RESUMO
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.
Assuntos
Amiloide/química , Amiloide/metabolismo , Amiloide/genética , Amiloidose/etiologia , Amiloidose/metabolismo , Amiloidose/patologia , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sobrevivência Celular , Humanos , Agregados Proteicos , Agregação Patológica de Proteínas , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Benzotiazóis/metabolismo , Biofilmes , Vermelho Congo/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Multimerização ProteicaRESUMO
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-ß (Aß) aggregation in vitro and suppress the Alzheimer's-like phenotypes in a transgenic mouse model of Aß 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.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Biofilmes/efeitos dos fármacos , Proteínas de Escherichia coli/química , Escherichia coli/efeitos dos fármacos , Pré-Albumina/farmacologia , Amiloide/antagonistas & inibidores , Amiloide/metabolismo , Proteínas Amiloidogênicas/antagonistas & inibidores , Proteínas Amiloidogênicas/genética , Proteínas Amiloidogênicas/metabolismo , Sítios de Ligação , Biofilmes/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inibidores , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Humanos , Cinética , Pré-Albumina/química , Pré-Albumina/metabolismo , Agregados Proteicos/efeitos dos fármacos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização ProteicaRESUMO
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 catabolite repressor protein (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 ΔcyaA and Δcrp 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 catabolite repressor protein (CRP)-cyclic AMP (cAMP) complex influences the transcription of â¼7% 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 ΔcyaA and Δcrp 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.
Assuntos
Biofilmes , Proteína Receptora de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transativadores/metabolismo , Escherichia coli Uropatogênica/fisiologia , Proteína Receptora de AMP Cíclico/genética , Proteínas de Escherichia coli/genética , Regiões Promotoras Genéticas , Ligação Proteica , Transativadores/genética , Escherichia coli Uropatogênica/genéticaRESUMO
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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Chaperonas Moleculares/metabolismo , Agregação Patológica de Proteínas , alfa-Sinucleína/metabolismo , Animais , Humanos , Camundongos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Multimerização Proteica , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , alfa-Sinucleína/química , alfa-Sinucleína/genéticaRESUMO
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-ß-sheet-rich conformation. Interestingly, CsgC inhibited amyloid assembly of human α-synuclein, but not Aß42, 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 Aß42. 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.
Assuntos
Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacologia , Escherichia coli/genética , Agregados Proteicos/efeitos dos fármacos , alfa-Sinucleína/metabolismo , Motivos de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Sequência de Bases , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Estrutura Secundária de Proteína , alfa-Sinucleína/químicaRESUMO
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.
Assuntos
Amiloide/química , Proteínas Amiloidogênicas/química , Proteínas de Bactérias/química , Transporte Proteico , Amiloide/metabolismo , Proteínas Amiloidogênicas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Dobramento de ProteínaRESUMO
Enteric 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."
Assuntos
Amiloide/química , Biofilmes/efeitos dos fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/farmacologia , Proteínas de Membrana Transportadoras/farmacologia , Chaperonas Moleculares/farmacologia , Piridonas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Amiloide/metabolismo , Biofilmes/crescimento & desenvolvimento , Desenho de Fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Proteínas de Escherichia coli/metabolismo , Cinética , Multimerização Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Piridonas/química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
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 Aß1-40) 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 Aß1-40 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.
RESUMO
Amyloid is a distinct ß-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.
Assuntos
Adesinas Bacterianas/metabolismo , Amiloide/metabolismo , Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Adesinas Bacterianas/química , Amiloide/química , Bactérias/química , Biodiversidade , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Dobramento de Proteína , Multimerização Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Amyloid formation is an ordered aggregation process, where ß-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.
Assuntos
Amiloide/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Periplásmicas/metabolismo , Amiloide/química , Amiloide/ultraestrutura , Proteínas de Bactérias/metabolismo , Escherichia coli/metabolismo , Espectrometria de FluorescênciaRESUMO
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 DNA replication, we examined the subcellular localization of MMR and DNA replication proteins fused to green fluorescent protein (GFP) in live cells, following an increase in DNA replication errors. We demonstrate that foci of the essential DNA polymerase DnaE-GFP decrease following mismatch incorporation and that loss of DnaE-GFP foci requires MutS. 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 DNA replication 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.
Assuntos
Bacillus subtilis/enzimologia , Bacillus subtilis/fisiologia , Reparo de Erro de Pareamento de DNA , DNA Polimerase III/metabolismo , Replicação do DNA , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , DNA Polimerase III/genética , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Gal4 is a prototypical eukaryotic transcriptional activator whose recruitment function is inhibited in the absence of galactose by the Gal80 protein through masking of its transcriptional activation domain (AD). A long-standing nondissociation model posits that galactose-activated Gal3 interacts with Gal4-bound Gal80 at the promoter, yielding a tripartite Gal3-Gal80-Gal4 complex with altered Gal80-Gal4 conformation to enable Gal4 AD activity. Some recent data challenge this model, whereas other recent data support the model. To address this controversy, we imaged fluorescent-protein-tagged Gal80, Gal4, and Gal3 in live cells containing a novel GAL gene array. We find that Gal80 rapidly dissociates from Gal4 in response to galactose. Importantly, this dissociation is Gal3 dependent and concurrent with Gal4-activated GAL gene expression. When galactose-triggered dissociation is followed by galactose depletion, preexisting Gal80 reassociates with Gal4, indicating that sequestration of Gal80 by Gal3 contributes to the observed Gal80-Gal4 dissociation. Moreover, the ratio of nuclear Gal80 to cytoplasmic Gal80 decreases in response to Gal80-Gal3 interaction. Taken together, these and other results provide strong support for a GAL gene switch model wherein Gal80 rapidly dissociates from Gal4 through a mechanism that involves sequestration of Gal80 by galactose-activated Gal3.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Galactoquinase/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Proteínas de Ligação a DNA/efeitos dos fármacos , Galactoquinase/efeitos dos fármacos , Galactose/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Proteínas Repressoras/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Fatores de Transcrição/efeitos dos fármacosRESUMO
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.