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During biofilm formation, Escherichia coli and other Enterobacteriaceae produce an extracellular matrix consisting of curli amyloid fibers and cellulose. The precursor of curli fibers is the amyloidogenic protein CsgA. The human systemic amyloid precursor protein transthyretin (TTR) is known to inhibit amyloid-β (Aβ) aggregation in vitro and suppress the Alzheimer’s-like phenotypes in a transgenic mouse model of Aβ deposition. We hypothesized that TTR might have broad antiamyloid activity because the biophysical properties of amyloids are largely conserved across species and kingdoms. Here, we report that both human WT tetrameric TTR (WT-TTR) and its engineered nontetramer-forming monomer (M-TTR, F87M/L110M) inhibit CsgA amyloid formation in vitro, with M-TTR being the more efficient inhibitor. Preincubation of WT-TTR with small molecules that occupy the T4 binding site eliminated the inhibitory capacity of the tetramer; however, they did not significantly compromise the ability of M-TTR to inhibit CsgA amyloidogenesis. TTR also inhibited amyloid-dependent biofilm formation in two different bacterial species with no apparent bactericidal or bacteriostatic effects. These discoveries suggest that TTR is an effective antibiofilm agent that could potentiate antibiotic efficacy in infections associated with significant biofilm formation.

Gram-negative bacteria, such as E. coli and Salmonella, contain proteinaceous, hair-like, cell surface filaments known as curli. Curli serve to facilitate cell-cell interactions and are essential for host cell colonization. Curli assembly involves six proteins, CsgA, CsgB, CsgC, CsgE, CsgF, and CsgG. CsgE and CsgF are thought to act as chaperones to help prevent the premature aggregation of CsgA and/or CsgB, and to help transport these proteins, through the outer-membrane protein CsgG, to the cell surface where they assemble to form Curli. It has been observed that CsgF is able to inhibit the aggregation of CsgA, the major protein component of Curli. This article describes CsgF’s ability to influence the aggregation of human islet amyloid polypeptide (hIAPP), an amyloidogenic polypeptide that is unrelated to Curli. In the presence of CsgF no increase in Thioflavin T fluorescence was observed for freshly solubilized hIAPP monitored as a function of time, suggesting that CsgF prevents the aggregation of hIAPP during the time period of observation. An analog of CsgF lacking the N-terminal unstructured region retained the ability to inhibit the aggregation of hIAPP. The nature of the CsgF-hIAPP interaction was probed via fluorescence quenching using a series of single cysteine mutants of CsgF labeled via the individual cysteine side chains with the fluorophore IAEDANS. In the presence of hIAPP, but not in the presence of the non-amyloidogenic rat islet amyloid polypeptide, the fluorophore attached to position of 23 of CsgF was found to be less exposed the quencher acrylamide suggesting that the interaction of hIAPP changes the solvent exposure of the N-terminus of CsgF. Taken together these data suggest that the structured region of CsgF, between residues 66 and 128, is involved in the protein’s interaction with hIAPP and that upon interaction structural changes make the N-terminus less solvent exposed.

Pilicides and curlicides are compounds that block the formation of the virulence factors pili and curli, respectively. To facilitate studies of the interaction between these compounds and the pili and curli assembly systems, fluorescent pilicides and curlicides have been synthesized. This was achieved by using a strategy based on structure-activity knowledge, in which key pilicide and curlicide substituents on the ring-fused dihydrothiazolo 2-pyridone central fragment were replaced by fluorophores. Several of the resulting fluorescent compounds had improved activities as measured in pili- and curli-dependent biofilm assays. We created fluorescent pilicides and curlicides by introducing coumarin and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores at two positions on the peptidomimetic pilicide and curlicide central fragment. Fluorescence images of the uropathogenic Escherichia coli (UPEC) strain UTI89 grown in the presence of these compounds shows that the compounds are strongly associated with the bacteria with a heterogeneous distribution.

Curli fibers are known to be involved in bacterial adhesion to surfaces, cell aggregation, and biofilm formation. Curli belong to a class of fibers known as amyloids. Curli assembly requires 5 proteins: CsgA, CsgB, CsgE, CsgF, and CsgG. CsgA and CsgB are the major structural components of curli. CsgG is an outer membrane protein responsible for the secretion of CsgA and CsgB to the extracellular surface. CsgE and CsgF are thought to be chaperon proteins which are responsible for the transport of CsgA and CsgB in the periplasm. It has been shown that CsgE inhibits the aggregation of CsgA, the main component of curli fibers. However, not much is known about the specific interactions between these two proteins. We employed a series of single cysteine mutants of CsgE and fluorescence quenching to investigate the interaction of CsgE with CsgA and with human islet amyloid polypeptide another amyloidogenic protein unrelated to curli formation.

Gram-negative bacteria assemble functional amyloid surface fibers called curli. CsgB nucleates the major curli subunit protein, CsgA, into a self-propagating amyloid fiber on the cell surface. The CsgG lipoprotein is sufficient for curlin transport across the outer membrane and is hypothesized to be the central molecule of the curli fiber secretion and assembly complex. We tested the hypothesis that the curli secretion protein, CsgG, was restricted to certain areas of the cell to promote the interaction of CsgA and CsgB during curli assembly. Here, electron microscopic analysis of curli-producing strains showed that relatively few cells in the population contacted curli fibers and that curli emanated from spatially discrete points on the cell surface. Microscopic analysis revealed that CsgG was surface exposed and spatially clustered around curli fibers. CsgG localization to the outer membrane and exposure of the surface domain were not dependent on any other csg -encoded protein, but the clustering of CsgG required the csg -encoded proteins CsgE, CsgF, CsgA, and CsgB. CsgG formed stable oligomers in all the csg mutant strains, but these oligomers were distinct from the CsgG complexes assembled in wild-type cells. Finally, we found that efficient fiber assembly was required for the spatial clustering of CsgG. These results suggest a new model where curli fiber formation is spatially coordinated with the CsgG assembly apparatus.

Summary Produced by many Enterobacteriaceae spp., curli are biologically important amyloid fibres that have been associated with biofilm formation, host cell adhesion and invasion, and immune system activation. CsgA is the major fibre subunit and CsgE, CsgF and CsgG are non-structural proteins involved in curli biogenesis. We have characterized the role of CsgG in curli sub- unit secretion across the outer membrane. Directed mutagenesis of CsgG confirmed that its activity is dependent on localization to the outer membrane. Rotary Shadow electron microscopy of purified CsgG suggested that this protein assembles into an oligo- meric complex with an apparent central pore. Oligo- meric CsgG complexes were confirmed using co- purification experiments. Antibiotic sensitivity assays demonstrated that overexpression of CsgG rendered Escherichia coli susceptible to the antibiotic erythro- mycin. A 22-amino-acid sequence at the N-terminus of CsgA was sufficient to direct heterologous proteins to the CsgG secretion apparatus. Finally, we deter- mined that CsgG participates in an outer membrane complex with two other curli assembly proteins, CsgE and CsgF.

Curli are functional amyloid fibers assembled by many Gram-negative bacteria as part of an extracellular matrix that encapsulates the bacteria within a biofilm. A multicomponent secretion system ensures the safe transport of the aggregation-prone curli subunits across the periplasm and outer membrane, and coordinates subunit self-assembly into surface-attached fibers. To avoid the build-up of potentially toxic intracellular protein aggregates, the timing and location of the interactions of the different curli proteins are of paramount importance. Here we review the structural and molecular biology of curli biogenesis, with a focus on the recent breakthroughs in our understanding of subunit chaperoning and secretion. The mechanistic insight into the curli assembly pathway will provide tools for new biotechnological applications and inform the design of targeted inhibitors of amyloid polymerization and biofilm formation.

Curli, an adhesive surface fibre produced by Escherichia coli and salmonellae, was proposed on the basis of genetic evidence to follow a distinct assembly pathway involving an extracellular intermediate of the fibre subunit CsgA, the polymerization of which can be induced at the cell surface by a 'nucleator' protein (CsgB). Here we show biochemically that CsgA is actively secreted to the extracellular milieu and that CsgB is surface located. We demonstrate that the putative curli assembly factor CsgG is an outer membrane-located lipoprotein. CsgG is highly resistant to protease digestion both in vivo and in vitro. During curli assembly, CsgG is required to maintain the stability of CsgA and CsgB. In line with this, it is possible to modulate the steady-state levels of CsgA and CsgB by varying intracellular levels of CsgG. This suggests that, in the absence of CsgG, CsgA and CsgB are proteolytically degraded. Moreover, curli production and steady-state levels of CsgA and CsgB can be increased above wild-type levels by overexpression of CsgG, meaning that the quantity of assembled curli fibres can be controlled by this lipoprotein.

SummaryEnteric bacteria assemble functional amyloid fibers, curli, on their surfaces that share structural and biochemical properties with disease-associated amyloids. Here, we test rationally designed 2-pyridone compounds for their ability to alter amyloid formation of the major curli subunit CsgA. We identified several compounds that discourage CsgA amyloid formation and several compounds that accelerate CsgA amyloid formation. The ability of inhibitor compounds to stop growing CsgA fibers was compared to the same property of the CsgA chaperone, CsgE. CsgE blocked CsgA amyloid assembly and arrested polymerization when added to actively polymerizing fibers. Additionally, CsgE and the 2-pyridone inhibitors prevented biofilm formation by Escherichia coli at the air-liquid interface of a static culture. We demonstrate that curli amyloid assembly and curli-dependent biofilm formation can be modulated not only by protein chaperones, but also by “chemical chaperones.”

Amyloids are highly aggregated proteinaceous fibers historically associated with neurodegenerative conditions including Alzheimers, Parkinsons, and prion-based encephalopathies. Polymerization of amyloidogenic proteins into ordered fibers can be accelerated by preformed amyloid aggregates derived from the same protein in a process called seeding. Seeding of disease-associated amyloids and prions is highly specific and cross-seeding is usually limited or prevented. Here we describe the first study on the cross-seeding potential of bacterial functional amyloids. Curli are produced on the surface of many Gram-negative bacteria where they facilitate surface attachment and biofilm development. Curli fibers are composed of the major subunit CsgA and the nucleator CsgB, which templates CsgA into fibers. Our results showed that curli subunit homologs from Escherichia coli, Salmonella typhimurium LT2, and Citrobacter koseri were able to cross-seed in vitro. The polymerization of Escherichia coli CsgA was also accelerated by fibers derived from a distant homolog in Shewanella oneidensis that shares less than 30% identity in primary sequence. Cross-seeding of curli proteins was also observed in mixed colony biofilms with E. coli and S. typhimurium. CsgA was secreted from E. coli csgB− mutants assembled into fibers on adjacent S. typhimurium that presented CsgB on its surfaces. Similarly, CsgA was secreted by S. typhimurium csgB− mutants formed curli on CsgB-presenting E. coli. This interspecies curli assembly enhanced bacterial attachment to agar surfaces and supported pellicle biofilm formation. Collectively, this work suggests that the seeding specificity among curli homologs is relaxed and that heterogeneous curli fibers can facilitate multispecies biofilm development.

Colonization on a solid surface is influenced by the cell surface appendages such as flagella and curli, of which expressions are regulated by rpoS gene encoding a sigma factor. In this study, we investigated the effect of rpoS or yggE (a rpoS-related and stress-responsive gene) deficiency on the colonization of Escherichia coli BW25113. Under a static condition, the deletion of rpoS or yggE induced 3.9- and 3.7-fold higher colonization as compared to wild-type cells, respectively, on the solid surfaces. However, under a liquid flow condition, only ΔyggE cells maintained the stable colonization on the surface, and the values of cell layer thickness and cell coverage on the surface were 17 and 9.2 times as high as those of wild-type cells, respectively. Gene expression analyses revealed that the deletion of rpoS or yggE positively impacted the expressions of genes involved in flagellum formation. On the other hand, curli assembly was severely prohibited by the rpoS deficiency. Here, we proposed that the plentiful flagella on the ΔrpoS and ΔyggE cell surfaces facilitated mainly the colonization under the static condition. Meanwhile, curli existing on the ΔyggE cell surface played an important role in keeping stable cell attachment and developing attached colonies under the flow stress condition.

Summary Bacteria produce functional amyloid fibers called curli in a controlled, noncytotoxic manner. These extracellular fimbriae enable biofilm formation and promote pathogenicity. Understanding curli biogenesis is important for appreciating microbial lifestyles and will offer clues as to how disease-associated human amyloid formation might be ameliorated. Proteins encoded by the curli specific genes (csgA-G) are required for curli production. We have determined the structure of CsgC and derived the first structural model of the outer-membrane subunit translocator CsgG. Unexpectedly, CsgC is related to the N-terminal domain of DsbD, both in structure and oxido-reductase capability. Furthermore, we show that CsgG belongs to the nascent class of helical outer-membrane macromolecular exporters. A cysteine in a CsgG transmembrane helix is a potential target of CsgC, and mutation of this residue influences curli assembly. Our study provides the first high-resolution structural insights into curli biogenesis., Graphical Abstract Highlights ► CsgC is related to the redox-active N-terminal domain of DsbD ► Outer-membrane transporter CsgG inserts into the membrane via an α-helical oligomer ► The transmembrane domain of CsgG is vital for pore forming and curli assembly ► CsgC appears to affect CsgG pore behavior and biofilm formation

Amyloids are proteinaceous fibers commonly associated with neurodegenerative diseases and prion-based encephalopathies. Many different polypeptides can form amyloid fibers, leading to the suggestion that amyloid is a primitive main chain-dominated structure. A growing body of evidence suggests that amino acid side chains dramatically influence amyloid formation. The specific role fulfilled by side chains in amyloid formation, especially in vivo, remains poorly understood. Here, we determined the role of internally conserved polar and aromatic residues in promoting amyloidogenesis of the functional amyloid protein CsgA, which is the major protein component of curli fibers assembled by enteric bacteria such as Escherichia coli and Salmonella spp. In vivo CsgA polymerization into an amyloid fiber requires the CsgB nucleator protein. The CsgA amyloid core region is composed of five repeating units, defined by regularly spaced Ser, Gln and Asn residues. The results of a comprehensive alanine scan mutagenesis screen showed that Gln and Asn residues at positions 49, 54, 139 and 144 were critical for curli assembly. Alanine substitution of Q49 or N144 impeded the ability of CsgA to respond to CsgB-mediated heteronucleation, and the ability of CsgA to self-polymerize in vitro. However, CsgA proteins harboring these mutations were still seeded by preformed wild-type CsgA fibers in vitro. This suggests that CsgA-fibril-mediated seeding and CsgB-mediated heteronucleation have distinguishable mechanisms. Remarkably, Gln residues at positions 49 and 139 could not be replaced by Asn residues without interfering with curli assembly, suggesting that the side chain requirements were especially stringent at these positions. This analysis demonstrates that bacterial amyloid formation is driven by specific side chain contacts, and provides a clear illustration of the essential roles of specific side chains in promoting amyloid formation.

Amyloid is associated with debilitating human ailments including Alzheimer's and prion diseases. Biochemical, biophysical, and imaging analyses revealed that fibers produced by Escherichia coli called curli were amyloid. The CsgA curlin subunit, purified in the absence of the CsgB nucleator, adopted a soluble, unstructured form that upon prolonged incubation assembled into fibers that were indistinguishable from curli. In vivo, curli biogenesis was dependent on the nucleation-precipitation machinery requiring the CsgE and CsgF chaperone-like and nucleator proteins, respectively. Unlike eukaryotic amyloid formation, curli biogenesis is a productive pathway requiring a specific assembly machinery.

Bacterial adhesion to other bacteria, to eukaryotic cells, and to extracellular matrix proteins is frequently mediated by cell surface-associated polymers (fimbriae) consisting of one or more subunit proteins. We have found that polymerization of curlin to fimbriae-like structures (curli) on the surface of Escherichia coli markedly differs from the prevailing model for fimbrial assembly in that it occurs extracellularly through a self-assembly process depending on a specific nucleator protein. The cell surface-bound nucleator primes the polymerization of curlin secreted by the nucleator-presenting cell or by adjacent cells. The addition of monomers to the growing filament seems to be driven by mass action and guided only by the diffusion gradient between the source of secreted monomer and the surface of monomer condensation.