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The term amyloid has historically been used to describe fibrillar aggregates formed as the result of protein misfolding and that are associated with a range of diseases broadly termed amyloidoses. The discovery of "functional amyloids" expanded the amyloid umbrella to encompass aggregates structurally similar to disease-associated amyloids but that engage in a variety of biologically useful tasks without incurring toxicity. The mechanisms by which functional amyloid systems ensure nontoxic assembly has provided insights into potential therapeutic strategies for treating amyloidoses. Some of the most-studied functional amyloids are ones produced by bacteria. Curli amyloids are extracellular fibers made by enteric bacteria that function to encase and protect bacterial communities during biofilm formation. Here we review recent studies highlighting microbial functional amyloid assembly systems that are tailored to enable the assembly of non-toxic amyloid aggregates.

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.

Many bacteria can assemble functional amyloid fibers on their cell surface. Most bacterial amyloids contribute to biofilm or other community behaviors where cells interact with a surface or with other cells. Bacterial amyloids, like all functional amyloids, share structural and biochemical properties with disease-associated eukaryotic amyloids. The general ability of amyloids to bind specific dyes, like Congo red and Thioflavin T, and their resistance to denaturation have provided useful tools for scoring and quantifying bacterial amyloid formation. Here, we present basic approaches to study bacterial amyloids by focusing on the well-studied curli amyloid fibers expressed by Enterobacteriaceae. These methods exploit the specific tinctorial and biophysical properties of amyloids. The methods described here are straightforward and can be easily applied by any modern molecular biology lab for the study of other bacterial amyloids.

Functional amyloids, like curli, contribute to biofilm development by uropathogenic Escherichia coli and other Gram-negative bacteria. Biofilms allow pathogens to subvert immune defenses and antibiotic treatments. Therefore, strategies to interrupt bacterial biofilm formation are urgently required. Here, we discovered that human transthyretin potently inhibits biofilm formation by E. coli and Bacillus subtilis. Transthyretin inhibits biofilm formation by preventing curli proteins from adopting the functional amyloid state that supports biofilm development. To our knowledge, this report of amyloid-dependent biofilm inhibition by a human protein with no significant bactericidal or bacteriostatic activity is unique. The observations indicate that a conformational relationship relevant to a set of specific protein–protein interactions, independent of primary sequence, can be functional across biological kingdoms.

The extracellular matrix protects Escherichia coli from immune cells, oxidative stress, predation, and other environmental stresses. Production of the E. coli extracellular matrix is regulated by transcription factors that are tuned to environmental conditions. The biofilm master regulator protein CsgD upregulates curli and cellulose, the two major polymers in the extracellular matrix of uropathogenic E. coli (UPEC) biofilms. We found that cyclic AMP (cAMP) regulates curli, cellulose, and UPEC biofilms through csgD . The alarmone cAMP is produced by adenylate cyclase (CyaA), and deletion of cyaA resulted in reduced extracellular matrix production and biofilm formation. The c atabolite r epressor p rotein (CRP) positively regulated csgD transcription, leading to curli and cellulose production in the UPEC isolate, UTI89. Glucose, a known inhibitor of CyaA activity, blocked extracellular matrix formation when added to the growth medium. The mutant strains Δ cyaA and Δ crp did not produce rugose biofilms, pellicles, curli, cellulose, or CsgD. Three putative CRP binding sites were identified within the csgD-csgB intergenic region, and purified CRP could gel shift the csgD-csgB intergenic region. Additionally, we found that CRP binded upstream of kpsMT , which encodes machinery for K1 capsule production. Together our work shows that cAMP and CRP influence E. coli biofilms through transcriptional regulation of csgD . IMPORTANCE The c atabolite r epressor p rotein (CRP)-cyclic AMP (cAMP) complex influences the transcription of ∼7% of genes on the Escherichia coli chromosome (D. Zheng, C. Constantinidou, J. L. Hobman, and S. D. Minchin, Nucleic Acids Res 32:5874–5893, 2004, https://dx.doi.org/10.1093/nar/gkh908 ). Glucose inhibits E. coli biofilm formation, and Δ cyaA and Δ crp mutants show impaired biofilm formation (D. W. Jackson, J.W. Simecka, and T. Romeo, J Bacteriol 184:3406–3410, 2002, https://dx.doi.org/10.1128/JB.184.12.3406-3410.2002 ). We determined that the cAMP-CRP complex regulates curli and cellulose production and the formation of rugose and pellicle biofilms through csgD . Additionally, we propose that cAMP may work as a signaling compound for uropathogenic E. coli (UPEC) to transition from the bladder lumen to inside epithelial cells for intracellular bacterial community formation through K1 capsule regulation.

Amyloid is a distinct β-sheet-rich fold that many proteins can acquire. Frequently associated with neurodegenerative diseases in humans, including Alzheimer's, Parkinson's and Huntington's diseases, amyloids are traditionally considered the product of protein misfolding. However, the amyloid fold is now recognized as a ubiquitous part of normal cellular biology. Functional amyloids have been identified in nearly all facets of cellular life, with microbial functional amyloids leading the way. Unlike disease-associated amyloids, functional amyloids are assembled by dedicated, directed pathways and ultimately perform a physiological function that benefits the organism. The evolved amyloid assembly and disassembly pathways of microbes have provided novel insights into how cells have harnessed the amyloid assembly process for productive means. An understanding of functional amyloid biogenesis promises to provide a fresh perspective on the molecular events that underlie disease-associated amyloidogenesis. Here, we review functional microbial amyloids with an emphasis on curli fibers and their role in promoting biofilm formation and other community behaviors.

Summary Mismatch repair (MMR) corrects DNA polymerase errors occurring during genome replication. MMR is critical for genome maintenance, and its loss increases mutation rates several hundred fold. Recent work has shown that the interaction between the mismatch recognition protein MutS and the replication processivity clamp is important for MMR in Bacillus subtilis. To further understand how MMR is coupled to DNAreplication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNAreplication errors. We demonstrate that foci of the essential DNA polymerase DnaE‐GFP decrease following mismatch incorporation and that lossofDnaE‐GFPfocirequiresMutS.Furthermore,we show that MutS and MutL bind DnaE in vitro, suggesting that DnaE is coupled to repair. We also found that DnaE‐GFP foci decrease in vivo following a DNA damage-independent arrest of DNA synthesis showing that loss of DnaE‐GFP foci is caused by perturbations to DNA replication. We propose that MutS directly contacts the DNAreplication machinery, causing a dynamic change in the organization of DnaE at the replication fork during MMR. Our results establish a striking and intimate connection between MMR and the replicating DNA polymerase complex in vivo.

Chromatin serves as a regulator of various nuclear processes, with post-translational modifications of histone proteins serving as modulators to influence chromatin function. We have previously shown that histone H3 K79 methylation is important for repair of UV-induced DNA damage in Saccharomyces cerevisiae, acting through multiple repair pathways. To evaluate the potential role of distinct K79 methylation states in DNA repair, we identified four mutations in histone H3 that confer sensitivity to UV, each of which also has a distinct effect on specific K79 methylation states. Epistasis analyses indicate that each mutation exerts its phenotypic effects through distinct subsets of the various DNA damage response pathways, suggesting the existence of discrete roles for histone H3 in DNA damage checkpoint and repair pathways. Furthermore, we find that the distribution of K79 methylation states is altered by mutation of the acetylatable N terminal lysines in histone H4. The combined results suggest that K79 methylation states may be modulated in response to UV damage via a trans-histone regulatory pathway, and that distinct methylation states may provide a means of coordinating specific DNA repair and damage checkpoint pathways.

Amyloid formation is historically associated with cytotoxicity, but many organisms produce functional amyloid fibers (e.g., curli) as a normal part of cell biology. Two E. coli genes in the curli operon encode the chaperone-like proteins CsgC and CsgE that both can reduce in vitro amyloid formation by CsgA. CsgC was also found to arrest amyloid formation of the human amyloidogenic protein α-synuclein, which is involved in Parkinson’s disease. Here, we report that the inhibitory effects of CsgC arise due to transient interactions that promote the formation of spherical α-synuclein oligomers. We find that CsgE also modulates α-synuclein amyloid formation through transient contacts but, in contrast to CsgC, CsgE accelerates α-synuclein amyloid formation. Our results demonstrate the significance of transient protein interactions in amyloid regulation and emphasize that the same protein may inhibit one type of amyloid while accelerating another.

Curli are extracellular functional amyloids that are assembled by enteric bacteria during biofilm formation and host colonization. An efficient secretion system and chaperone network ensures that the major curli fiber subunit, CsgA, does not form intracellular amyloid aggregates. We discovered that the periplasmic protein CsgC was a highly effective inhibitor of CsgA amyloid formation. In the absence of CsgC, CsgA formed toxic intracellular aggregates. In vitro, CsgC inhibited CsgA amyloid formation at substoichiometric concentrations and maintained CsgA in a non-β-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aβ42, in vitro. We identified a common D-Q-Φ-X0,1-G-K-N-ζ-E motif in CsgC client proteins that is not found in Aβ42. CsgC is therefore both an efficient and selective amyloid inhibitor. Dedicated functional amyloid inhibitors may be a key feature that distinguishes functional amyloids from disease-associated amyloids.

Many bacteria assemble extracellular amyloid fibers on their cell surface. Secretion of proteins across membranes and the assembly of complex macromolecular structures must be highly coordinated to avoid the accumulation of potentially toxic intracellular protein aggregates. Extracellular amyloid fiber assembly poses an even greater threat to cellular health due to the highly aggregative nature of amyloids and the inherent toxicity of amyloid assembly intermediates. Therefore, temporal and spatial control of amyloid protein secretion is paramount. The biogenesis and assembly of the extracellular bacterial amyloid curli is an ideal system for studying how bacteria cope with the many challenges of controlled and ordered amyloid assembly. Here, we review the recent progress in the curli field that has made curli biogenesis one of the best-understood functional amyloid assembly pathways. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

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.”

Fragments of prostatic acid phosphatase (PAP248-286) in human semen dramatically increase HIV infection efficiency by increasing virus adhesion to target cells. PAP248-286 only enhances HIV infection in the form of amyloid aggregates termed SEVI (Semen Enhancer of Viral Infection), however monomeric PAP248-286 aggregates very slowly in isolation. It has therefore been suggested that SEVI fiber formation in vivo may be promoted by exogenous factors. We show here that a bacterially-produced extracellular amyloid (curli or Csg) acts as a catalytic agent for SEVI formation from PAP248-286 at low concentrations in vitro, producing fibers that retain the ability to enhance HIV (Human Immunodeficiency Virus) infection. Kinetic analysis of the cross-seeding effect shows an unusual pattern. Cross-seeding PAP248-286 with curli only moderately affects the nucleation rate while significantly enhancing the growth of fibers from existing nuclei. This pattern is in contrast to most previous observations of cross-seeding, which show cross-seeding partially bypasses the nucleation step but has little effect on fiber elongation. Seeding other amyloidogenic proteins (IAPP (islet amyloid polypeptide) and Aβ1-40) with curli showed varied results. Curli cross-seeding decreased the lag-time of IAPP amyloid formation but strongly inhibited IAPP elongation. Curli cross-seeding exerted a complicated concentration dependent effect on Aβ1-40 fibrillogenesis kinetics. Combined, these results suggest that the interaction of amyloidogenic proteins with preformed fibers of a different type can take a variety of forms and is not limited to epitaxial nucleation between proteins of similar sequence. The ability of curli fibers to interact with proteins of dissimilar sequences suggests cross-seeding may be a more general phenomenon than previously supposed.

Amyloid formation is an ordered aggregation process, where β-sheet rich polymers are assembled from unstructured or partially folded monomers. We examined how two Escherichia coli cytosolic chaperones, DnaK and Hsp33, and a more recently characterized periplasmic chaperone, Spy, modulate the aggregation of a functional amyloid protein, CsgA. We found that DnaK, the Hsp70 homologue in E. coli, and Hsp33, a redox-regulated holdase, potently inhibited CsgA amyloidogenesis. The Hsp33 anti-amyloidogenesis activity was oxidation dependent, as oxidized Hsp33 was significantly more efficient than reduced Hsp33 at preventing CsgA aggregation. When soluble CsgA was seeded with preformed amyloid fibers, neither Hsp33 nor DnaK were able to efficiently prevent soluble CsgA from adopting the amyloid conformation. Moreover, both DnaK and Hsp33 increased the time that CsgA was reactive with the amyloid oligomer conformation-specific A11 antibody. Since CsgA must also pass through the periplasm during secretion, we assessed the ability of the periplasmic chaperone Spy to inhibit CsgA polymerization. Like DnaK and Hsp33, Spy also inhibited CsgA polymerization in vitro. Overexpression of Spy resulted in increased chaperone activity in periplasmic extracts and in reduced curli biogenesis in vivo. We propose that DnaK, Hsp33 and Spy exert their effects during the nucleation stages of CsgA fibrillation. Thus, both housekeeping and stress induced cytosolic and periplasmic chaperones may be involved in discouraging premature CsgA interactions during curli biogenesis.

Direct masking of the activation domain (AD) of a transcriptional activator by an inhibitory protein and relief of such masking in response to signals is typical for several eukaryotic gene regulatory systems. Such is the case for the transcriptional inhibitors RB, MDM4/MDMX, ZFM1, Opi1, and Gal80, all of which target DNA-binding transcription activators to exert their inhibitory effect (5, 6, 16, 33). In the case of RB, it binds to a site embedded in the transactivation domain of the E2F protein. Phosphorylation of RB lowers its binding affinity to E2F and results in gene activation (6). For the Opi1 protein in Saccharomyces cerevisiae, signal-responsive tethering of Opi1 to the ER membrane physically separates Opi1 and its target, Ino2, the transcriptional activator protein involved in the inositol pathway (12). For the yeast Gal80 protein, its ability to mask the AD of the transcriptional activator Gal4 is somehow overcome through interaction with the galactose-activated form of the Gal3 protein. For each of these systems, the molecular activities that cause the unmasking event constitute the overall signal-responsive gene activation mechanism. In our efforts to elucidate the mechanism of GAL gene activation in yeast, we have focused on how galactose relieves Gal80 masking of the Gal4 AD. Gal4 is a prototypical acidic transcriptional activator that binds to a 17-bp upstream activation sequence within the GAL gene promoters (UASGAL) (3, 7). In the absence of galactose, Gal80 masks the Gal4 AD, preventing recruitment of the transcriptional machinery (13-15). In the presence of galactose and ATP, Gal3 physically interacts with Gal80, and such interaction is required for relieving the Gal80 inhibition of Gal4, resulting in induced GAL gene expression (2, 26). However, how the Gal3-Gal80 interaction leads to Gal4 activation has been unresolved. There are two contrasting bodies of evidence concerning how the Gal80-Gal3 interaction relieves Gal80 inhibition of Gal4. Historically, two independent studies led to the view that Gal80 remains associated with Gal4 at the promoter in galactose-induced cells (11, 21). The initial evidence came from experiments that used a Gal80-VP16 hybrid protein in which the transcriptional AD of VP16 was fused to Gal80. Gal80-VP16 was found to stimulate transcription of a GAL reporter gene in the presence of galactose, indicating that Gal80 stayed bound to Gal4 at the GAL gene promoter after induction (11). Later, others using a constitutive mutant of Gal3 protein at a 30× excess relative to Gal80 detected a complex of Gal3-Gal80-Gal4 associated with a UASGAL-containing DNA fragment in an electrophoresis mobility shift assay (21). Taken together, these data supported a nondissociation model proposing that galactose-activated Gal3 binds to Gal4-associated Gal80 at the GAL gene promoter in the nucleus and causes the Gal80-Gal4 complex to adopt a conformation that exposes the Gal4 AD (21). Challenging the nondissociation model is a more recent body of evidence that points to dissociation of Gal80 from Gal4 after induction. Gal3 was detectable by two different methods only in the cytoplasm, and cells in which Gal3 was tethered to membranes outside the nucleus exhibited a magnitude of induction similar to that exhibited by wild-type cells (18). In addition, chromatin immunoprecipitation experiments revealed reduced binding of Gal80 to Gal4 after galactose induction (19). Accordingly, a dissociation model was proposed, in which the binding of Gal80 to cytoplasm-localized Gal3 results in a decrease of Gal80 content in the nucleus, leading to its dissociation from Gal4 (19). However, Gal80 dissociation from Gal4 has in turn been called into question by the report of a fluorescence resonance energy transfer (FRET) between Gal80-enhanced cyan fluorescent protein (ECFP) and Gal4-enhanced yellow fluorescent protein (EYFP) in galactose-grown yeast (1). Here we present the results of new experiments aimed at resolving the conflicting models for how galactose triggers relief of Gal80 inhibition of Gal4. We demonstrate with the use of a GAL1 promoter-controlled reporter gene array and live-cell imaging that Gal80 rapidly dissociates from Gal4 in response to galactose. Our results further show that such dissociation depends on interaction between Gal3 and Gal80 and is temporally correlated with GAL reporter gene expression. We also find that Gal80 is able to reassociate with Gal4 when galactose is depleted and protein synthesis is blocked, suggesting that reversible binding of Gal80 by Gal3 contributes to the galactose-triggered Gal80-Gal4 dissociation event. We also detect a modest redistribution of Gal80 from the nucleus to the cytoplasm by 15 to 25 min following galactose addition. Finally, we provide here the first evidence that Gal3 is detectable within the nucleus before and after galactose addition. Based on these results, we conclude that the dissociation of Gal80 from Gal4 by Gal80 interaction with Gal3 is the event that initiates the active state of Gal4.