What can AlphaFold do for antimicrobial amyloids?

Amyloids, protein, and peptide assemblies in various organisms are crucial in physiological and pathological processes. Their intricate structures, however, present significant challenges, limiting our understanding of their functions, regulatory mechanisms, and potential applications in biomedicine and technology. This study evaluated the AlphaFold2 ColabFold method's structure predictions for antimicrobial amyloids, using eight antimicrobial peptides (AMPs), including those with experimentally determined structures and AMPs known for their distinct amyloidogenic morphological features. Additionally, two well-known human amyloids, amyloid-ß and islet amyloid polypeptide, were included in the analysis due to their disease relevance, short sequences, and antimicrobial properties. Amyloids typically exhibit tightly mated ß-strand sheets forming a cross-ß configuration. However, certain amphipathic α-helical subunits can also form amyloid fibrils adopting a cross-α structure. Some AMPs in the study exhibited a combination of cross-α and cross-ß amyloid fibrils, adding complexity to structure prediction. The results showed that the AlphaFold2 ColabFold models favored α-helical structures in the tested amyloids, successfully predicting the presence of α-helical mated sheets and a hydrophobic core resembling the cross-α configuration. This implies that the AI-based algorithms prefer assemblies of the monomeric state, which was frequently predicted as helical, or capture an α-helical membrane-active form of toxic peptides, which is triggered upon interaction with lipid membranes.

Staphylococcus aureus functional amyloids catalyze degradation of ß-lactam antibiotics.

Antibiotic resistance of bacteria is considered one of the most alarming developments in modern medicine. While varied pathways for bacteria acquiring antibiotic resistance have been identified, there still are open questions concerning the mechanisms underlying resistance. Here, we show that alpha phenol-soluble modulins (PSMαs), functional bacterial amyloids secreted by Staphylococcus aureus, catalyze hydrolysis of ß-lactams, a prominent class of antibiotic compounds. Specifically, we show that PSMα2 and, particularly, PSMα3 catalyze hydrolysis of the amide-like bond of the four membered ß-lactam ring of nitrocefin, an antibiotic ß-lactam surrogate. Examination of the catalytic activities of several PSMα3 variants allowed mapping of the active sites on the amyloid fibrils' surface, specifically underscoring the key roles of the cross-α fibril organization, and the combined electrostatic and nucleophilic functions of the lysine arrays. Molecular dynamics simulations further illuminate the structural features of ß-lactam association upon the fibril surface. Complementary experimental data underscore the generality of the functional amyloid-mediated catalytic phenomenon, demonstrating hydrolysis of clinically employed ß-lactams by PSMα3 fibrils, and illustrating antibiotic degradation in actual S. aureus biofilms and live bacteria environments. Overall, this study unveils functional amyloids as catalytic agents inducing degradation of ß-lactam antibiotics, underlying possible antibiotic resistance mechanisms associated with bacterial biofilms.

Differential fibril morphologies and thermostability determine functional roles of Staphylococcus aureus PSMα1 and PSMα3.

Phenol-soluble modulins (PSMs) are virulent peptides secreted by staphylococci that undergo self-assembly into amyloid fibrils. This study focuses on Staphylococcus aureus PSMα1 and PSMα3, which share homologous sequences but exhibit distinct amyloid fibril structures. Upon subjecting PSMα1 to an 80°C heat shock, it fibrillates into cross-ß structures, resulting in the loss of cytotoxic activity. Conversely, PSMα3 cross-α fibrils undergo reversible disaggregation upon heat shock, leading to the recovery of cytotoxicity. The differential thermostability probably arises from the presence of hydrogen bonds along the ß-strands within the ß-sheets of the cross-ß fibrils. We propose that the breakdown of PSMα3 fibrils into soluble species, potentially co-aggregating with membrane lipids, is crucial for its toxic process and enables the reversible modulation of its biological activity under stress conditions. In contrast, the formation of robust and irreversible cross-ß fibrils by PSMα1 corresponds to its role in biofilm stability. These findings emphasize how the unique fibril morphologies and thermostability of PSMα1 and PSMα3 shape their functional roles in various environments of S. aureus.

Beyond One-Trick Ponies: The Multifunctional Marvels of Microbial Functional Amyloids.

Various organisms, including bacteria, protists, fungi, plants, and animals, secrete proteins and peptides that self-assemble into ordered amyloid fibrils that perform different physiological functions [...].

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.

Natural Antimicrobial Peptides Self-assemble as α/ß Chameleon Amyloids.

Amyloid protein fibrils and some antimicrobial peptides (AMPs) share biophysical and structural properties. This observation suggests that ordered self-assembly can act as an AMP-regulating mechanism, and, vice versa, that human amyloids play a role in host defense against pathogens, as opposed to their common association with neurodegenerative and systemic diseases. Based on previous structural information on toxic amyloid peptides, we developed a sequence-based bioinformatics platform and, led by its predictions, experimentally identified 14 fibril-forming AMPs (ffAMPs) from living organisms, which demonstrated cross-ß and cross-α amyloid properties. The results support the amyloid-antimicrobial link. The high prevalence of ffAMPs produced by amphibians and marine creatures among other species suggests that they confer unique advantageous properties in distinctive environments, potentially providing stability and adherence properties. Most of the newly identified 14 ffAMPs showed lipid-induced and/or time-dependent secondary structure transitions in the fibril form, indicating structural and functional cross-α/ß chameleons. Specifically, ffAMP cytotoxicity against human cells correlated with the inherent or lipid-induced α-helical fibril structure. The findings raise hypotheses about the role of fibril secondary structure switching in regulation of processes, such as the transition between a stable storage conformation and an active state with toxicity against specific cell types.

Structure and Conservation of Amyloid Spines From the Candida albicans Als5 Adhesin.

Candida Als family adhesins mediate adhesion to biological and abiotic substrates, as well as fungal cell aggregation, fungal-bacterial co-aggregation and biofilm formation. The activity of at least two family members, Als5 and Als1, is dependent on amyloid-like protein aggregation that is initiated by shear force. Each Als adhesin has a ∼300-residue N-terminal Ig-like/invasin region. The following 108-residue, low complexity, threonine-rich (T) domain unfolds under shear force to expose a critical amyloid-forming segment 322SNGIVIVATTRTV334 at the interface between the Ig-like/invasin domain 2 and the T domain of Candida albicans Als5. Amyloid prediction programs identified six potential amyloidogenic sequences in the Ig-like/invasin region and three others in the T domain of C. albicans Als5. Peptides derived from four of these sequences formed fibrils that bound thioflavin T, the amyloid indicator dye, and three of these revealed atomic-resolution structures of cross-ß spines. These are the first atomic-level structures for fungal adhesins. One of these segments, from the T domain, revealed kinked ß-sheets, similarly to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked segments) found in human functional amyloids. Based on the cross-ß structures in Als proteins, we use evolutionary arguments to identify functional amyloidogenic sequences in other fungal adhesins, including adhesins from Candida auris. Thus, cross-ß structures are often involved in fungal pathogenesis and potentially in antifungal therapy.

The Cryo-EM structures of two amphibian antimicrobial cross-ß amyloid fibrils.

The amyloid-antimicrobial link hypothesis is based on antimicrobial properties found in human amyloids involved in neurodegenerative and systemic diseases, along with amyloidal structural properties found in antimicrobial peptides (AMPs). Supporting this hypothesis, we here determined the fibril structure of two AMPs from amphibians, uperin 3.5 and aurein 3.3, by cryogenic electron microscopy (cryo-EM), revealing amyloid cross-ß fibrils of mated ß-sheets at atomic resolution. Uperin 3.5 formed a 3-blade symmetrical propeller of nine peptides per fibril layer including tight ß-sheet interfaces. This cross-ß cryo-EM structure complements the cross-α fibril conformation previously determined by crystallography, substantiating a secondary structure switch mechanism of uperin 3.5. The aurein 3.3 arrangement consisted of six peptides per fibril layer, all showing kinked ß-sheets allowing a rounded compactness of the fibril. The kinked ß-sheets are similar to LARKS (Low-complexity, Amyloid-like, Reversible, Kinked Segments) found in human functional amyloids.

Structural Mimicry in Microbial and Antimicrobial Amyloids.

The remarkable variety of microbial species of human pathogens and microbiomes generates significant quantities of secreted amyloids, which are structured protein fibrils that serve diverse functions related to virulence and interactions with the host. Human amyloids are associated largely with fatal neurodegenerative and systemic aggregation diseases, and current research has put forward the hypothesis that the interspecies amyloid interactome has physiological and pathological significance. Moreover, functional and molecular-level connections between antimicrobial activity and amyloid structures suggest a neuroimmune role for amyloids that are otherwise known to be pathological. Compared to the extensive structural information that has been accumulated for human amyloids, high-resolution structures of microbial and antimicrobial amyloids are only emerging. These recent structures reveal both similarities and surprising departures from the typical amyloid motif, in accordance with their diverse activities, and advance the discovery of novel antivirulence and antimicrobial agents. In addition, the structural information has led researchers to postulate that amyloidogenic sequences are natural targets for structural mimicry, for instance in host-microbe interactions. Microbial amyloid research could ultimately be used to fight aggressive infections and possibly processes leading to autoimmune and neurodegenerative diseases.

Rare by Natural Selection: Disulfide-Bonded Supramolecular Antimicrobial Peptides.

Human LL-3717-29 is an antimicrobial peptide forming thermostable supramolecular fibrils that surround bacterial cells. The crystal structure of LL-3717-29 bearing an I24C substitution of most buried position in the fibril revealed disulfide-bonded dimers that further assembled into a fibrillar structure of densely packed helices. We further demonstrated the position-dependent controllable antibacterial activity of LL-3717-29 I24C and other cysteine mutants, mediated by regulation of intermolecular disulfide bonds and their role in the formation of supramolecular structures. The morphology of the fibrils and their antibacterial mechanism of action might be dependent on their interactions with specific bacteria. The significant effect of disulfide bonds on the assembly into supramolecular structures and their sensitivity to reducing/oxidizing conditions may explain why short helical antimicrobial peptides with a single cysteine and an odd number of cysteines are selected against in nature.

Signal inhibitory receptor on leukocytes-1 recognizes bacterial and endogenous amphipathic α-helical peptides.

Signal inhibitory receptor on leukocytes-1 (SIRL-1) is a negative regulator of myeloid cell function and dampens antimicrobial responses. We here show that different species of the genus Staphylococcus secrete SIRL-1-engaging factors. By screening a library of single-gene transposon mutants in Staphylococcus aureus, we identified these factors as phenol-soluble modulins (PSMs). PSMs are amphipathic α-helical peptides involved in multiple aspects of staphylococcal virulence and physiology. They are cytotoxic and activate the chemotactic formyl peptide receptor 2 (FPR2) on immune cells. Human cathelicidin LL-37 is also an amphipathic α-helical peptide with antimicrobial and chemotactic activities, structurally and functionally similar to α-type PSMs. We demonstrate that α-type PSMs from multiple staphylococcal species as well as human cathelicidin LL-37 activate SIRL-1, suggesting that SIRL-1 recognizes α-helical peptides with an amphipathic arrangement of hydrophobicity, although we were not able to show direct binding to SIRL-1. Upon rational peptide design, we identified artificial peptides in which the capacity to ligate SIRL-1 is segregated from cytotoxic and FPR2-activating properties, allowing specific engagement of SIRL-1. In conclusion, we propose staphylococcal PSMs and human LL-37 as a potential new class of natural ligands for SIRL-1.

Structural and Functional Insights into the Biofilm-Associated BceF Tyrosine Kinase Domain from Burkholderia cepacia.

BceF is a bacterial tyrosine kinase (BY-kinase) from Burkholderia cepacia, a Gram-negative bacterium accountable for respiratory infections in immunocompromised and cystic fibrosis patients. BceF is involved in the production of exopolysaccharides secreted to the biofilm matrix and promotes resistant and aggressive infections. BY-kinases share no homology with mammalian kinases, and thereby offer a means to develop novel and specific antivirulence drugs. Here, we report the crystal structure of the BceF kinase domain at 1.85 Å resolution. The isolated BceF kinase domain is assembled as a dimer in solution and crystallized as a dimer in the asymmetric unit with endogenous adenosine-diphosphate bound at the active sites. The low enzymatic efficiency measured in solution may be explained by the partial obstruction of the active sites at the crystallographic dimer interface. This study provides insights into self-assembly and the specific activity of isolated catalytic domains. Several unique variations around the active site compared to other BY-kinases may allow for structure-based design of specific inhibitors to target Burkholderia cepacia virulence.

Integrative Structural Biology in the Era of Accurate Structure Prediction.

Characterizing the three-dimensional structure of macromolecules is central to understanding their function. Traditionally, structures of proteins and their complexes have been determined using experimental techniques such as X-ray crystallography, NMR, or cryo-electron microscopy-applied individually or in an integrative manner. Meanwhile, however, computational methods for protein structure prediction have been improving their accuracy, gradually, then suddenly, with the breakthrough advance by AlphaFold2, whose models of monomeric proteins are often as accurate as experimental structures. This breakthrough foreshadows a new era of computational methods that can build accurate models for most monomeric proteins. Here, we envision how such accurate modeling methods can combine with experimental structural biology techniques, enhancing integrative structural biology. We highlight the challenges that arise when considering multiple structural conformations, protein complexes, and polymorphic assemblies. These challenges will motivate further developments, both in modeling programs and in methods to solve experimental structures, towards better and quicker investigation of structure-function relationships.

Inhibition of Staphylococcus aureus biofilm-forming functional amyloid by molecular tweezers.

Biofilms are rigid and largely impenetrable three-dimensional matrices constituting virulence determinants of various pathogenic bacteria. Here, we demonstrate that molecular tweezers, unique supramolecular artificial receptors, modulate biofilm formation of Staphylococcus aureus. In particular, the tweezers affect the structural and assembly properties of phenol-soluble modulin α1 (PSMα1), a biofilm-scaffolding functional amyloid peptide secreted by S. aureus. The data reveal that CLR01, a diphosphate tweezer, exhibits significant S. aureus biofilm inhibition and disrupts PSMα1 self-assembly and fibrillation, likely through inclusion of lysine side chains of the peptide. In comparison, different peptide binding occurs in the case of CLR05, a tweezer containing methylenecarboxylate units, which exhibits lower affinity for the lysine residues yet disrupts S. aureus biofilm more strongly than CLR01. Our study points to a possible role for molecular tweezers as potent biofilm inhibitors and antibacterial agents, particularly against untreatable biofilm-forming and PSM-producing bacteria, such as methicillin-resistant S. aureus.

Functional and pathological amyloid structures in the eyes of 2020 cryo-EM.

The amyloid state of protein aggregation is associated with neurodegenerative and systemic diseases but can play physiological roles in many organisms, including as stress granules and virulence determinants. The recent resolution revolution in cryogenic electron microscopy (cryo-EM) has significantly expanded the repertoire of high-resolution amyloid structures, to include, for the first-time, fibrils extracted ex vivo in addition to those formed, or seeded, in vitro. Here, we review recently solved cryo-EM amyloid structures, and compare amino acid prevalence, in efforts to systematically distinguish between pathological and functional amyloids, even though such structural classification is hindered by extensive polymorphism even among fibrils of the same protein, and by dual functioning of some human amyloids in both physiological activities and disease mechanisms. Forthcoming structures of bacterial amyloids may expose specific, evolutionary-designed properties specific to functional fibrils.

The amphibian antimicrobial peptide uperin 3.5 is a cross-α/cross-ß chameleon functional amyloid.

Antimicrobial activity is being increasingly linked to amyloid fibril formation, suggesting physiological roles for some human amyloids, which have historically been viewed as strictly pathological agents. This work reports on formation of functional cross-α amyloid fibrils of the amphibian antimicrobial peptide uperin 3.5 at atomic resolution, an architecture initially discovered in the bacterial PSMα3 cytotoxin. The fibrils of uperin 3.5 and PSMα3 comprised antiparallel and parallel helical sheets, respectively, recapitulating properties of ß-sheets. Uperin 3.5 demonstrated chameleon properties of a secondary structure switch, forming mostly cross-ß fibrils in the absence of lipids. Uperin 3.5 helical fibril formation was largely induced by, and formed on, bacterial cells or membrane mimetics, and led to membrane damage and cell death. These findings suggest a regulation mechanism, which includes storage of inactive peptides as well as environmentally induced activation of uperin 3.5, via chameleon cross-α/ß amyloid fibrils.

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.

The Human LL-37(17-29) antimicrobial peptide reveals a functional supramolecular structure.

Here, we demonstrate the self-assembly of the antimicrobial human LL-37 active core (residues 17-29) into a protein fibril of densely packed helices. The surface of the fibril encompasses alternating hydrophobic and positively charged zigzagged belts, which likely underlie interactions with and subsequent disruption of negatively charged lipid bilayers, such as bacterial membranes. LL-3717-29 correspondingly forms wide, ribbon-like, thermostable fibrils in solution, which co-localize with bacterial cells. Structure-guided mutagenesis analyses supports the role of self-assembly in antibacterial activity. LL-3717-29 resembles, in sequence and in the ability to form amphipathic helical fibrils, the bacterial cytotoxic PSMα3 peptide that assembles into cross-α amyloid fibrils. This argues helical, self-assembling, basic building blocks across kingdoms of life and points to potential structural mimicry mechanisms. The findings expose a protein fibril which performs a biological activity, and offer a scaffold for functional and durable biomaterials for a wide range of medical and technological applications.

Staphylococcus aureus PSMα3 Cross-α Fibril Polymorphism and Determinants of Cytotoxicity.

The phenol-soluble modulin (PSM) peptide family, secreted by Staphylococcus aureus, performs various virulence activities, some mediated by the formation of amyloid fibrils of diverse architectures. Specifically, PSMα1 and PSMα4 structure the S. aureus biofilm by assembling into robust cross-ß amyloid fibrils. PSMα3, the most cytotoxic member of the family, assembles into cross-α fibrils in which α helices stack into tightly mated sheets, mimicking the cross-ß architecture. Here we demonstrate that massive T cell deformation and death are linked with PSMα3 aggregation and co-localization with cell membranes. Our extensive mutagenesis analyses support the role of positive charges, and especially Lys17, in interactions with the membrane and suggest their regulation by inter- and intra-helical electrostatic interactions within the cross-α fibril. We hypothesize that PSMα3 cytotoxicity is governed by the ability to form cross-α fibrils and involves a dynamic process of co-aggregation with the cell membrane, rupturing it.

Structural Insights into Curli CsgA Cross-ß Fibril Architecture Inspire Repurposing of Anti-amyloid Compounds as Anti-biofilm Agents.

Curli amyloid fibrils secreted by Enterobacteriaceae mediate host cell adhesion and contribute to biofilm formation, thereby promoting bacterial resistance to environmental stressors. Here, we present crystal structures of amyloid-forming segments from the major curli subunit, CsgA, revealing steric zipper fibrils of tightly mated ß-sheets, demonstrating a structural link between curli and human pathological amyloids. D-enantiomeric peptides, originally developed to interfere with Alzheimer's disease-associated amyloid-ß, inhibited CsgA fibrillation and reduced biofilm formation in Salmonella typhimurium. Moreover, as previously shown, CsgA fibrils cross-seeded fibrillation of amyloid-ß, providing support for the proposed structural resemblance and potential for cross-species amyloid interactions. The presented findings provide structural insights into amyloidogenic regions important for curli formation, suggest a novel strategy for disrupting amyloid-structured biofilms, and hypothesize on the formation of self-propagating prion-like species originating from a microbial source that could influence neurodegenerative diseases.