The Role of Functional Amyloids in Bacterial Virulence

Amyloid fibrils are best known as a product of human and animal protein misfolding disorders, where amyloid formation is associated with cytotoxicity and disease. It is now evident that for some proteins, the amyloid state constitutes the native structure and serves a functional role. These functional amyloids are proving widespread in bacteria and fungi, fulfilling diverse functions as structural components in biofilms or spore coats, as toxins and surface-active fibers, as epigenetic material, peptide reservoirs or adhesins mediating binding to and internalization into host cells. In this review, we will focus on the role of functional amyloids in bacterial pathogenesis. The role of functional amyloids as virulence factor is diverse but mostly indirect. Nevertheless, functional amyloid pathways deserve consideration for the acute and long-term effects of the infectious disease process and may form valid antimicrobial targets.     

Prion-based nanomaterials and their emerging applications

ABSTRACT

Protein misfolding and aggregation into highly ordered fibrillar structures have been traditionally associated with pathological processes. Nevertheless, nature has taken advantage of the particular properties of amyloids for functional purposes, like in the protection of organisms against environmental changing conditions. Over the last decades, these fibrillar structures have inspired the design of new nanomaterials with intriguing applications in biomedicine and nanotechnology such as tissue engineering, drug delivery, adhesive materials, biodegradable nanocomposites, nanowires or biosensors. Prion and prion-like proteins, which are considered a subclass of amyloids, are becoming ideal candidates for the design of new and tunable nanomaterials. In this review, we discuss the particular properties of this kind of proteins, and the current advances on the design of new materials based on prion sequences.     

When microbial biotechnology meets material engineering

Bacterial biopolymers such as bacterial cellulose (BC), alginate or polyhydroxyalkanotes (PHAs) have aroused the interest of researchers in many fields, for instance biomedicine and packaging, due to their being biodegradable, biocompatible and renewable. Their properties can easily be tuned by means of microbial biotechnology strategies combined with materials science. This provides them with highly diverse properties, conferring them non-native features. Herein we highlight the enormous structural diversity of these macromolecules, how are they produced, as well as their wide range of potential applications in our daily lives. The emergence of new technologies, such as synthetic biology, enables the creation of next-generation-advanced materials presenting smart functional properties, for example the ability to sense and respond to stimuli as well as the capacity for self-repair. All this has given rise to the recent emergence of biohybrid materials, in which a synthetic component is brought to life with living organisms. Two different subfields have recently garnered particular attention: hybrid living materials (HLMs), such as encapsulation or bioprinting, and engineered living materials (ELMs), in which the material is created bottom-up with the use of microbial biotechnology tools. Early studies showed the strong potential of alginate and PHAs as HLMs, whilst BC constituted the most currently promising material for the creation of ELMs.     

Engineering biological systems with synthetic RNA molecules

RNA molecules play diverse functional roles in natural biological systems. There has been growing interest in designing synthetic RNA counterparts for programming biological function. The design of synthetic RNA molecules that exhibit diverse activities, including sensing, regulatory, information processing, and scaffolding activities, has highlighted the advantages of RNA as a programmable design substrate. Recent advances in implementing these engineered RNA molecules as key control elements in synthetic genetic networks are highlighting the functional relevance of this class of synthetic elements in programming cellular behaviors.     

合成生物学技术在材料科学中的应用

材料是人类赖以生存与发展的物质基础,科技和社会的进步都离不开材料技术的发展,未来先进材料的合成和制备必然朝着绿色可持续、低耗高产出、精细可调控、高效多功能的方向发展。以"基因调控·工程设计"为核心的合成生物学技术从分子、细胞层面极大地推动了生命科学的发展,也已经并继续为材料科学的发展注入新的思路和活力。本文将围绕合成生物学技术在材料科学中的应用,以基因回路设计为核心,概念应用为线索,重点介绍合成生物学技术在高分子生物材料和无机纳米材料领域的开发和生产,细胞展示和蛋白定向进化战略对分子材料的筛选和优化,"活体"功能材料、工程菌调节的人工光合系统功能材料体系以及基因回路在材料科学中的应用。     

Engineered living materials (ELMs) design: From function allocation to dynamic behavior modulation

Natural materials possess many distinctive “living” attributes, such as self-growth, self-healing, environmental responsiveness, and evolvability, that are beyond the reach of many existing synthetic materials. The emerging field of engineered living materials (ELMs) takes inspiration from nature and harnesses engineered living systems to produce dynamic and responsive materials with genetically programmable functionalities. Here, we identify and review two main directions for the rational design of ELMs: first, engineering of living materials with enhanced performances by incorporating functional material modules, including engineered biological building blocks (proteins, polysaccharides, and nucleic acids) or well-defined artificial materials; second, engineering of smart ELMs that can sense and respond to their surroundings by programming dynamic cellular behaviors regulated via cell–cell or cell–environment interactions. We next discuss the strengths and challenges of current ELMs and conclude by providing a perspective of future directions in this promising area.     

Precision materials: Computational design methods of accurate protein materials

Nature has evolved a vast repertoire of structures and functions based on an ordered, orchestrated, protein building-blocks assembly. For decades these sophisticated materials have been studied, mimicked, and repurposed, yet recently, computational protein engineering methods provided an alternative route: creating protein materials de-novo, surpassing evolutionary constraints and optimized for specific tasks. We highlight two areas of research that fundamentally accelerate design of structurally well-defined programmable protein materials. First, implementations of hierarchical assembly and geometric sampling (docking) strategies to create designable backbones under pre-specified symmetry constraints. Second, progress in protein–protein interfaces and sequence design methods, using Rosetta, that drive programmable supramolecular assemblies. These approaches have proven effective in generating diverse protein assemblies in 0-, 1-, 2-, and 3-dimensional architectures (constituting single or multiple components), and as part of a synthetic or a biological system. We expect these methods shall transform the toolbox of protein designers developing next generation synthetic and biological materials.     

Amyloid by Design: Intrinsic Regulation of Microbial Amyloid Assembly

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.     

In vitro polymerization of a functional Escherichia coli amyloid protein

Amyloid formation is characterized by the conversion of soluble proteins into biochemically and structurally distinct fibers. Although amyloid formation is traditionally associated with diseases such as Alzheimer disease, a number of biologically functional amyloids have recently been described. Curli are amyloid fibers produced by Escherichia coli that contribute to biofilm formation and other important physiological processes. We characterized the polymerization properties of the major curli subunit protein CsgA. CsgA polymerizes into an amyloid fiber in a sigmoidal kinetic fashion with a distinct lag, growth, and stationary phase. Adding sonicated preformed CsgA fibers to the polymerization reaction can significantly shorten the duration of the lag phase. We also demonstrate that the conversion of soluble CsgA into an insoluble fiber involves the transient formation of an intermediate similar to that characterized for several disease-associated amyloids. The CsgA core amyloid domain can be divided into five repeating units that share sequence and structural hallmarks. We show that peptides representing three of these repeating units are amyloidogenic in vitro. Although the defining characteristics of CsgA polymerization appear conserved with disease-associated amyloids, these proteins evolved in diverse systems and for different purposes. Therefore, amyloidogenesis appears to be an innate protein folding pathway that can be capitalized on to fulfill normal physiological tasks.     

SynAggreg: A Multifunctional High-Throughput Technology for Precision Study of Amyloid Aggregation and Systematic Discovery of Synergistic Inhibitor Compounds

Numerous proteins can coalesce into amyloid self-assemblies, which are responsible for a class of diseases called amyloidoses, but which can also fulfill important biological functions and are of great interest for biotechnology. Amyloid aggregation is a complex multi-step process, poorly prone to detailed structural studies. Therefore, small molecules interacting with amyloids are often used as tools to probe the amyloid aggregation pathway and in some cases to treat amyloidoses as they prevent pathogenic protein aggregation. Here, we report on SynAggreg, an in vitro high-throughput (HT) platform dedicated to the precision study of amyloid aggregation and the effect of modulator compounds. SynAggreg relies on an accurate bi-fluorescent amyloid-tracer readout that overcomes some limitations of existing HT methods. It allows addressing diverse aspects of aggregation modulation that are critical for pathomechanistic studies, such as the specificity of compounds toward various amyloids and their effects on aggregation kinetics, as well as the co-assembly propensity of distinct amyloids and the influence of prion-like seeding on self-assembly. Furthermore, SynAggreg is the first HT technology that integrates tailored methodology to systematically identify synergistic compound combinations—an emerging strategy to improve fatal amyloidoses by targeting multiple steps of the aggregation pathway. To this end, we apply analytical combinatorial scores to rank the inhibition efficiency of couples of compounds and to readily detect synergism. Finally, the SynAggreg platform should be suited for the characterization of a broad class of amyloids, whether of interest for drug development purposes, for fundamental research on amyloid functions, or for biotechnological applications.     

Diversity, biogenesis and function of microbial amyloids

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.     

Designed inhibitors to reduce amyloid virulence and cytotoxicity and combat neurodegenerative and infectious diseases

The review highlights the role of amyloids in various diseases and the challenges associated with targeting human amyloids in therapeutic development. However, due to the better understanding of microbial amyloids' role as virulence factors, there is a growing interest in repurposing and designing anti-amyloid compounds for antivirulence therapy. The identification of amyloid inhibitors has not only significant clinical implications but also provides valuable insights into the structure and function of amyloids. The review showcases small molecules and peptides that specifically target amyloids in both humans and microbes, reducing cytotoxicity and biofilm formation, respectively. The review emphasizes the importance of further research on amyloid structures, mechanisms, and interactions across all life forms to yield new drug targets and improve the design of selective treatments. Overall, the review highlights the potential for amyloid inhibitors in therapeutic development for both human diseases and microbial infections.     

Amyloids of multiple species: are they helpful in survival?

Amyloids are primarily known for their roles in neurodegenerative disorders, as well as in systemic diseases like diabetes. Evolutionary forces tend to maintain a healthy set of heritable characteristics, while eliminating toxic or unfavourable elements; but amyloids seem to represent an exception to this fundamental concept. In addition to their presence in mammals, amyloids also persist in the proteome of many lower organisms that may be linked with possible roles in survival, which are still unexplored. Herein, we address some unanswered questions regarding amyloids: are these well‐structured proteinaceous aggregates a by‐product of inefficient folding events, or have they been retained in our protein repertoire for as yet unknown functional roles; and how do protein misfolding and associated disorders originate, despite the presence of protein quality‐control systems inside the cells? This review aims to extend our current understanding about the multifaceted useful properties of amyloids and their functional interactions with other molecular pathways in various species; this may provide new insights to identify novel therapeutic strategies for ageing and neurodegenerative diseases.     

On the evolutionary trajectories of signal-transducing amyloids in fungi and beyond

In the last decade, multiple reports have established that amyloids can bear important functional roles in a variety of biological processes and in distant taxonomic clades. In filamentous fungi, amyloids are involved in a signal transducing mechanism in which a group of NOD-like receptors (NLRs) controls downstream effector proteins to induce a programmed cell death reaction. A structurally characterized example of fungal signal-transducing amyloid is the prion-forming domain (PFD) of the HET-S toxin from Podospora anserina. Amyloid-mediated programmed cell death is equally reported in metazoans in the context of innate immunity and antiviral response. The cell death reaction, described as programmed necrosis, is dependent on an amyloid-forming RHIM motif (RIP homotypic interaction motif). An evolutionary link between the RHIM and the PFD signaling amyloids has been previously reported. Our recent study ties further the signaling amyloids in fungi and metazoans, reporting a fungal signal-transducing domain with amyloid and prion-like properties, which shows significant sequence similarity to the metazoan RHIM motif. Here, I discuss the expanding class of the signal-transducing amyloids and reflect on the possible evolutionary scenarios of their diversification.     

Reverse β-oxidation pathways for efficient chemical production

Microbial production of fuels, chemicals, and materials has the potential to reduce greenhouse gas emissions and contribute to a sustainable bioeconomy. While synthetic biology allows readjusting of native metabolic pathways for the synthesis of desired products, often these native pathways do not support maximum efficiency and are affected by complex regulatory mechanisms. A synthetic or engineered pathway that allows modular synthesis of versatile bioproducts with minimal enzyme requirement and regulation while achieving high carbon and energy efficiency could be an alternative solution to address these issues. The reverse β-oxidation (rBOX) pathways enable iterative non-decarboxylative elongation of carbon molecules of varying chain lengths and functional groups with only four core enzymes and no ATP requirement. Here, we describe recent developments in rBOX pathway engineering to produce alcohols and carboxylic acids with diverse functional groups, along with other commercially important molecules such as polyketides. We discuss the application of rBOX beyond the pathway itself by its interfacing with various carbon-utilization pathways and deployment in different organisms, which allows feedstock diversification from sugars to glycerol, carbon dioxide, methane, and other substrates.     

Polymerizing the fibre between bacteria and host cells: the biogenesis of functional amyloid fibres

Amyloid fibres are proteinaceous aggregates associated with several human diseases, including Alzheimer's, Huntington's and Creutzfeldt Jakob's. Disease-associated amyloid formation is the result of proteins that misfold and aggregate into β sheet-rich fibre polymers. Cellular toxicity is readily associated with amyloidogenesis, although the molecular mechanism of toxicity remains unknown. Recently, a new class of 'functional' amyloid fibres was discovered that demonstrates that amyloids can be utilized as a productive part of cellular biology. These functional amyloids will provide unique insights into how amyloid formation can be controlled and made less cytotoxic. Bacteria produce some of the best-characterized functional amyloids, including a surface amyloid fibre called curli. Assembled by enteric bacteria, curli fibres mediate attachment to surfaces and host tissues. Some bacterial amyloids, like harpins and microcinE492, have exploited amyloid toxicity in a directed and functional manner. Here, we review and discuss the functional amyloids assembled by bacteria. Special emphasis will be paid to the biology of functional amyloid synthesis and the connections between bacterial physiology and pathology.     

Functional diversity of protein fibrillar aggregates from physiology to RNA granules to neurodegenerative diseases

Many proteins exhibit propensities to form fibrillar aggregates called amyloids that are rich in β-sheet structures. Abnormal accumulation of amyloids in the brain and spinal cords is well known as a major pathological change in neurodegenerative diseases; therefore, amyloids have long been considered as disease culprits formed via protein misfolding and should be avoided in healthy cells. Recently, however, increasing numbers of proteins have been identified that require formation of fibrillar states for exertion of their physiological functions, and the critical roles of such functional amyloids include a molecular switch for environmental adaptation, a structural template for catalysis, and a regulator of intracellular signaling. Protein amyloids will, therefore, be more prevailed in our physiologies than we have expected so far. Here, we have reviewed recent studies on such regulatory roles of protein fibrillar aggregates in various physiologies and further discussed possible relations of functional to pathological amyloids.     

AlphaFold and the amyloid landscape

Protein aggregation is a widespread phenomenon with important implications in many scientific areas. Although amyloid formation is typically considered as detrimental, functional amyloids that perform physiological roles have been identified in all kingdoms of life. Despite their functional and pathological relevance, the structural details of the majority of molecular species involved in the amyloidogenic process remains elusive. Here, we explore the application of AlphaFold, a highly accurate protein structure predictor, in the field of protein aggregation. While we envision a straightforward application of AlphaFold in assisting the design of globular proteins with improved solubility for biomedical and industrial purposes, the use of this algorithm for predicting the structure of aggregated species seems far from trivial. First, in amyloid diseases, the presence of multiple amyloid polymorphs and the heterogeneity of aggregation intermediates challenges the “one sequence, one structure” paradigm, inherent to sequence-based predictions. Second, aberrant aggregation is not the subject of positive selective pressure, precluding the use of evolutionary-based approaches, which are the core of the AlphaFold pipeline. Instead, amyloid polymorphism seems to be constrained by the need for a defined structure-activity relationship in functional amyloids. They may thus provide a starting point for the application of AlphaFold in the amyloid landscape.     

Synthetic biology advancing clinical applications

The 'omics' era, with its identification of genetic and protein components, has combined with systems biology, which provided insights into network structures, to set the stage for synthetic biology, an emerging interdisciplinary life science that uses engineering principles. By capitalizing on an iterative design cycle that involves molecular and computational biology tools to assemble functional designer devices from a comprehensive catalogue of standardized biological components with predictable functions, synthetic biology has significantly advanced our understanding of complex control dynamics that program living systems. Such insights, collected over the past decade, are priming a variety of synthetic biology-inspired biomedical applications that have the potential to revolutionize drug discovery and production technologies, as well as treatment strategies for infectious diseases and metabolic disorders.     

Degradation of fungal prion HET-s(218-289) induces formation of a generic amyloid fold

The prion-forming domain of the fungal prion protein HET-s, HET-s(218-289), is known from solid-state NMR studies to have a β-solenoidal structure; the β-solenoid has the cross-β structure characteristic of all amyloids, but is inherently more complex than the generic stacked β-sheets found in studies of small synthetic peptides. At low pH HET-s(218-289) has also been reported to form an alternative structure, which has not been characterized. We have confirmed by x-ray fiber diffraction that HET-s(218-289) adopts a β-solenoidal structure at neutral pH, and shown that at low pH, it forms either a β-solenoid or a stacked β-sheet structure, depending on the integrity of the protein and the conditions of fibrillization. The low pH stacked-sheet structure is usually formed only by proteolyzed HET-s(218-289), but intact HET-s(218-289) can form stacked sheets when seeded with proteolyzed stacked-sheet HET-s(218-289). The polymorphism of HET-s parallels the structural differences between the infectious brain-derived and the much less infectious recombinant mammalian prion protein PrP. Taken together, these observations suggest that the functional or pathological forms of amyloid proteins are more complex than the simple generic stacked-sheet amyloids commonly formed by short peptides.