Bacterial Amyloid Research Articles

Molecular Architecture and Helicity of Bacterial Amyloid Nanofibers: Implications for the Design of Nanoscale Antibiotics

Molecular Architecture and Helicity of Bacterial Amyloid Nanofibers: Implications for the Design of Nanoscale Antibiotics

The Amyloid Region of Hfq Riboregulator Promotes DsrA:rpoS RNAs Annealing.

Simple SummaryRNA:amyloid protein interactions have been observed in the past few years. Nevertheless, the molecular basis and physiological implications of these interactions are still poorly understood. Here we focus on a bacterial amyloid protein, Hfq. This protein is a pleiotropic bacterial regulator that mediates many aspects of RNA metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small noncoding regulatory RNAs. This regulation contributes to bacterial adaptation to stresses. Our results show that the amyloid region of Hfq significantly influences the efficiency of annealing between DsrA small noncoding RNA to its target mRNA. This unexpected result opens perspectives for a novel physiological role of amyloids, including those associated with neurodegenerative diseases.Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed.

Designing multi-epitope vaccine candidates against functional amyloids in Pseudomonas aeruginosa through immunoinformatic and structural bioinformatics approach.

Pseudomonas aeruginosa (P. aeruginosa) displays high drug resistance and biofilm-mediated adaptability, which makes its infections difficult to treat. Alternative intervention methods and targets have made such infections treatment manageable. One of the biofilm components, functional amyloids of Pseudomonas (Fap) is correlated positively with virulence and mucoidy phenotype found in infection in cystic fibrosis (CF) patients. Extracellular accessibility, conservation across P. aeruginosa isolates and linkage with lung infections phenotype in CF patients, makes Fap a promising intervention target. Furthermore, the reported effect of bacterial amyloid on neuronal function and immune response makes it a targetable candidate. In the current study, Fap C protein and its immediate interactions were explored to extract antigenic T-cell and B-cell epitopes. A combination of epitopes and peptide adjuvants has been linked to derive vaccine candidate structures. The vaccine candidates were validated for antigenicity, allergenicity, physiochemical properties, stability and interactions with TLRs and MHC alleles. Immunosimulation studies have demonstrated that vaccines elicit Th1 dominated response, which can assist in good prognosis of infection in CF patients.

Genome-wide screen identifies curli amyloid fibril as a bacterial component promoting host neurodegeneration

Growing evidence indicates that gut microbiota play a critical role in regulating the progression of neurodegenerative diseases such as Parkinson's disease. The molecular mechanism underlying such microbe-host interaction is unclear. In this study, by feeding Caenorhabditis elegans expressing human α-syn with Escherichia coli knockout mutants, we conducted a genome-wide screen to identify bacterial genes that promote host neurodegeneration. The screen yielded 38 genes that fall into several genetic pathways including curli formation, lipopolysaccharide assembly, and adenosylcobalamin synthesis among others. We then focused on the curli amyloid fibril and found that genetically deleting or pharmacologically inhibiting the curli major subunit CsgA in E. coli reduced α-syn-induced neuronal death, restored mitochondrial health, and improved neuronal functions. CsgA secreted by the bacteria colocalized with α-syn inside neurons and promoted α-syn aggregation through cross-seeding. Similarly, curli also promoted neurodegeneration in C. elegans models of Alzheimer's disease, amyotrophic lateral sclerosis, and Huntington's disease and in human neuroblastoma cells.

Human Fibrinogen Inhibits Amyloid Assembly of Most Phenol-Soluble Modulins from Staphylococcus aureus.

Functional amyloids are highly organized protein/peptide structures that inter alia promote biofilm formation in different bacteria. One such example is provided by a family of 20–45 residue-long peptides called phenol-soluble modulins (PSMs) from Staphylococcus aureus. External components such as eukaryotic host proteins, which alter self-assembly of bacterial amyloids, can affect the biofilm matrix. Here, we studied the effect of the highly prevalent human plasma protein fibrinogen (Fg) on fibrillation of PSMs. Fg inhibits or suppresses fibrillation of most PSMs tested (PSMα1, PSMβ1, and PSMβ2) except for PSMα3, whose already rapid aggregation is accelerated even further by Fg but leads to amorphous β-rich aggregates rather than fibrils. Fg also induces PSMβ2 to form amorphous aggregates and diverts PSMα1 into off-pathway oligomers which consist of both Fg and PSMα1 and cannot seed fibrillation. Peptide arrays showed that Fg bound to the N-terminus of PSMα1, while it bound to the entire length of PSMα3 (except the C terminus) and to the C-termini of PSMβ1 and PSMβ2. The latter peptides are all positively charged, while Fg is negatively charged at physiological pH. The positive charges complement Fg’s net negative charge of −7.6 at pH 7.4. Fg’s ability to inhibit PSM fibrillation reveals a potential host-defense mechanism to prevent bacterial biofilm growth and infections in the human body.

Design and Engineering of Amyloid Aggregation‐Prone Fragments and Their Antimicrobial Conjugates with Multi‐Target Functionality (Adv. Funct. Mater. 32/2021)

Amyloid Aggregation In article number 2102978, Jie Zheng and co-workers provide a new “kill two birds with one stone” model to not only reconcile with both the “amyloid cascade hypothesis” and “microbial infection hypothesis”, but also reveal the functional and structural correlations between antimicrobial peptides and amyloid proteins with their built-in bacterial killing and amyloid inhibition functions.

Anti-Biofilm Molecules Targeting Functional Amyloids.

The choice of an effective therapeutic strategy in the treatment of biofilm-related infections is a significant issue. Amyloids, which have been historically related to human diseases, are now considered to be prevailing structural components of the biofilm matrix in a wide range of bacteria. This assumption creates the potential for an exciting research area, in which functional amyloids are considered to be attractive targets for drug development to dissemble biofilm structures. The present review describes the best-characterized bacterial functional amyloids and focuses on anti-biofilm agents that target intrinsic and facultative amyloids. This study provides a better understanding of the different modes of actions of the anti-amyloid molecules to inhibit biofilm formation. This information can be further exploited to improve the therapeutic strategies to combat biofilm-related infections.

Interrelationship between the 5-lipoxygenase pathway and microbial dysbiosis in the progression of Alzheimer's disease

Alzheimer's disease (AD) is an age-related neurodegenerative disorder involving neurofibrillary tangles and amyloid plaques. The tau phosphorylation responsible for neurofibrillary tangles and amyloid deposition which causes plaques are both accelerated through the activity of 5-lipoxygenase (5-LO). In addition to these pathological pathways, 5-LO has also been linked to the neuro-inflammation associated with disease progression as well as to dysbiosis in the gut. Interestingly, gut dysbiosis itself has been correlated to AD development. Not only do gut metabolites have direct effects on the brain, but pro-inflammatory mediators such as LPS, BMAA and bacterial amyloids produced in the gut due to dysbiosis reach the brain causing increased neuro-inflammation. While microbial dysbiosis and 5-LO exert detrimental effects in the brain, the cause/effect relationship between these factors remain unknown. These issues may be addressed using mouse models of AD in the context of different knockout mice in the 5-LO pathway in specific pathogen-free, germ-free as well as gnotobiotic conditions.

Development of a Novel Point of Care Test for Toxic Amyloids in Patients Recovering from Hospital Acquired Pneumonia

Amyloids have autofluorescence properties that may be exploited for their detection in patients recovering from hospital acquired pneumonia (HAP). Recovering HAP patients have cytotoxic amyloids in their bronchoalveolar lavage fluid, blood, and cerebrospinal fluid, and these amyloids may be the cause of the cognitive decline and secondary organ failure patients encounter after HAP. Infection of pulmonary microvascular endothelial cells (PMVECs) produces bacterial strain-dependent cytotoxic or antimicrobial amyloids. Currently, there are no detection methods for pulmonary amyloids at the bedside. Here, we hypothesize that cytotoxic and antimicrobial amyloids can be detected and distinguished by their autofluorescence properties. PMVECS were infected with mutants of gram-negative bacterium Pseudomonas aeruginosa, including virulent ExoY+ and avirulent ∆PcrV, to generate amyloids with distinctive phenotypes. Infection with ExoY+ for 7 hours produced cytotoxic amyloids, while infection with ∆PcrV for 4 hours yielded antimicrobial amyloids. To resolve whether the autofluorescence properties are concentration-dependent, protein concentrations of the supernatants were standardized to 50 μg/mL. To assess whether fibrillar or oligomeric amyloids are responsible for the autofluorescent signature of ExoY+ and ∆PcrV supernatants, the supernatants were immunodepleted with the amyloid fibril-specific OC or amyloid oligomer-specific A11 antibody. Fluorescence spectroscopy was performed to obtain the fluorescence spectra of the unstandardized, standardized, and immunodepleted ExoY+ and ΔPcrV supernatants. The unstandardized fluorescence spectra of ExoY+ and ΔPcrV were indistinguishable; however, when standardized to 50 μg/mL, the fluorescence intensity of ΔPcrV supernatant containing antimicrobial amyloids was significantly greater than ExoY+ supernatant containing cytotoxic amyloids. Immunodepleting the supernatant with the OC antibody eliminated the fluorescence intensity of ΔPcrV supernatant but did not diminish the fluorescence intensity of the ExoY+ supernatant. However, fluorescence of the ExoY+ supernatant was eradicated after immunodepletion with the A11 antibody. Our work suggests that the intrinsic fluorescence of amyloids can be developed as a point-of-care diagnostic tool in HAP. Antimicrobial amyloids may be detected by the immunodepletion of amyloid fibrils whereas cytotoxic amyloids can be distinguished by immunodepleting oligomeric amyloids. In the future we will apply this method to patient samples to assess its use as a diagnostic tool.

Searching for universal model of amyloid signaling motifs using probabilistic context-free grammars

BackgroundAmyloid signaling motifs are a class of protein motifs which share basic structural and functional features despite the lack of clear sequence homology. They are hard to detect in large sequence databases either with the alignment-based profile methods (due to short length and diversity) or with generic amyloid- and prion-finding tools (due to insufficient discriminative power). We propose to address the challenge with a machine learning grammatical model capable of generalizing over diverse collections of unaligned yet related motifs.ResultsFirst, we introduce and test improvements to our probabilistic context-free grammar framework for protein sequences that allow for inferring more sophisticated models achieving high sensitivity at low false positive rates. Then, we infer universal grammars for a collection of recently identified bacterial amyloid signaling motifs and demonstrate that the method is capable of generalizing by successfully searching for related motifs in fungi. The results are compared to available alternative methods. Finally, we conduct spectroscopy and staining analyses of selected peptides to verify their structural and functional relationship.ConclusionsWhile the profile HMMs remain the method of choice for modeling homologous sets of sequences, PCFGs seem more suitable for building meta-family descriptors and extrapolating beyond the seed sample.

AB013. CXCL4-DNA immune complexes drive inflammation in systemic sclerosis by amplifying TLR9-mediated interferon-α production

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by inflammation-driven tissue fibrosis and vasculopathy. CXCL4 is a chemokine overexpressed in SSc and represents an early serum biomarker of severe disease. CXCL4 likely contributes to inflammation via chemokine signaling pathways, but the exact role of CXCL4 in SSc pathogenesis is unclear. Here, we combine X-ray scattering, confocal microscopy, immune cell experiments, and analysis of SSc skin biopsies to elucidate an unanticipated mechanism for CXCL4-mediated immune amplification in SSc. We find that CXCL4 organizes “self” and microbial DNA into liquid crystalline immune complexes that drastically amplify Toll-like receptor 9 (TLR9)-mediated plasmacytoid dendritic cell (pDC) hyperactivation and interferon-α production. Surprisingly, this immunomodulatory activity is independent of CXCR3, the CXCL4 receptor. Importantly, we find that CXCL4-DNA complexes are present in vivo and correlate with type I interferon (IFN-I) levels in SSc blood, and that CXCL4-positive skin pDCs coexpress IFN-I-related genes. Taken together, we establish a direct link between CXCL4 overexpression and the IFN-I-gene signature in SSc and identify a potential therapeutic opportunity to disrupt inflammation in SSc by inhibiting the self-assembly of CXCL4-DNA complexes. Our findings are consistent with an emerging general paradigm in which nucleic acid-scaffolding proteins like antimicrobial peptides, bacterial amyloids, and chemokines can drive autoimmunity by modulating innate immune receptors without being direct agonists.

Microbiome or Infections: Amyloid-Containing Biofilms as a Trigger for Complex Human Diseases.

The human microbiota is the community of microorganisms that live upon or within their human host. The microbiota consists of various microorganisms including bacteria, fungi, viruses, and archaea; the gut microbiota is comprised mostly of bacteria. Many bacterial species within the gut microbiome grow as biofilms, which are multicellular communities embedded in an extracellular matrix. Studies have shown that the relative abundances of bacterial species, and therefore biofilms and bacterial byproducts, change during progression of a variety of human diseases including gastrointestinal, autoimmune, neurodegenerative, and cancer. Studies have shown the location and proximity of the biofilms within the gastrointestinal tract might impact disease outcome. Gram-negative enteric bacteria secrete the amyloid curli, which makes up as much as 85% of the extracellular matrix of enteric biofilms. Curli mediates cell-cell attachment and attachment to various surfaces including extracellular matrix components such as fibronectin and laminin. Structurally, curli is strikingly similar to pathological and immunomodulatory human amyloids such as amyloid-β, which has been implicated in Alzheimer's disease, α-synuclein, which is involved in Parkinson's disease, and serum amyloid A, which is secreted during the acute phase of inflammation. The immune system recognizes both bacterial amyloid curli and human amyloids utilizing the same receptors, so curli also induces inflammation. Moreover, recent work indicates that curli can participate in the self-assembly process of pathological human amyloids. Curli is found within biofilms of commensal enteric bacteria as well as invasive pathogens; therefore, evidence suggests that curli contributes to complex human diseases. In this review, we summarize the recent findings on how bacterial biofilms containing curli participate in the pathological and immunological processes in gastrointestinal diseases, systemic autoimmune diseases, and neurodegenerative diseases.

The Role of Gut Bacterial Metabolites in Brain Development, Aging and Disease.

In the last decade, emerging evidence has reported correlations between the gut microbiome and human health and disease, including those affecting the brain. We performed a systematic assessment of the available literature focusing on gut bacterial metabolites and their associations with diseases of the central nervous system (CNS). The bacterial metabolites short-chain fatty acids (SCFAs) as well as non-SCFAs like amino acid metabolites (AAMs) and bacterial amyloids are described in particular. We found significantly altered SCFA levels in patients with autism spectrum disorder (ASD), affective disorders, multiple sclerosis (MS) and Parkinson’s disease (PD). Non-SCFAs yielded less significantly distinct changes in faecal levels of patients and healthy controls, with the majority of findings were derived from urinary and blood samples. Preclinical studies have implicated different bacterial metabolites with potentially beneficial as well as detrimental mechanisms in brain diseases. Examples include immunomodulation and changes in catecholamine production by histone deacetylase inhibition, anti-inflammatory effects through activity on the aryl hydrocarbon receptor and involvement in protein misfolding. Overall, our findings highlight the existence of altered bacterial metabolites in patients across various brain diseases, as well as potential neuroactive effects by which gut-derived SCFAs, p-cresol, indole derivatives and bacterial amyloids could impact disease development and progression. The findings summarized in this review could lead to further insights into the gut–brain–axis and thus into potential diagnostic, therapeutic or preventive strategies in brain diseases.

Accelerated Amyloid Beta Pathogenesis by Bacterial Amyloid FapC

Self-assembly of amyloid proteins and peptides into amyloid fibrils is usually associated with many humane diseases such as Alzheimer's, Parkinson's diseases and type 2 diabetes. Functional amyloids such as bacterial amyloids, on the other hand, also have vital functions including strengthening bacterial attachment to surfaces and enhancing the stability and stiffness of biofilms. FapC is the precursor protein of a functional amyloid expressed in Pseudomonas. FapC sequence is comprised of repeat motifs that are separated by linker motifs and are highly conserved among the genera, which have been postulated to play important roles in amyloid formation, but little is known about their exact roles of in FapC fibrillization process.

Identification and characterization of the Hfq bacterial amyloid region DNA interactions

Nucleic acid amyloid proteins interactions have been observed in the past few years. These interactions often promote protein aggregation. Nevertheless, molecular basis and physiological consequences of these interactions are still poorly understood. Additionally, it is unknown whether the nucleic acid promotes the formation of self-assembly due to direct interactions or indirectly via sequences surrounding the amyloid region. Here we focus our attention on a bacterial amyloid, Hfq. This protein is a pleiotropic bacterial regulator that mediates many aspects of nucleic acids metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small non coding regulatory RNA. In addition, Hfq, thanks to its amyloid C-terminal region, binds and compacts DNA. A combination of experimental methodologies, including synchrotron radiation circular dichroism (SRCD), gel shift assay and infrared (FTIR) spectroscopy have been used to probe the interaction of Hfq C-terminal region with DNA. We clearly identify important amino acids in this region involved in DNA binding and polymerization properties. This allows to understand better how this bacterial amyloid interacts with DNA. Possible functional consequence to answer to stresses are discussed.

Potential roles of functional bacterial amyloid proteins, bacterial biosurfactants and other putative gut microbiota products in the etiopathogeny of Parkinson’s Disease

An increasing number of studies provide evidence for the existence of a microbiota-gut-brain axis and its potential involvement in the development of sporadic Parkinson’s disease and other neurodegenerative conditions. The neuropathologic hallmark of Parkinson’s disease is the presence of brain intraneuronal aggregates of misfolded alpha-synuclein, known as Lewy bodies. Some gut microbiota products may trigger alpha-synuclein conformational changes in the neurons of the enteric nervous system, which can then spread to the brain in a prion-like fashion through the vagus nerve. Others may interfere with neuroinflammatory pathways and susceptibility to neurodegeneration. In this review, we assess the potential role of putative gut microbiota products in the etiopathogeny of Parkinson’s disease, with a special emphasis on functional bacterial amyloid proteins, bacterial biosurfactants, endotoxins and short-chain fatty acids. The possible roles of molecular hydrogen, a common byproduct of bacterial fermentation, are also addressed.

The Microbiota-Gut-Brain Axis and Alzheimer's Disease: Neuroinflammation Is to Blame?

For years, it has been reported that Alzheimer’s disease (AD) is the most common cause of dementia. Various external and internal factors may contribute to the early onset of AD. This review highlights a contribution of the disturbances in the microbiota–gut–brain (MGB) axis to the development of AD. Alteration in the gut microbiota composition is determined by increase in the permeability of the gut barrier and immune cell activation, leading to impairment in the blood–brain barrier function that promotes neuroinflammation, neuronal loss, neural injury, and ultimately AD. Numerous studies have shown that the gut microbiota plays a crucial role in brain function and changes in the behavior of individuals and the formation of bacterial amyloids. Lipopolysaccharides and bacterial amyloids synthesized by the gut microbiota can trigger the immune cells residing in the brain and can activate the immune response leading to neuroinflammation. Growing experimental and clinical data indicate the prominent role of gut dysbiosis and microbiota–host interactions in AD. Modulation of the gut microbiota with antibiotics or probiotic supplementation may create new preventive and therapeutic options in AD. Accumulating evidences affirm that research on MGB involvement in AD is necessary for new treatment targets and therapies for AD.

Emerging Roles of Functional Bacterial Amyloids in Gene Regulation, Toxicity, and Immunomodulation.

Bacteria often reside in multicellular communities, called biofilms, held together by an extracellular matrix. In many bacteria, the major proteinaceous component of the biofilm are amyloid fibers. Amyloids are highly stable and structured protein aggregates which were known mostly to be associated with neurodegenerative diseases, such as Alzheimer's, Parkinson's, and Huntington's diseases. In recent years, microbial amyloids were identified also in other species and shown to play major roles in microbial physiology and virulence. For example, amyloid fibers assemble on the bacterial cell surface as a part of the extracellular matrix and are extremely important to the scaffolding and structural integrity of biofilms, which contribute to microbial resilience and resistance. Furthermore, microbial amyloids play fundamental nonscaffold roles that contribute to the development of biofilms underlying numerous persistent infections. Here, we review several nonscaffold roles of bacterial amyloid proteins, including bridging cells during collective migration, acting as regulators of cell fate, as toxins against other bacteria or against host immune cells, and as modulators of the hosts' immune system. These overall points on the complexity of the amyloid fold in encoding numerous activities, which offer approaches for the development of a novel repertoire of antivirulence therapeutics.

Implications of the Human Gut-Brain and Gut-Cancer Axes for Future Nanomedicine.

Recent clinical and pathological evidence have implicated the gut microbiota as a nexus for modulating the homeostasis of the human body, impacting conditions from cancer and dementia to obesity and social behavior. The connections between microbiota and human diseases offer numerous opportunities in medicine, most of which have limited or no therapeutic solutions available. In light of this paradigm-setting trend in science, this review aims to provide a comprehensive and timely summary of the mechanistic pathways governing the gut microbiota and their implications for nanomedicines targeting cancer and neurodegenerative diseases. Specifically, we discuss in parallel the beneficial and pathogenic relationship of the gut microbiota along the gut-brain and gut-cancer axes, elaborate on the impact of dysbiosis and the gastrointestinal corona on the efficacy of nanomedicines, and highlight a molecular mimicry that manipulates the universal cross-β backbone of bacterial amyloid to accelerate neurological disorders. This review further offers a forward-looking section on the rational design of cancer and dementia nanomedicines exploiting the gut-brain and gut-cancer axes.

Identification of NLR-associated Amyloid Signaling Motifs in Bacterial Genomes

In filamentous fungi, amyloid signaling sequences allow Nod-like receptors (NLRs) to activate downstream cell-death inducing proteins with HeLo and HeLo-like (HELL) domains and amyloid RHIM and RHIM-related motifs control immune defense pathways in mammals and flies. Herein, we show bioinformatically that analogous amyloid signaling motifs exist in bacteria. These short motifs are found at the N terminus of NLRs and at the C terminus of proteins with a domain we term BELL. The corresponding NLR and BELL proteins are encoded by adjacent genes. We identify 10 families of such bacterial amyloid signaling sequences (BASS), one of which (BASS3) is homologous to RHIM and a fungal amyloid motif termed PP. BASS motifs occur nearly exclusively in bacteria forming multicellular structures (mainly in Actinobacteria and Cyanobacteria). We analyze experimentally a subset of seven of these motifs (from the most common BASS1 family and the RHIM-related BASS3 family) and find that these sequences form fibrils in vitro. Using a fungal in vivo model, we show that all tested BASS-motifs form prions and that the NLR-side motifs seed prion-formation of the corresponding BELL-side motif. We find that BASS3 motifs show partial prion cross-seeding with mammalian RHIM and fungal PP-motifs and that proline mutations on key positions of the BASS3 core motif, conserved in RHIM and PP-motifs, abolish prion formation. This work expands the paradigm of prion amyloid signaling to multicellular prokaryotes and suggests a long-term evolutionary conservation of these motifs from bacteria, to fungi and animals.