functional amyloid: turning swords into plowshares. Prion 4:256 –264. http://dx.doi.org/10.4161/pri.4.4.13676. 4. Sawyer EB, Claessen D, Haas M, Hurgobin B, Gras SL. 2011. The


9. 10. 11.

assembly of individual chaplin peptides from Streptomyces coelicolor into functional amyloid fibrils. PLoS One 6:e18839. http://dx.doi.org/10.1371 /journal.pone.0018839. Sawyer EB, Claessen D, Gras SL, Perrett S. 2012. Exploiting amyloid: how and why bacteria use cross-␤ fibrils. Biochem Soc Trans 40:728 –734. http://dx.doi.org/10.1042/BST20120013. Alteri CJ, Xicohténcatl-Cortes J, Hess S, Caballero-Olín G, Girón JA, Friedman RL. 2007. Mycobacterium tuberculosis produces pili during human infection. Proc Natl Acad Sci U S A 104:5145–5150. http://dx.doi.org /10.1073/pnas.0602304104. Shahnawaz M, Soto C. 2012. Microcin amyloid fibrils A are reservoir of toxic oligomeric species. J Biol Chem 287:11665–11676. http://dx.doi.org /10.1074/jbc.M111.282533. Bavdek A, Kostanjšek R, Antonini V, Lakey JH, Dalla Serra M, Gilbert RJC, Anderluh G. 2012. pH dependence of listeriolysin O aggregation and pore-forming ability. FEBS J 279:126 –141. http://dx.doi.org/10.1111/j .1742-4658.2011.08405.x. Romero D, Kolter R. 2014. Functional amyloids in bacteria. Int Microbiol 17:65–73. http://dx.doi.org/10.2436/20.1501.01.208. Boesen T, Nielsen LP. 2013. Molecular dissection of bacterial nanowires. mBio 4:e00270-13. http://dx.doi.org/10.1128/mBio.00270-13. Larsen P, Nielsen JL, Dueholm MS, Wetzel R, Otzen D, Nielsen PH. 2007. Amyloid adhesins are abundant in natural biofilms. Environ Microbiol 9:3077–3090. http://dx.doi.org/10.1111/j.1462-2920.2007.01418.x.


jb.asm.org

2585

Meeting Review

12. DePas WH, Chapman MR. 2012. Microbial manipulation of the amyloid fold. Res Microbiol 163:592– 606. http://dx.doi.org/10.1016/j.resmic.2012 .10.009. 13. Hammar M, Arnqvist A, Bian Z, Olsen A, Normark S. 1995. Expression of two csg operons is required for production of fibronectin- and congo red-binding curli polymers in Escherichia coli K-12. Mol Microbiol 18: 661– 670. http://dx.doi.org/10.1111/j.1365-2958.1995.mmi_18040661.x. 14. Hammer ND, McGuffie BA, Zhou Y, Badtke MP, Reinke AA, Brännström K, Gestwicki JE, Olofsson A, Almqvist F, Chapman MR. 2012. The C-terminal repeating units of CsgB direct bacterial functional amyloid nucleation. J Mol Biol 422:376 –389. http://dx.doi.org/10.1016/j.jmb .2012.05.043. 15. Hobley L, Harkins C, Macphee CE, Stanley-Wall NR. 2015. Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes. FEMS Microbiol Rev 39:649 – 669. http://dx .doi.org/10.1093/femsre/fuv015. 16. Van Gerven N, Klein RD, Hultgren SJ, Remaut H. 2015. Bacterial amyloid formation: structural insights into curli biogensis. Trends Microbiol 23:693–706. http://dx.doi.org/10.1016/j.tim.2015.07.010. 17. Bian Z, Normark S. 1997. Nucleator function of CsgB for the assembly of adhesive surface organelles in Escherichia coli. EMBO J 16:5827–5836. http://dx.doi.org/10.1093/emboj/16.19.5827. 18. Robinson LS, Ashman EM, Hultgren SJ, Chapman MR. 2006. Secretion of curli fibre subunits is mediated by the outer membrane-localized CsgG protein. Mol Microbiol 59:870 – 881. http://dx.doi.org/10.1111/j.1365 -2958.2005.04997.x. 19. Nenninger AA, Robinson LS, Hammer ND, Epstein EA, Badtke MP, Hultgren SJ, Chapman MR. 2011. CsgE is a curli secretion specificity factor that prevents amyloid fibre aggregation. Mol Microbiol 81:486 – 499. http://dx.doi.org/10.1111/j.1365-2958.2011.07706.x. 20. Taylor JD, Zhou Y, Salgado PS, Patwardhan A, McGuffie M, Pape T, Grabe G, Ashman E, Constable SC, Simpson PJ, Lee W-C, Cota E, Chapman MR, Matthews SJ. 2011. Atomic resolution insights into curli fiber biogenesis. Structure 19:1307–1316. http://dx.doi.org/10.1016/j.str .2011.05.015. 21. Evans ML, Chorell E, Taylor JD, Åden J, Götheson A, Li F, Koch M, Sefer L, Matthews SJ, Wittung-Stafshede P, Almqvist F, Chapman MR. 2015. The bacterial curli system possesses a potent and selective inhibitor of amyloid formation. Mol Cell 57:445– 455. http://dx.doi.org/10.1016/j .molcel.2014.12.025. 22. Römling U, Rohde M, Olsen A, Normark S, Reinköster J. 2000. AgfD, the checkpoint of multicellular and aggregative behaviour in Salmonella typhimurium regulates at least two independent pathways. Mol Microbiol 36:10 –23. http://dx.doi.org/10.1046/j.1365-2958.2000.01822.x. 23. Simm R, Morr M, Kader A, Nimtz M, Römling U. 2004. GGDEF and EAL domains inversely regulate cyclic di-GMP levels and transition from sessility to motility. Mol Microbiol 53:1123–1134. http://dx.doi.org/10 .1111/j.1365-2958.2004.04206.x. 24. Brombacher E, Baratto A, Dorel C, Landini P. 2006. Gene expression regulation by the curli activator CsgD protein: modulation of cellulose biosynthesis and control of negative determinants for microbial adhesion. J Bacteriol 188:2027–2037. http://dx.doi.org/10.1128/JB.188.6.2027-2037 .2006. 25. Zakikhany K, Harrington CR, Nimtz M, Hinton JCD, Römling U. 2010. Unphosphorylated CsgD controls biofilm formation in Salmonella enterica serovar Typhimurium. Mol Microbiol 77:771–786. http://dx.doi .org/10.1111/j.1365-2958.2010.07247.x. 26. Prigent-Combaret C, Brombacher E, Vidal O, Ambert A, Lejeune P, Landini P, Dorel C. 2001. Complex regulatory network controls initial adhesion and biofilm formation in Escherichia coli via regulation of the csgD gene. J Bacteriol 183:7213–7223. http://dx.doi.org/10.1128/JB.183 .24.7213-7223.2001. 27. Jubelin G, Vianney A, Beloin C, Ghigo J-M, Lazzaroni J-C, Lejeune P, Dorel C. 2005. CpxR/OmpR interplay regulates curli gene expression in response to osmolarity in Escherichia coli. J Bacteriol 187:2038 –2049. http: //dx.doi.org/10.1128/JB.187.6.2038-2049.2005. 28. Gophna U, Oelschlaeger TA, Hacker J, Ron EZ. 2002. Role of fibronectin in curli-mediated internalization. FEMS Microbiol Lett 212:55–58. http: //dx.doi.org/10.1111/j.1574-6968.2002.tb11244.x. 29. Boyer RR, Sumner SS, Williams RC, Pierson MD, Popham DL, Kniel KE. 2007. Influence of curli expression by Escherichia coli O157:H7 on the cell’s overall hydrophobicity, charge, and ability to attach to lettuce. J Food Prot 70:1339 –1345.

2586

jb.asm.org

30. Pawar DM, Rossman ML, Chen J. 2005. Role of curli fimbriae in mediating the cells of enterohaemorrhagic Escherichia coli to attach to abiotic surfaces. J Appl Microbiol 99:418 – 425. http://dx.doi.org/10.1111/j.1365 -2672.2005.02499.x. 31. Saldaña Z, Xicohténcatl-Cortes J, Avelino F, Phillips AD, Kaper JB, Puente JL, Girón JA. 2009. Synergistic role of curli and cellulose in cell adherence and biofilm formation of attaching and effacing Escherichia coli and identification of Fis as a negative regulator of curli. Environ Microbiol 11:992–1006. http://dx.doi.org/10.1111/j.1462-2920.2008.01824.x. 32. Goulter-Thorsen RM, Taran E, Gentle IR, Gobius KS, Dykes GA. 2011. CsgA production by Escherichia coli O157:H7 alters attachment to abiotic surfaces in some growth environments. Appl Environ Microbiol 77:7339 – 7344. http://dx.doi.org/10.1128/AEM.00277-11. 33. Zhou Y, Smith DR, Hufnagel DA, Chapman MR. 2013. Experimental manipulation of the microbial functional amyloid called curli. Methods Mol Biol 966:53–75. http://dx.doi.org/10.1007/978-1-62703-245-2_4. 34. Zogaj X, Bokranz W, Nimtz M, Römling U. 2003. Production of cellulose and curli fimbriae by members of the family Enterobacteriaceae isolated from the human gastrointestinal tract. Infect Immun 71:4151– 4158. http://dx.doi.org/10.1128/IAI.71.7.4151-4158.2003. 35. Dueholm MS, Albertsen M, Otzen D, Nielsen PH. 2012. Curli functional amyloid systems are phylogenetically widespread and display large diversity in operon and protein structure. PLoS One 7:e51274. http://dx.doi.org /10.1371/journal.pone.0051274. 36. Dueholm MS, Søndergaard MT, Nilsson M, Christiansen G, Stensballe A, Overgaard MT, Givskov M, Tolker-Nielsen T, Otzen DE, Nielsen PH. 2013. Expression of Fap amyloids in Pseudomonas aeruginosa, P. fluorescens, and P. putida results in aggregation and increased biofilm formation Microbiologyopen 2:365–382. http://dx.doi.org/10.1002/mbo3.81. 37. Dueholm MS, Petersen SV, Sønderkaer M, Larsen P, Christiansen G, Hein KL, Enghild JJ, Nielsen JL, Nielsen KL, Nielsen PH, Otzen DE. 2010. Functional amyloid in Pseudomonas. Mol Microbiol 77:1009 – 1020. http://dx.doi.org/10.1111/j.1365-2958.2010.07269.x. 38. Dueholm MS, Otzen D, Nielsen PHR. 2013. Evolutionary insight into the functional amyloids of the pseudomonads. PLoS One 8:e76630. http: //dx.doi.org/10.1371/journal.pone.0076630. 39. Fowler DM, Koulov AV, Balch WE, Kelly JW. 2007. Functional amyloid—from bacteria to humans. Trends Biochem Sci 32:217–224. http: //dx.doi.org/10.1016/j.tibs.2007.03.003. 40. Seviour T, Hansen SH, Yang L, Yau YH, Wang VB, Stenvang MR, Christiansen G, Marsili E, Givskov M, Chen Y, Otzen DE, Nielsen PH, Geifman-Shochat S, Kjelleberg S, Dueholm MS. 2015. Functional amyloids keep quorum-sensing molecules in check. J Biol Chem 290:6457– 6469. http://dx.doi.org/10.1074/jbc.M114.613810. 41. Branda SS, Chu F, Kearns DB, Losick R, Kolter R. 2006. A major protein component of the Bacillus subtilis biofilm matrix. Mol Microbiol 59:1229 – 1238. http://dx.doi.org/10.1111/j.1365-2958.2005.05020.x. 42. Romero D, Aguilar C, Losick R, Kolter R. 2010. Amyloid fibers provide structural integrity to Bacillus subtilis biofilms. Proc Natl Acad Sci U S A 107:2230 –2234. http://dx.doi.org/10.1073/pnas.0910560107. 43. Romero D, Vlamakis H, Losick R, Kolter R. 2011. An accessory protein required for anchoring and assembly of amyloid fibers in B. subtilis biofilms. Mol Microbiol 80:1155–1168. http://dx.doi.org/10.1111/j.1365 -2958.2011.07653.x. 44. Romero D, Vlamakis H, Losick R, Kolter R. 2014. Functional analysis of the accessory protein TapA in Bacillus subtilis amyloid fiber assembly. J Bacteriol 196:1505–1513. http://dx.doi.org/10.1128/JB.01363-13. 45. Kearns DB, Chu F, Branda SS, Kolter R, Losick R. 2005. A master regulator for biofilm formation by Bacillus subtilis. Mol Microbiol 55: 739 –749. http://dx.doi.org/10.1111/j.1365-2958.2004.04440.x. 46. Terra R, Stanley-Wall NR, Cao G, Lazazzera BA. 2012. Identification of Bacillus subtilis SipW as a bifunctional signal peptidase that controls surface-adhered biofilm formation. J Bacteriol 194:2781–2790. http://dx.doi .org/10.1128/JB.06780-11. 47. Schwartz K, Syed AK, Stephenson RE, Rickard AH, Boles BR. 2012. Functional amyloids composed of phenol soluble modulins stabilize Staphylococcus aureus biofilms. PLoS Pathog 8:e1002744. http://dx.doi .org/10.1371/journal.ppat.1002744. 48. Cheung GYC, Kretschmer D, Queck SY, Joo H-S, Wang R, Duong AC, Nguyen TH, Bach T-HL, Porter AR, DeLeo FR, Peschel A, Otto M. 2014. Insight into structure-function relationship in phenol-soluble modulins using an alanine screen of the phenol-soluble modulin (PSM) ␣3 peptide. FASEB J 28:153–161. http://dx.doi.org/10.1096/fj.13-232041.



Meeting Review

49. Chatterjee SS, Joo H-S, Duong AC, Dieringer TD, Tan VY, Song Y, Fischer ER, Cheung GYC, Li M, Otto M. 2013. Essential Staphylococcus aureus toxin export system. Nat Med 19:364 –367. http://dx.doi.org/10 .1038/nm.3047. 50. Queck SY, Jameson-Lee M, Villaruz AE, Bach T-HL, Khan BA, Sturdevant DE, Ricklefs SM, Li M, Otto M. 2008. RNAIII-independent target gene control by the agr quorum-sensing system: insight into the evolution of virulence regulation in Staphylococcus aureus. Mol Cell 32:150 –158. http://dx.doi.org/10.1016/j.molcel.2008.08.005. 51. Vuong C, Götz F, Otto M. 2000. Construction and characterization of an agr deletion mutant of Staphylococcus epidermidis. Infect Immun 68: 1048 –1053. http://dx.doi.org/10.1128/IAI.68.3.1048-1053.2000. 52. Syed AK, Boles BR. 2014. Fold modulating function: bacterial toxins to functional amyloids. Front Microbiol 5:401. http://dx.doi.org/10.3389 /fmicb.2014.00401. 53. Schwartz K, Sekedat MD, Syed AK, O’Hara B, Payne DE, Lamb A, Boles BR. 2014. The AgrD N-terminal leader peptide of Staphylococcus aureus has cytolytic and amyloidogenic properties. Infect Immun 82:3837–3844. http://dx.doi.org/10.1128/IAI.02111-14. 54. Oli MW, Otoo HN, Crowley PJ, Heim KP, Nascimento MM, Ramsook CB, Lipke PN, Brady LJ. 2012. Functional amyloid formation by Streptococcus mutans. Microbiology 158:2903–2916. http://dx.doi.org/10.1099 /mic.0.060855-0. 55. Knudsen SK, Stensballe A, Franzmann M, Westergaard UB, Otzen DE. 2008. Effect of glycosylation on the extracellular domain of the Ag43 bacterial autotransporter: enhanced stability and reduced cellular aggregation. Biochem J 412:563–577. http://dx.doi.org/10.1042/BJ20071497. 56. Otzen DE (ed). 2013. Amyloid fibrils and prefibrillar aggregates: molecular and biological properties. Wiley, Hoboken, NJ. 57. Cucarella C, Solano C, Valle J, Amorena B, Lasa I, Penadés JR. 2001. Bap, a Staphylococcus aureus surface protein involved in biofilm formation. J Bacteriol 183:2888 –2896. http://dx.doi.org/10.1128/JB.183.9.2888 -2896.2001. 58. Tormo MA, Knecht E, Götz F, Lasa I, Penadés JR. 2005. Bap-dependent biofilm formation by pathogenic species of Staphylococcus: evidence of horizontal gene transfer? Microbiology 151:2465–2475. http://dx.doi.org /10.1099/mic.0.27865-0. 59. Arrizubieta MJ, Toledo-Arana A, Amorena B, Penadés JR, Lasa I. 2004. Calcium inhibits Bap-dependent multicellular behavior in Staphylococcus aureus. J Bacteriol 186:7490 –7498. http://dx.doi.org/10.1128/JB.186.22 .7490-7498.2004. 60. Geoghegan JA, Corrigan RM, Gruszka DT, Speziale P, O’Gara JP, Potts JR, Foster TJ. 2010. Role of surface protein SasG in biofilm formation by Staphylococcus aureus. J Bacteriol 192:5663–5673. http://dx.doi.org/10 .1128/JB.00628-10. 61. Barbu EM, Mackenzie C, Foster TJ, Höök M. 2014. SdrC induces staphylococcal biofilm formation through a homophilic interaction. Mol Microbiol 94:172–185. http://dx.doi.org/10.1111/mmi.12750. 62. Missineo A, Di Poto A, Geoghegan JA, Rindi S, Heilbronner S, Gianotti V, Arciola CR, Foster TJ, Speziale P, Pietrocola G. 2014. IsdC from Staphylococcus lugdunensis induces biofilm formation in low-iron growth conditions. Infect Immun 82:2448 –2459. http://dx.doi.org/10 .1128/IAI.01542-14. 63. Conrady DG, Brescia CC, Horii K, Weiss AA, Hassett DJ, Herr AB. 2008. A zinc-dependent adhesion module is responsible for intercellular adhesion in staphylococcal biofilms. Proc Natl Acad Sci U S A 105:19456 – 19461. http://dx.doi.org/10.1073/pnas.0807717105. 64. Herman-Bausier P, El-Kirat-Chatel S, Foster TJ, Geoghegan JA, Dufrêne YF. 2015. Staphylococcus aureus fibronectin-binding protein A mediates cell-cell adhesion through low-affinity homophilic bonds. mBio 6:e00413-15. http://dx.doi.org/10.1128/mBio.00413-15. 65. Formosa-Dague C, Speziale P, Foster TJ, Geoghegan JA, Dufrêne YF. 2016. Zinc-dependent mechanical properties of Staphylococcus aureus biofilm-forming surface protein SasG. Proc Natl Acad Sci U S A 113:410 – 415. http://dx.doi.org/10.1073/pnas.1519265113. 66. Taglialegna A, Navarro S, Ventura S, Garnett JA, Matthews SJ, Penadés JR, Lasa I, Valle J. 2016. Staphylococcal Bap proteins build amyloid scaffold biofilm matrices in response to environmental signals. PLoS Pathog 12:e1005711. http://dx.doi.org/10.1371/journal.ppat.1005711. 67. Lasa I, Penadés JR. 2006. Bap: a family of surface proteins involved in biofilm formation. Res Microbiol 157:99 –107. http://dx.doi.org/10.1016 /j.resmic.2005.11.003. 68. Larson MR, Rajashankar KR, Patel MH, Robinette RA, Crowley PJ,


69.

70.

71.

72. 73.

74.

75.

76. 77. 78.

79.

80.

81.

82. 83.

84.

85.

86.

Michalek S, Brady LJ, Deivanayagam C. 2010. Elongated fibrillar structure of a streptococcal adhesin assembled by the high-affinity association of alpha- and PPII-helices. Proc Natl Acad Sci U S A 107:5983–5988. http: //dx.doi.org/10.1073/pnas.0912293107. Troffer-Charlier N, Ogier J, Moras D, Cavarelli J. 2002. Crystal structure of the V-region of Streptococcus mutans antigen I/II at 2.4 A resolution suggests a sugar preformed binding site. J Mol Biol 318:179 –188. http://dx .doi.org/10.1016/S0022-2836(02)00025-6. Heras B, Totsika M, Peters KM, Paxman JJ, Gee CL, Jarrott RJ, Perugini MA, Whitten AE, Schembri MA. 2014. The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping. Proc Natl Acad Sci U S A 111:457– 462. http://dx.doi.org /10.1073/pnas.1311592111. Klemm P, Hjerrild L, Gjermansen M, Schembri MA. 2004. Structurefunction analysis of the self-recognizing antigen 43 autotransporter protein from Escherichia coli. Mol Microbiol 51:283–296. http://dx.doi.org /10.1046/j.1365-2958.2003.03833.x. Otoo HN, Kyeng GL, Qiu W, Lipke PN. 2008. Candida albicans Als adhesins have conserved amyloid-forming sequences. Eukaryot Cell 7:776 –782. http://dx.doi.org/10.1128/EC.00309-07. Klis FM, Sosinska GJ, de Groot PWJ, Brul S. 2009. Covalently linked cell wall proteins of Candida albicans and their role in fitness and virulence. FEMS Yeast Res 9:1013–1028. http://dx.doi.org/10.1111/j.1567-1364 .2009.00541.x. Lipke PN, Ramsook C, Garcia-Sherman MC, Jackson DN, Chan CXJ, Bois M, Klotz SA. 2014. Between amyloids and aggregation lies a connection with strength and adhesion. New J Sci 2014:815102. http://dx.doi.org /10.1155/2014/815102. Rauceo JM, Gaur NK, Lee KG, Edwards JE, Klotz SA, Lipke PN. 2004. Global cell surface conformational shift mediated by a Candida albicans adhesin. Infect Immun 72:4948 – 4955. http://dx.doi.org/10.1128/IAI.72.9 .4948-4955.2004. Alsteens D, Ramsook CB, Lipke PN, Dufrêne YF. 2012. Unzipping a functional microbial amyloid. ACS Nano 6:7703–7711. http://dx.doi.org /10.1021/nn3025699. Garcia MC, Lee JT, Ramsook CB, Alsteens D, Dufrêne YF, Lipke PN. 2011. A role for amyloid in cell aggregation and biofilm formation. PLoS One 6:e17632. http://dx.doi.org/10.1371/journal.pone.0017632. Lipke PN, Garcia MC, Alsteens D, Ramsook CB, Klotz SA, Dufrêne YF. 2012. Strengthening relationships: amyloids create adhesion nanodomains in yeasts. Trends Microbiol 20:59 – 65. http://dx.doi.org/10.1016/j .tim.2011.10.002. Buell AK, Galvagnion C, Gaspar R, Sparr E, Vendruscolo M, Knowles TPJ, Linse S, Dobson CM. 2014. Solution conditions determine the relative importance of nucleation and growth processes in ␣-synuclein aggregation. Proc Natl Acad Sci U S A 111:7671–7676. http://dx.doi.org /10.1073/pnas.1315346111. Gallo PM, Rapsinski GJ, Wilson RP, Oppong GO, Sriram U, Goulian M, Buttaro B, Caricchio R, Gallucci S, Tükel C. 2015. Amyloid-DNA composites of bacterial biofilms stimulate autoimmunity. Immunity 42: 1171–1184. http://dx.doi.org/10.1016/j.immuni.2015.06.002. Schwartz K, Ganesan M, Payne DE, Solomon MJ, Boles BR. 2016. Extracellular DNA facilitates the formation of functional amyloids in Staphylococcus aureus biofilms. Mol Microbiol 99:123–134. http://dx.doi .org/10.1111/mmi.13219. Zhao Y, Lukiw WJ. 2015. Microbiome-generated amyloid and potential impact on amyloidogenesis in Alzheimer’s disease (AD). J Nat Sci 1:e138. Tükel C, Raffatellu M, Humphries AD, Wilson RP, Andrews-Polymenis HL, Gull T, Figueiredo JF, Wong MH, Michelsen KS, Akçelik M, Adams LG, Bäumler AJ. 2005. CsgA is a pathogen-associated molecular pattern of Salmonella enterica serotype Typhimurium that is recognized by Tolllike receptor 2. Mol Microbiol 58:289 –304. http://dx.doi.org/10.1111/j .1365-2958.2005.04825.x. Tükel C, Wilson RP, Nishimori JH, Pezeshki M, Chromy BA, Bäumler AJ. 2009. Responses to amyloids of microbial and host origin are mediated through toll-like receptor 2. Cell Host Microbe 6:45–53. http://dx.doi.org /10.1016/j.chom.2009.05.020. Tükel C, Nishimori JH, Wilson RP, Winter MG, Keestra AM, van Putten JPM, Bäumler AJ. 2010. Toll-like receptors 1 and 2 cooperatively mediate immune responses to curli, a common amyloid from enterobacterial biofilms. Cell Microbiol 12:1495–1505. http://dx.doi.org/10.1111/j .1462-5822.2010.01485.x. Oppong GO, Rapsinski GJ, Tursi SA, Biesecker SG, Klein-Szanto AJ,


jb.asm.org

2587

Meeting Review

87.

88. 89.

90.

91.

92.

Goulian M, McCauley C, Healy C, Wilson PR, Tükel C. 2015. Biofilm-associated bacterial amyloids dampen inflammationin the gut: oral treatment with curli fibres reduces the severity of hapten-induced colitis in mice. Biofilms Microbiomes 1:15019. http://dx.doi.org/10 .1038/npjbiofilms.2015.19. Cegelski L, Pinkner JS, Hammer ND, Cusumano CK, Hung CS, Chorell E, Aberg V, Walker JN, Seed PC, Almqvist F, Chapman MR, Hultgren SJ. 2009. Small-molecule inhibitors target Escherichia coli amyloid biogenesis and biofilm formation. Nat Chem Biol 5:913–919. http://dx.doi.org /10.1038/nchembio.242. Romero D, Sanabria-Valentín E, Vlamakis H, Kolter R. 2013. Biofilm inhibitors that target amyloid proteins. Chem Biol 20:102–110. http://dx .doi.org/10.1016/j.chembiol.2012.10.021. Porat Y, Abramowitz A, Gazit E. 2006. Inhibition of amyloid fibril formation by polyphenols: structural similarity and aromatic interactions as a common inhibition mechanism. Chem Biol Drug Des 67:27–37. http: //dx.doi.org/10.1111/j.1747-0285.2005.00318.x. Bieschke J, Russ J, Friedrich RP, Ehrnhoefer DE, Wobst H, Neugebauer K, Wanker EE. 2010. EGCG remodels mature alpha-synuclein and amyloid-beta fibrils and reduces cellular toxicity. Proc Natl Acad Sci U S A 107:7710 –7715. http://dx.doi.org/10.1073/pnas.0910723107. Ehrnhoefer DE, Bieschke J, Boeddrich A, Herbst M, Masino L, Lurz R, Engemann S, Pastore A, Wanker EE. 2008. EGCG redirects amyloidogenic polypeptides into unstructured, off-pathway oligomers. Nat Struct Mol Biol 15:558 –566. http://dx.doi.org/10.1038/nsmb.1437. Maher MC, Lim JY, Gunawan C, Cegelski L. 2015. Cell-based high-

2588

jb.asm.org

93. 94.

95.

96.

97. 98.

99.

throughput screening identifies rifapentine as an inhibitor of amyloid and biofilm formation in Escherichia coli. ACS Infect Dis 1:460 – 468. http://dx .doi.org/10.1021/acsinfecdis.5b00055. Ventura S. 2016. Curing bacterial infections with protein aggregates. Mol Microbiol 99:827– 830. http://dx.doi.org/10.1111/mmi.13293. Bednarska NG, Van Eldere J, Gallardo R, Ganesan A, Ramakers M, Vogel I, Baatsen P, Staes A, Goethals M, Hammarström P, Nilsson KPR, Gevaert K, Schymkowitz J, Rousseau F. 2016. Protein aggregation as an antibiotic design strategy. Mol Microbiol 99:849 – 868. http://dx.doi .org/10.1111/mmi.13269. Cherny I, Gazit E. 2008. Amyloids: not only pathological agents but also ordered nanomaterials. Angew Chem Int Ed Engl 47:4062– 4069. http://dx .doi.org/10.1002/anie.200703133. Hauser CAE, Maurer-Stroh S, Martins IC. 2014. Amyloid-based nanosensors and nanodevices. Chem Soc Rev 43:5326. http://dx.doi.org/10 .1039/C4CS00082J. Chen AY, Zhong C, Lu TK. 2015. Engineering living functional materials. ACS Synth Biol 4:8 –11. http://dx.doi.org/10.1021/sb500113b. Nguyen PQ, Botyanszki Z, Tay PKR, Joshi NS. 2014. Programmable biofilm-based materials from engineered curli nanofibres. Nat Commun 5:4945. http://dx.doi.org/10.1038/ncomms5945. Botyanszki Z, Tay PKR, Nguyen PQ, Nussbaumer MG, Joshi NS. 2015. Engineered catalytic biofilms: site-specific enzyme immobilization onto E. coli curli nanofibers. Biotechnol Bioeng 112:2016 –2024. http://dx.doi.org /10.1002/bit.25638.



Copper-Containing Anti-Biofilm Nanofiber Scaffolds as a Wound Dressing Material.

Yeast synthetic biology platform generates novel chemical structures as scaffolds for drug discovery.

Injectable Extracellular Matrix Hydrogels as Scaffolds for Spinal Cord Injury Repair.

Multicomponent dipeptide hydrogels as extracellular matrix-mimetic scaffolds for cell culture applications.

Identification of Poly-N-acetylglucosamine as a Major Polysaccharide Component of the Bacillus subtilis Biofilm Matrix.

Matrix Metalloproteinase Inhibitor as an Antimicrobial Agent to Eradicate Enterococcus faecalis Biofilm.

Fibroblast extracellular matrix and adhesion on microtextured polydimethylsiloxane scaffolds.

Immunomodulatory effects of amniotic membrane matrix incorporated into collagen scaffolds.

Multipotent Stromal Cells Outperform Chondrocytes on Cartilage-Derived Matrix Scaffolds.

Electrospun cartilage-derived matrix scaffolds for cartilage tissue engineering.

Novel xeno-free human heart matrix-derived three-dimensional scaffolds.

Crosslinking strategies for preparation of extracellular matrix-derived cardiovascular scaffolds.

Salmonella Extracellular Matrix Components Influence Biofilm Formation and Gallbladder Colonization.

Biofilm matrix and its regulation in Pseudomonas aeruginosa.

Biofilm-specific extracellular matrix proteins of nontypeable Haemophilus influenzae.

The Candida albicans Biofilm Matrix: Composition, Structure and Function.

Community participation in biofilm matrix assembly and function.

Conformational selection in amyloid-based immunotherapy: Survey of crystal structures of antibody-amyloid complexes.

Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production.

Glutamate receptors function as scaffolds for the regulation of β-amyloid and cellular prion protein signaling complexes.

Rapidly progressive Alzheimer's disease features distinct structures of amyloid-β.

Matrix Metalloproteinase-9 Protects Islets from Amyloid-induced Toxicity.

Characterization of amyloid in equine recurrent uveitis as AA amyloid.

A unique biofilm in human deep mycoses: fungal amyloid is bound by host serum amyloid P component.