WO2025201633A1 - An agent for reducing amyloid formation - Google Patents

Abstract

The present invention relates to uses of an agent for reducing and/or preventing and/or reversing amyloid formation.

Description

AN AGENT FOR REDUCING AMYLOID FORMATION

The present invention relates to uses of an agent for reducing and/or preventing and/or reversing amyloid formation, wherein the agent comprises, or consists of, the N- terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation, uses and methods for reducing and/or preventing and/or reversing microbial biofilm formation, uses and methods for preventing and/or treating a disease or condition characterised by amyloid formation, and combinations and compositions comprising the agent.

Protein aggregation and amyloid formation have historically been linked with various diseases such as Alzheimer's and Parkinson's disease. These pathological amyloids are insoluble, fibrous protein aggregates that accumulate abnormally in various tissues and organs, contributing to the pathogenesis of amyloid diseases. Unlike normal proteins, which fold into specific three-dimensional structures to perform their biological functions, pathological amyloids undergo misfolding and aggregation, forming p-sheet- rich fibrils that are resistant to degradation. Alzheimer's disease (AD) is the most prevalent form of dementia, resulting in progressive memory loss and profound cognitive dysfunction, producing a considerable societal burden. At the neuropathological level, the brains of AD patients exhibit amyloid-p (A[3) plaques, neurofibrillary tangles, and neuroinflammation (Sala Frigerio and De Strooper, (2016) Alzheimer's disease mechanisms and emerging roads to novel therapeutics. Annu Rev Neurosci 39:57-79). The growing number of individuals affected with AD underscores the pressing need for the development of effective treatments, and a cure remains elusive.

Biofilms are microbial communities where microorganisms live in a matrix of hydrated extracellular polymeric substances (EPS) that forms the scaffold of the biofilm. Biofilm formation by bacteria enables retention of nutrients and water, adhesion to surfaces and other bacteria cells, and it additionally acts as a protective barrier. Biofilm- associated pathogenic microbes are protected from anti-microbial agents and host immune system attacks, as a result they are more infectious and difficult to treat. Eighty percent of all chronic infections are related to bacterial biofilms. Aggregated proteins in the form of amyloid fibrils play a key role in maintaining the structural integrity of biofilm and when these proteins are mutated, the biofilm is disrupted and bacteria become exposed to, for example, antibiotic treatments. Not all amyloids are pathological as such, and some can have biological functions in the organism producing them, hence the term "functional amyloids" has been coined. Such functional amyloids can strengthen biofilms and are thus a major threat to human health, since the chronic infections they cause are difficult to treat due to the biofilm structural integrity and insufficient penetration of drugs, thus promoting antibiotic resistance. Targeting biofilms could be a novel approach to fight antibiotic resistance, which results in millions of casualties per year, and is estimated to cause more death than cancer by 2050. However, very little structural information exists about biofilms and their fibrillar components and how these components interact with other proteins.

Against this background, the inventors have surprisingly found that a portion of the CagA (cytotoxin-associated gene A) protein secreted by Helicobacter pylori has broad anti-amyloid activity and therefore provides a new approach for combating bacterial infections and/or human protein misfolding diseases.

In an aspect, the invention provides use of an agent for reducing and/or preventing and/or reversing amyloid formation, wherein the agent comprises, or consists of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

In an aspect, the invention provides a method for reducing and/or preventing and/or reversing amyloid formation in a subject, the method comprising administering an effective amount of an agent comprising, or consisting of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid fibril formation to a subject.

In an aspect, the invention provides a method for reducing and/or preventing and/or reversing amyloid formation on a non-living surface, the method comprising contacting an amyloid with an agent comprising, or consisting of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid fibril formation. Examples of surfaces are described herein. By a "non-living surface" we include the meaning of a surface outside of the human or animal body.

The basic structural composition of amyloid is the fibril. An amyloid fibril is built up by twisted protofilaments. Amyloid fibrils may be formed from 1, 2, 3, 4, or many such protofilaments. Protofilaments are bound to each other in a parallel fashion via their sidechains. By "amyloid formation" we include the meaning of the process by which certain proteins misfold and aggregate, forming insoluble fibrillar structures typically characterized by a cross- 0-sheet structure known as amyloid fibrils. Such fibrils exhibit the characteristic fibrillary ultra-structure, X-ray crystallographic diffraction patterns, and the binding of dyes such as thioflavin T and Congo red. Typical birefringence after staining with the latter dye is also seen. These fibrils can be deposited extracellularly and can accumulate in various tissues and organs, leading to tissue damage and dysfunction. Amyloid fibrils are associated with several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease, as well as other conditions such as type 2 diabetes. We also include the formation of "functional" or non-pathologic amyloids from amyloid fibrils. In nature 0-sheet fibrils are adapted for many functions. Certain polypeptide hormones are stored in p-sheet conformation, perhaps not as regular fibrils; melanin is bound to the 0-sheet fibrillar carrier (p-mell7) in melanosomes. The strength of 0-sheet fibrils is used by several bacterial and fungal organisms. Bacteria make several different structures, such as biofilms, that have 0-sheet fibrillary compositions. The presence of amyloid fibrils in the biofilm matrix can protect microbial cells from environmental stresses, including antimicrobial agents, host immune responses, and fluctuations in nutrient availability. The fungus Neurospora crassa expresses the hydrophobin EAS which forms amphipathic amyloid monolayers that facilitate spore formation and dispersal. These are all examples of a functional amyloid.

By "reducing and/or preventing and/or reversing amyloid formation", we include the meaning of inhibiting the aggregation of proteins into amyloid fibrils or destabilizing existing fibrils thereby reducing or halting the progression of diseases associated with amyloid deposition, and/or reducing or halting biofilm formation. For example, the amount of amyloid formation will be reduced and/or prevented and/or reversed in a subject who has received the agent described herein compared to the amount of amyloid formation in a subject who has not received the agent. For example, amyloid formation in a subject who has received the agent as described herein may be reduced by 10%, 20%, 30% or 40%, such as by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% when compared to amyloid formation in a subject who has not received the agent. In an embodiment the use for reducing and/or preventing and/or reversing amyloid formation is carried out outside of the human or animal body. Measuring amyloid formation in a patient typically involves a combination of clinical assessments, imaging techniques, and laboratory tests. Common methods used to detect and quantify amyloid deposition include the clinical assessment by a healthcare provider which may involve evaluating symptoms and signs suggestive of amyloid- related diseases, such as cognitive decline in Alzheimer's disease or cardiac involvement in systemic amyloidosis. Imaging techniques such as Positron Emission Tomography (PET) which uses radiotracers such as Pittsburgh Compound B (PiB) or Florbetapir to visualize amyloid deposits in the brain of patients suspected of having Alzheimer's disease. Cardiac Magnetic Resonance Imaging (MRI) with late gadolinium enhancement (LGE) can detect myocardial amyloid deposits in patients with cardiac amyloidosis. Scintigraphy with radiotracers such as technetium-99m-labeled bisphosphonates can detect amyloid deposits in bone marrow in patients with systemic amyloidosis. Tissue biopsy, such as from the skin, gastrointestinal tract, or affected organs, followed by histopathological examination and staining techniques (e.g., Congo red staining with apple-green birefringence under polarized light) can confirm the presence of amyloid deposits and identify the specific type of amyloid protein. Serum and Urine Protein Electrophoresis can detect abnormal protein bands suggestive of monoclonal gammopathies, which may be associated with systemic amyloidosis. Immunohistochemical staining of tissue samples can identify the specific amyloid protein involved, such as amyloid beta in Alzheimer's disease or immunoglobulin light chains in systemic amyloidosis. Blood or cerebrospinal fluid biomarker assays, such as measurements of amyloid beta 42 (A[342) and tau protein, can be used in research settings to assess amyloid burden and neuronal injury in Alzheimer's disease.

By "effective amount" as used herein, we include the meaning of a quantity sufficient to achieve a desired effect. In the context of therapeutic or prophylactic applications, the effective amount will depend on the type and severity of the condition at issue and the characteristics of the individual subject, such as general health, age, sex, body weight, and tolerance to pharmaceutical compositions. The skilled artisan will be able to determine appropriate amounts depending on these and other factors. In the case of an in vitro application, in some embodiments the effective amount will depend on the size and nature of the application in question. It will also depend on the nature and sensitivity of the in vitro target and the methods in use. The skilled artisan will be able to determine the effective amount based on these and other considerations. The effective amount may comprise one or more administrations of a composition depending on the embodiment. By "CagA" as used herein, we include the meaning of the CagA protein which is a virulence factor secreted by Helicobacter pylori. An exemplary full length CagA of H. pylori has amino acids 1-1186 as set out in SEQ ID NO: 1 (Fig. lb). Sequence numbering corresponds to CagA from H. pylori strain 26695 (Tomb JF, et al. Nature. 1997;388:539-547). CagA consists of multiple domains/regions: an N-terminal structured region and a disordered C-terminal tail for perturbing host signal transduction (Fig. la and b) (Hatakeyama, M. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 93, 196-219 (2017); and Kaplan-Turkoz, B. et al. Proc. Natl. Acad. Sci. U. S. A. 109, 14640-14645 (2012)).

In an embodiment, CagA comprises, or consists of, the sequence of amino acids 1- 1186 of H. pylori strain 26695, or an orthologue thereof, optionally wherein CagA has the amino acid sequence of SEQ ID NO: 1 In an embodiment, CagA has the amino acid sequence of SEQ ID: NO: 1. In an embodiment, CagA has sequence of amino acids 1-1186 of H. pylori strain 26695, or an orthologue thereof, such as from H. pylori strain G27 (Bagnoli F. et al., (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16339-16344), V225 (Olbermann P, et al. PLoS Genet. 2010;6:el001069), HP-No20, and HP-No31 (Truong BX, et al. J Clin Microbiol. 2009;47:4021-4028). By "orthologue", we include the meaning of a gene found in different species that shares a common evolutionary origin. The skilled person is able to determine whether a sequence is an orthologue of CagA or CagA^N from H. pylori strain 26695 using methods known in the art. For example, by using various computational and experimental methods, including, determining sequence similarity, since orthologous genes often exhibit high sequence similarity across species. Computational tools such as BLAST (Basic Local Alignment Search Tool) can be used to search for sequences that are homologous between different species. Phylogenetic analysis can be used and so by constructing phylogenetic trees based on the sequences of genes from different species, the skilled person can identify orthologous genes by observing patterns of gene divergence and speciation events. Synteny analysis may be used as orthologous genes are often located in similar genomic contexts (synteny) across species, and so comparing the genomic organization of genes in different species can help identify orthologues. Functional analysis can be used as orthologous genes tend to perform similar functions across species, therefore experimental studies, such as gene knockout experiments or functional assays, can be used to validate the functional conservation of orthologous genes.

In an embodiment, the agent comprises the N-terminal region of CagA (CagA^N). In an embodiment, the agent consists of the N-terminal region of CagA (CagA^N). By "the N- terminal region of CagA" we include the meaning of the N-terminal region spanning from position 1 to 884 of CagA, designated "CagA^N". The CagA protein's size ranges from 130 to 145 kDa due to structural polymorphism in the C-terminal region (Hatakeyama, M. Proc. Jpn. Acad. Ser. B Phys. Biol. Sci. 93, 196-219 (2017)).

In an embodiment, the agent comprises, or consists of, a portion of the N-terminal region of CagA (CagA^N). By "portion of the N-terminal region of CagA (CagA^N)", we include the meaning of a segment or fragment of CagA^N's primary structure, which is the linear sequence of amino acids that make up the protein chain. A portion of a protein can vary in size and can range from a short sequence of amino acids, such as a peptide or a domain, to larger segments that encompass multiple domains or functional regions within the protein. The specific portion of a CagA^N must be capable of reducing and/or preventing and/or reversing amyloid formation.

In an embodiment, the agent comprises, or consists of, a variant of the N-terminal region of CagA (CagA^N). By "variant of the N-terminal region of CagA (CagA^N)", we include the meaning of a polypeptide derived from CagA^N comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (several) positions that maintains the ability to reduce and/or prevent and/or reverse amyloid formation (i.e. it is functionally equivalent). It is understood that functional equivalents or variants of the CagA^N also are within the scope of this invention, for example, those having conservative amino acid substitutions of the amino acids. A conservative substitution is typically the substitution of one amino acid for another that is a member of the same class. For example, it is well known in the art of protein biochemistry that an amino acid belonging to a grouping of amino acids having a particular size or characteristic (such as charge, hydrophobicity, and/or hydrophilicity) may generally be substituted for another amino acid without substantially altering the secondary and/or tertiary structure of a polypeptide. For the purposes of this invention, conservative amino acid substitutions are defined to result from exchange of amino acids residues from within one of the following classes of residues: Class I: Gly, Ala, Vai, Leu, and He (representing aliphatic side chains); Class II: Gly, Ala, Vai, Leu, He, Ser, and Thr (representing aliphatic and aliphatic hydroxyl side chains); Class III: Tyr, Ser, and Thr (representing hydroxyl side chains); Class IV: Cys and Met (representing sulphur- containing side chains); Class V: Glu, Asp, Asn and Gin (carboxyl or amide group containing side chains); Class VI: His, Arg and Lys (representing basic side chains); Class VII: Gly, Ala, Pro, Trp, Tyr, lie, Vai, Leu, Phe and Met (representing hydrophobic side chains); Class VIII: Phe, Trp, and Tyr (representing aromatic side chains); and Class IX: Asn and Gin (representing amide side chains). The classes are not limited to naturally occurring amino acids, but also include artificial amino acids, such as beta or gamma amino acids and those containing non-natural side chains, and/or other similar monomers such as hydroxy acids. For example, the amino acid sequence of the variant may have the sequence of CagA^N or a portion thereof, in which one or more amino acids of CagA^N or the portion thereof are changed. The variant polypeptide sequence is preferably one which is not found in nature.

Methods of making a variant of a protein are well known in the art and include genetic engineering approaches such as introducing mutations into the gene encoding the protein of interest using techniques such as site-directed mutagenesis or random mutagenesis. Site-directed mutagenesis allows precise control over the location and nature of the amino acid substitutions, insertions, or deletions introduced into the protein sequence. Random mutagenesis techniques, such as error-prone PCR or chemical mutagenesis, generate a library of mutant proteins with diverse amino acid changes, which can be screened for the required function. Protein variants can be designed and engineered with the required properties using rational design or directed evolution approaches. Rational design involves making informed changes to the protein sequence based on structural and functional knowledge. Directed evolution methods, such as phage display or yeast surface display, involve generating libraries of protein variants and selecting for those with desired characteristics through iterative rounds of screening or selection.

Variants and portions of CagA^N can be tested for their ability to reduce and/or prevent and/or reverse amyloid formation using the methods described herein, such as the Thioflavin T (ThT) fluorescence assay.

In an embodiment, the N-terminal region of CagA (CagA^N) comprises, or consists of, amino acids 1-884 of CagA from strain 26695, or an orthologue thereof, optionally wherein CagA^N comprises, or consists of, the amino acid sequence of SEQ ID NO: 2. Accordingly, in an embodiment, the portion or variant of CagA^N is a portion or variant of amino acids 1-884 of CagA from strain 26695, optionally the portion or variant of CagA^N is a portion or variant of the amino acid sequence of SEQ ID NO: 2. SEQ ID NO: 2 is shown in the sequence listing table herein. The inventors have surprisingly found that the N-terminal region spanning of CagA, designated "CagA^N" herein, has potent anti-amyloid activity. In an embodiment, the portion or variant of the N-terminal region of CagA (CagA^N) that is capable of reducing and/or preventing and/or reversing amyloid formation is between 20 and 400 amino acids in length.

In an embodiment, the portion or variant of the N-terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is between 20 and 400 amino acids in lengths, such as between 20 and 350, 20 and 300, 20 and 250, 20 and 200, 20 and 150, 20 and 100, or between 20 and 50 amino acids in length. In an embodiment, the portion or variant of the N-terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is between 100 and 400 amino acids in lengths, such as between 100 and 350, 100 and 300, 100 and 250, 100 and 200, or 100 and 150 amino acids in length. In an embodiment, the portion or variant of the N-terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is between 200 and 400 amino acids in lengths, such as between 200 and 350, 200 and 300, or 200 and 250 amino acids in length. In an embodiment, the portion or variant of the N-terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is between 250 and 400 amino acids in lengths, such as between 250 and 350, or between 250 and 300 amino acids in length. In an embodiment, the portion or variant of the N-terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is between 300 and 400 amino acids in lengths, such as between 300 and 350 amino acids in length. In an embodiment, the portion or variant of the N-terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is between 350 and 400 amino acids in lengths.

In an embodiment, the portion or variant of the N-terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is at least 50 amino acids in length, such as at least 60, 70, 90, 110, 130, 150, 170, 190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, 650, 670, 690, 710, 730, 750, 770, 790, 810, 830, 850, 870, or at least 880 amino acids in length. In an embodiment, the portion or variant of the N- terminal region of CagA that is capable of reducing and/or preventing and/or reversing amyloid formation is at least 300 amino acids in length.

CagA^N can be divided into Domain I (DI), Domain II (D2) (SEQ ID NO: 3), and Domain III (D3). Domain I comprises 10 o-helices, Domain II (D2) forms the central core of CagA, spanning residues 300-644 of CagA of H. pylori (SEQ ID NO: 1) and features a substantial anti-parallel 0-sheet, encompassing a subdomain with five o-helices and two short 0-sheets, and Domain III adopts a four-helix bundle structure with the C- terminal region from a long o-helix connecting Domain II and III (Fig. lb). As can be seen in the accompanying Examples, the inventors have surprisingly found that Domain II (D2) is an effective inhibitor of amyloid fibril formation. In an embodiment, the portion of the N-terminal region of CagA (CagA^N) comprises Domain II (D2) of CagA^N. In an embodiment, the portion of the N-terminal region of CagA (CagA^N) consists of Domain II (D2) of CagA^N.

In an embodiment, the variant has at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%,

56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,

70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,

84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99%, or has 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 and maintains at least one of the major properties of CagA^N or a similar tertiary structure.

A polypeptide or protein having a certain percentage (for example, 80%, 85%, 90%>, or 95%) of "sequence identity" to another sequence means that, when aligned, that percentage of amino acids are the same in comparing the two sequences. For the purposes of the present invention, the sequence identity between two amino acid sequences may be determined using the Needleman-Wunsch algorithm (Needleman & Wunsch, 1970, J Mol Biol 48(3) :443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al, 2000, Trends Genet 16(6) :276-277), preferably version 5.0.0 or later. For Example, the D2 domain of CagA^N shares 39% sequence identity with CagA^N.

The term "protein", "peptide" and "polypeptide" are used interchangeably and in their broadest sense include a compound of two or more subunit amino acids, amino acid analogs or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunit may be linked by other bonds, e.g., ester, ether, etc. A protein or peptide must contain at least two amino acids and no limitation is placed on the maximum number of amino acids which may comprise a protein's or peptide's sequence. As used herein the term "amino acid" refers to either natural and/or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogues and peptidomimetics. In an embodiment, the portion comprises SEQ ID NO: 3. In an embodiment, the portion consists of a polypeptide having the sequence of SEQ ID NO: 3.

In an embodiment, the agent comprises SEQ ID NO: 2. In an embodiment, the agent consists of a polypeptide having the sequence of SEQ ID NO: 2.

In amyloid research the designation nucleation (primary) is used for a concentrationdependent stochastic event by which misfolded proteins bind to each other, thereby shifting the equilibrium and allowing the attraction of additional structurally identical/related molecules. This creates a protofilament which grows by addition of new identical molecules to fibril ends. The process templates identical misfolding of the subunits. The resulting fibrils do not necessarily adopt the exact same misfolding as the parent fibril (Merrill D. Benson, et al., (2020) Amyloid nomenclature 2020: update and recommendations by the International Society of Amyloidosis (ISA) nomenclature committee, Amyloid, 27:4, 217-222). In an embodiment, the agent reduces and/or prevents and/or reverses amyloid formation by inhibiting fibril formation by impeding primary nucleation, secondary nucleation, and the elongation process. The kinetics of amyloid aggregation involves distinct stages as described in Meisl, G., et al,. Kinetic Analysis of Amyloid Formation. Methods Mol. Biol. 1779, 181-196 (2018)). Specifically, in the primary nucleation phase, monomers come together to create a nucleus (primary nucleation, k_n), from which a fibril can start to elongate (elongation, k+) simultaneously, during secondary nucleation (/Q), monomers adhere to the fibril's surface, catalyzing the development of a new nucleus and facilitating exponential fibril growth (see the schematic representation in Figure 5 (a) and (b) for a visual depiction of this process. As can be seen from the accompanying Examples, the inventors have surprisingly found that CagA^N is capable of impeding primary nucleation, secondary nucleation, and the elongation process.

In an embodiment, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, is fused to another protein having anti-amyloid activity, such as a BRICHOS domain. In an embodiment, the protein having anti-amyloid activity is BRICHOS domain (or a mutant thereof) such as those of SEQ ID NO: 4 or 5.

BRICHOS is an about 100-residue domain found in 10 human protein families, and the name is derived from three of these, Bri2 (associated with dementia and brain amyloid), chondromodulin (chondrosarcoma), and prosurfactant protein C (proSP-C; interstitial lung disease and amyloid). All BRICHOS-containing proproteins have well- conserved regions that are prone to form p-strands. BRICHOS has been proposed to assist the amyloid-prone region of their respective proprotein to fold correctly during biosynthesis (Chen et al., Nature Communications volume 8, Article number: 2081 (2017)). Bri2 BRICHOS domain (SEQ ID NO: 4) can pass the blood brain barrier with a ratio from 0.5 to 1% after intravenous injection (Blood-brain and blood-cerebrospinal fluid passage of BRICHOS domains from two molecular chaperones in mice, J Biol Chem. 2019 Feb 22;294(8):2606-2615).

In an embodiment, the capability of the agent to reduce and/or prevent and/or reverse amyloid formation is determined using a Thioflavin T (ThT) fluorescence assay, optionally using a ThT assay as described in the accompanying Examples.

Thioflavin T (ThT) is a benzothiazole dye that exhibits enhanced fluorescence upon binding to amyloid fibrils and is commonly used to diagnose amyloid fibrils, both ex vivo and in vitro. It binds to amyloid structures with a resulting increase of the fluorescence intensity (see Khurana, R. et al. Mechanism of thioflavin T binding to amyloid fibrils. J. Struct. Biol. 151, 229-238 (2005)). A ThT assay is well known in the art and is described in detail in the accompanying Examples. It is possible to monitor the aggregation kinetics as a function of time at a range of different initial monomer concentrations.

When an agent can inhibit amyloid formation, the aggregation kinetics (Y for ThT fluorescence and for amyloid fibril mass; X for Time) shift to the right, indicating that the amyloid substrate will need more time (unit: h) to form amyloid, i.e., an inhibition effect. Alternatively, if an agent is inactive or can promote amyloid formation, then the kinetic for the amyloid substrate will remain the same or shift to the left, indicating that in the presence of the agent the amyloid substrates will form amyloid fibril as normal or need less time to form amyloid (i.e. promotion effects). The results of the ThT assay are presented as the half-time (n/2), which is the time needed to reach to halfway point of the amyloid forming plateau.

In an embodiment, the amyloid is formed from amyloid fibril proteins and/or amyloid protein inclusions.

By "amyloid fibril protein" we include the meaning of elongated, insoluble protein aggregates, typically characterized by a cross-p-sheet secondary structure. An amyloid fibril protein can occur in tissue deposits as rigid, non-branching fibrils approximately 10 nm in diameter. The fibrils bind the dye Congo red and exhibit green, yellow or orange birefringence when the stained deposits are viewed by polarization microscopy. When isolated from tissues and analyzed by X-ray diffraction, the fibrils exhibit a characteristic cross [3 diffraction pattern. To carry out chemical identification of an amyloid fibril protein, immunohistochemistry, Western blotting, mass spectrometry, after or without combination with laser capture, amino acid sequencing, and immune- electron microscopy are useful techniques (see Table 1 for examples and Joel N. Buxbaum, et al., (2022) Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee, Amyloid, 29:4, 213-219). According to the International Society of Amyloidosis (ISA) Nomenclature Committee, the following different classes of amyloid fibril exist and will be known to the person skilled in the art: i. In vivo and ex vivo disease-related fibrils; ii. In vivo and ex vivo functional fibrils; iii. Recombinant fibrils of disease-related proteins and of functional amyloid proteins; iv. Fibrils of synthetic or non-disease related peptides; and v. Fibrils from condensates and hydrogels that give the cross-0 diffraction pattern.

Members of all amyloid fibril classes are included herein by the term "amyloid fibril protein" (Joel N. Buxbaum, et al., (2022) Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee, Amyloid, 29:4, 213-219).

By "amyloid fibril protein" we also include amyloid-like fibrils, such as p53. P53 is a tumour suppressor protein, very often mutated in different malignancies and leading to its loss-of-function. P53 can aggregate into fibrillary forms with an aggregation pattern similar to that of other amyloids. It has therefore been proposed as an amyloid fibril protein. However, no characteristic 0-sheet structure appears to have been shown for the in vivo p53 fibrils. Fibrillar fragments of prostatic acidic phosphatase (PAP) have been termed Semen-derived Enhancer of Virus Infection (SEVI). PAP fragments, are believed to play an active role in human immunodeficiency virus infections and have been shown to form in vivo. Accordingly, amyloid-like fibrils may not have the characteristic 0-sheet structure but still aggregate into fibrillary forms with an aggregation pattern similar to that of other amyloids.

We also include functional amyloid fibril proteins which form a non-pathologic amyloid. In nature 0-sheet fibrils are adapted for many functions. Certain polypeptide hormones are stored in 0-sheet conformation, perhaps not as regular fibrils; melanin is bound to the 0-sheet fibrillar carrier (p-mell7) in melanosomes. The strength of 0-sheet fibrils is used by several bacterial and fungal organisms. Bacteria make several different structures, such as biofilms, that have 0-sheet fibrillary compositions. The fungus Neurospora crassa expresses the hydrophobin EAS which forms amphipathic amyloid monolayers that facilitate spore formation and dispersal. These are all examples of what functional amyloid is (Joel N. Buxbaum, et al., (2022) Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee, Amyloid, 29:4, 213-219)

We also include intracellular protein inclusions such as neurofibrillary tangles that have been described to have fibrillar structure, a cross p-sheet X-ray diffraction pattern and bind Congo red with green birefringence.

By "amyloid protein inclusions" we include the meaning of intracellular protein inclusions such as neurofibrillary tangles which have fibrillar structure, a cross 0-sheet X-ray diffraction pattern and bind Congo red with green birefringence. Lewy bodies in the brains of Parkinson's disease patients with dementia consist of fine amyloid-like fibrils, which sometimes, stain with Congo red and exhibit birefringence (see Table 2 for examples and Joel N. Buxbaum, etal., (2022) Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee, Amyloid, 29:4, 213-219).

In an embodiment, the amyloid is formed from amyloid fibril proteins and/or amyloid protein inclusions; preferably selected from those recited in Tables 1 and 2.

Table 1: Amyloid fibril proteins and their precursors in human.^aProteins are listed, when possible, according to relationship. Thus, apolipoproteins are grouped together, as are polypeptide hormones.^bADan is the product of the same gene as ABri.^cAlso called amylin. (Joel N. Buxbaum, et al., (2022) Amyloid nomenclature 2022: update, novel proteins, and recommendations by the International Society of Amyloidosis (ISA) Nomenclature Committee, Amyloid, 29:4, 213-219)

Table 2. Intracellular inclusions with known biochemical composition.^aSimplified - additional components may exist.^bAlso included in Table 1 since deposits may appear extracellularly.^CALS Amyotrophic lateral sclerosis, FTD Frontotemporal dementia.

In an embodiment, the amyloid is formed from a plurality of mammalian amyloid fibril proteins, such as human amyloid fibril proteins.

In an embodiment, the amyloid is formed from a plurality of microbial amyloid fibril proteins, selected from the group comprising: curli proteins; CsgA proteins; FapC proteins, TasA proteins, Biofilm-associated protein (Bap); PSM; SuhB; CdrA; CsgA; Pl; WapA; SMU_63C; Microcin E492; RbmA; TasA; MTP; Tu Elongation factor; HpaG; BE- AM1; Rodlins; Chaplins; Het-s; HET-s-like proteins (HET-s-like), Sup35, Ure2, HIV-1 Tat protein, Hepatitis C virus (HCV) core protein, Influenza A virus (IAV) matrix protein Ml, and Polyomavirus capsid proteins.

Curli proteins (CsgA and CsgB) are major components of extracellular amyloid fibrils produced by several Gram-negative bacteria, including Escherichia coli and Salmonella enterica. The major curli subunit, CsgA, forms the amyloid fibrils, while CsgB acts as a nucleator for CsgA polymerization. Curli fibers are involved in biofilm formation and adhesion to surfaces. Biofilm-associated protein (Bap) is produced by Staphylococcus aureus and certain other bacteria. Bap forms amyloid-like fibrils that contribute to biofilm formation and adherence to surfaces, enhancing bacterial colonization and persistence.

TasA is a protein produced by Bacillus subtilis that forms amyloid-like fibrils known as TasA fibers. These fibrils are involved in the formation and stability of biofilms produced by B. subtilis, contributing to surface adhesion and community development.

Pseudomonas aeruginosa produces amyloid fibrils composed of the FapC protein, which contribute to biofilm formation and persistence in chronic infections. FapC amyloids are associated with the development of antibiotic resistance and immune evasion in P. aeruginosa biofilms.

Het-s is a protein from the fungus Podospora anserina that forms amyloid fibrils involved in a programmed cell death process called heterokaryon incompatibility. HET- s-like proteins (HET-s-like), similar to Het-s, these proteins are found in other fungi and also play roles in programmed cell death and fungal self-recognition processes. Sup35 (Prion domain) is a protein found in the yeast Saccharomyces cerevisiae. Its prion domain can convert between soluble and amyloid forms, leading to the formation of the [PSI+] prion, which alters the yeast's phenotype. Ure2 (Prion domain) is another yeast protein from Saccharomyces cerevisiae. Its prion domain can also convert between soluble and amyloid forms, leading to the formation of the [UR.E3] prion, which alters nitrogen catabolite repression.

The transactivator of transcription (Tat) protein from human immunodeficiency virus type 1 (HIV-1) has been reported to form amyloid-like fibrils in vitro. These fibrils exhibit structural and functional similarities to amyloid fibrils associated with neurodegenerative diseases. Studies have suggested that the core protein of hepatitis C virus (HCV) can undergo amyloid-like aggregation under certain conditions. These aggregates may contribute to liver pathogenesis in chronic HCV infection. The matrix protein Ml of influenza A virus (IAV) has been shown to form amyloid-like fibrils in vitro. These fibrils may play a role in viral assembly and budding. Some studies have suggested that capsid proteins from certain polyomaviruses, such as the JC virus (JCV) and the BK virus (BKV), can form amyloid-like fibrils under certain conditions. These fibrils may contribute to viral persistence and pathogenesis in infected individuals.

In an embodiment, the plurality of microbial amyloid fibril-forming proteins are a plurality of bacterial, fungal or viral amyloidogenic proteins. In an embodiment, the plurality of microbial amyloidogenic proteins are a plurality of bacterial amyloid fibril proteins.

In the in vitro experiments described herein, a dose from nanomolar to few micromolar level is sufficient to prevent/reduce/reverse biofilm formation. The concentration range for administering the agent can vary widely depending on factors, for example, the route of administration, the patient's age, weight, and medical condition, as well as the desired therapeutic effect. For oral administration, typically, concentrations range from micrograms to milligrams per milliliter (pg/mL to mg/mL). For example, a common concentration might be 5 mg/mL for a liquid oral medication. For intravenous (IV) administration, concentrations can range from very low concentrations, such as those used for continuous infusions (e.g., 1-10 pg/mL for certain medications), to higher concentrations for bolus doses (e.g., 1-20 mg/mL). Intravenous medications are often delivered in standardized concentrations or diluted to achieve the desired dose. For topical administration (including to a surface) concentrations vary widely depending on the intended use and the specific formulation. For example, a topical formulation might have a concentration of 0.01-0.5% (weight/volume) of the active ingredient, such as 10 mg/ml - 50 mg/ml. For subcutaneous or intramuscular Injection concentrations can range from micrograms to milligrams per milliliter (pg/mL to mg/mL), similar to IV administration. However, the concentrations may be adjusted based on factors such as injection volume and desired absorption rate. These are just general ranges, and specific concentrations should always be determined by a healthcare professional based on the individual patient's needs.

In an aspect, the invention provides use of an agent for reducing and/or preventing and/or reversing microbial biofilm formation, wherein the agent comprises, or consists of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

In an aspect, the invention provides use of an agent for reducing and/or preventing and/or reversing microbial biofilm formation, wherein the agent comprises, or consists of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

In an aspect, the invention provides a method for reducing and/or preventing and/or reversing microbial biofilm formation, the method comprising the step of contacting a biofilm, or a surface susceptible to biofilm formation with an agent comprising, or consisting of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

In an embodiment, the above uses and methods are carried out outside of the human or animal body.

In an aspect, the invention provides a method for reducing and/or preventing and/or reversing microbial biofilm formation in a subject, the method comprising the step of administering an agent comprising, or consisting of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation to the subject.

In an aspect, the invention provides an agent comprising, or consisting of, the N- terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation for use in reducing and/or preventing and/or reversing microbial biofilm formation in a subject.

By "reducing and/or preventing and/or reversing microbial biofilm formation" we include the meaning of a reduction in the formation of the amyloid fibrils that is a component of a microbial biofilm. For example, biofilm formation will be reduced and/or prevented and/or reversed following contact with the agent described herein compared to biofilm formation in the absence of agent. For example, biofilm formation on a surface contacted with the agent as described herein may be reduced and/or prevented and/or reversed by 10%, 20%, 30% or 40%, such as by at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% when compared to biofilm formation on a surface which has not been contacted with the agent.

By "a surface which may be susceptible to microbial biofilm formation", we include the meaning of an surface outside of the human or animal body that provides favourable conditions for microorganisms to adhere and form biofilms but which does not yet comprise biofilm formation. Microorganisms tend to adhere more readily to surfaces with irregularities or roughness, as these provide more opportunities for attachment. Surfaces with organic or inorganic nutrients present can support microbial growth and biofilm formation. For example, surfaces in contact with water, food residues, bodily fluids, or organic matter are often susceptible. Moist surfaces provide an environment conducive to microbial growth and biofilm formation. Surfaces that come into contact with microorganisms, either from the environment or through direct contamination, are more likely to support biofilm formation. Examples of surfaces susceptible to microbial biofilm formation are described herein.

Methods for assessing biofilm formation are well known in the art and are descried herein. Crystal violet is commonly used in microbiology to stain and visualize biofilms; an exemplary assay is as follows:

Preparation of Peg Lid Plates

The peg lid plates consist of a multi-well plate where each well contains pegs (small cylindrical plastic projections) instead of a flat bottom. These pegs provide a surface for biofilm formation. The peg lid is designed to fit into the corresponding wells of a standard microtiter plate.

Inoculation of Microorganisms

The microorganisms of interest, typically bacteria or fungi, are inoculated into each well of a microtiter plate containing a suitable growth medium. Each well of the microtiter plate corresponds to a peg on the peg lid. The microorganisms are allowed to adhere to and form biofilms on the surface of the pegs.

Incubation

The peg lid plate is then inverted and placed onto the microtiter plate containing the inoculated microorganisms, ensuring that each peg is submerged in the culture medium. The combined assembly is incubated under conditions conducive to biofilm formation, allowing the microorganisms to grow and form biofilms on the surface of the pegs.

Treatment:

Treatments such as anti-microbial agents or inhibitors can be added to the wells containing the microorganisms before or during incubation to assess their effects on biofilm formation.

Crystal Violet Staining

After an appropriate incubation period (typically 24-48 hours), the peg lid plate is carefully removed from the microtiter plate, and the pegs are gently rinsed to remove any non-adherent cells. The pegs are then immersed in a solution of crystal violet, which stains the biofilms formed by the adherent microorganisms.

Destaininq

Excess crystal violet is washed away, and the pegs are gently rinsed to remove any unbound dye. The biofilm-bound crystal violet is retained on the surface of the pegs.

Quantification

The pegs are transferred to a clean microtiter plate, and the crystal violet bound to the biofilms is solubilized using a suitable solvent, typically ethanol or acetic acid. The absorbance of the solubilized crystal violet solution is then measured spectrophotometrically at an appropriate wavelength (usually around 590 nm). The absorbance values obtained are proportional to the biomass of the biofilms formed on the pegs, allowing for the quantification of biofilm formation.

By using peg lids and crystal violet staining, it is possible to measure biofilm formation in a high-throughput manner, allowing for the screening of multiple conditions, treatments, or microbial strains simultaneously. By comparing the absorbance values of the treated samples with those of untreated control samples, it is possible to determine the efficacy of an agent in inhibiting biofilm formation. The degree of activity can be quantified as the percentage reduction in absorbance/biomass. For instance, an agent might be considered to have significant biofilm-inhibitory activity if there is a substantial reduction in absorbance at certain concentrations compared to untreated controls.

Biofilms are not exclusive to bacteria; many other microorganisms, as well as some multicellular organisms, can also produce biofilms. Some examples include various species of fungi, including Candida albicans and Aspergillus fumigatus, are capable of forming biofilms. These fungal biofilms are often associated with infections, particularly in immunocompromised individuals. Certain protozoa, such as those belonging to the genus Giardia and Entamoeba, can form biofilms in aquatic environments and within the host gastrointestinal tract. Algae, such as species of diatoms and green algae, are capable of forming biofilms on surfaces submerged in water, including rocks, aquatic plants, and artificial substrates. Some viruses have been shown to form biofilms under certain conditions. For example, bacteriophages (viruses that infect bacteria) can adhere to surfaces and form biofilms composed of viral particles. Certain multicellular organisms, such as marine invertebrates like corals, sponges, and mussels, can form biofilms with the help of symbiotic bacteria. These biofilms play important roles in the ecology and physiology of these organisms. Biofilms produced by non-bacterial organisms often have similar characteristics to bacterial biofilms, including the formation of extracellular polymeric substances (EPS) that provide structural support and protection to the community of organisms within the biofilm.

In an embodiment, the agent is effective at reducing and/or preventing and/or reversing microbial biofilm formation in microorganisms which have the capacity to generate amyloid fibrils. In an embodiment, the microorganism is selected from the group comprising bacteria, fungi, protozoa, algae, viruses, and multicellular organisms. In an embodiment, the microorganism is bacteria. In an embodiment, the agent is effective at reducing and/or preventing and/or reversing bacterial biofilm formation. Exemplary microorganisms are described herein.

In an embodiment, the agent is effective at reducing and/or preventing and/or reversing bacterial biofilm formation in bacteria which have the capacity to generate amyloid fibrils. For example, E. coli can produce curli amyloid fibrils, which are involved in cell-cell adhesion and biofilm formation. These fibrils are composed of the major curli subunit protein, CsgA. Similar to E. coli, Salmonella enterica can produce curli amyloid fibrils, contributing to biofilm formation and host colonization. Staphylococcus species such as Staphylococcus aureus can generate amyloid-like fibrils composed of the protein Bap (Biofilm-associated protein), which facilitates biofilm formation and adherence to surfaces. Bacillus species can produce amyloid-like fibrils known as TasA fibrils, which are involved in biofilm formation and stability. Pseudomonas aeruginosa is known to produce amyloid fibrils composed of the protein FapC, contributing to biofilm formation and persistence in chronic infections. Klebsiella pneumoniae is known to produce amyloid component Microcin E492. Streptococcus is known to produce amyloid component Pl, WapA, and SMU_63C. Vibrio cholerae is known to produce amyloid component RbmA. Mycobacterium tuberculosis is known to produce amyloid component MTP. Gallibacterium anatis is known to produce amyloid component Tu Elongation factor. Xanthomonas anxidopidos is known to produce amyloid component HpaG. Solibacillus Silvestris AMI is known to produce amyloid component BE-AM1. Streptomyces coelicolor is known to produce amyloid component Rodlins, and Chaplins.

In an embodiment, the bacteria which have the capacity to generate amyloid fibrils are selected from the group comprising: Staphylococcus species, Salmonella enterica, Pseudomonas species, E. coli, Streptococcus mutans, Klebsiella pneumoniae, Legionella pneumophila, Vibrio cholerae, Bacillus species, Mycobacterium tuberculosis, Gallibacterium anatis, Xanthomonas anxidopidos, Soli bacillus Silvestris AMI, Streptomyces coelicolor.

It will be appreciated that by inhibiting amyloid formation in a microorganism, the agent is capable of disrupting the integrity of the biofilm in the microorganism, thus making the microorganism susceptible to treatment, such as with antibiotics.

In an embodiment, the methods and uses for reducing and/or preventing and/or reversing microbial biofilm formation further comprise contacting the microbial biofilm, or contacting a surface which may be susceptible to microbial biofilm formation with a further anti-microbial agent. Examples of anti-microbial agents are described herein.

In an embodiment, the agent is capable of reducing and/or preventing and/or reversing bacterial biofilm formation across different bacterial species.

In an embodiment, reducing and/or preventing and/or reversing microbial biofilm formation comprises contacting a biofilm present on a surface, or contacting a surface which may be susceptible to biofilm formation, with an effective amount of the agent. In an embodiment, the surface is outside of the human or animal body. The surface can be impregnated with the agent, coated with the agent, or a combination thereof. A surface can be biological or non-biotic. Examples of biological surfaces include, but are not limited to, a surface of an animal or a plant. Examples of non-biotic surfaces include any medium, e.g., plastic, glass, or metal, used in an article, for instance, prosthetics, floors, counters, soil, dental instruments and equipment (such as dental water lines, dental tools, dentures, dental retainers, dental braces), medical instruments, medical devices (e.g., such as catheters, implants, and surgical instruments), contact lenses and lens cases, bandages, tissue dressings, surfaces (e.g., tabletop, countertop, bathtub, tile, filters (e.g., water filter), membranes (e.g., reverse osmosis membrane, etc.), fabrics (e.g., anti-odour fabric), tubing, drains, pipes (such as water pipes, gas pipes, oil pipes, drilling pipes, fracking pipes, sewage pipes, drainage pipes), water storage tanks, hoses, fish tanks, showers, children's toys, boat hulls, cooling- and heating-water systems including cooling towers,

In some embodiments, the number of the bacterial colony forming units (CFUs) on a surface following contact with the agent may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or reduced by 100% compared to the number of CFUs on the surface immediately before contact with the agent. Methods for determining the number of bacterial colony-forming units (CFUs) on a surface are well known in the art. A general protocol is provided herein.

(1) Collect a sample from the surface of interest using a sterile swab, loop, or other suitable means. Ensure that the sampling method does not introduce contamination.

(2) Transfer the collected sample into a sterile liquid medium, such as saline solution or sterile broth, to suspend the bacteria.

(3) To reduce the number of bacteria in the sample to a countable range, perform serial dilutions. This involves diluting the sample multiple times with a sterile diluent. Each dilution is then plated onto an agar plate.

(4) After dilution, plate aliquots of the diluted sample onto agar plates using techniques such as spread plating or pour plating. Ensure that the agar plates are appropriate for the type of bacteria you are trying to count and that they provide optimal conditions for bacterial growth.

(5) Incubate the agar plates at the appropriate temperature for the bacteria being studied. This could be at room temperature, body temperature, or another specific temperature depending on the bacterial species.

(6) After the appropriate incubation period (usually 24-48 hours), count the number of visible bacterial colonies on the agar plates. Each colony represents a single viable bacterial cell from the original sample.

(7) Calculate the number of CFUs per unit area or volume based on the dilution factor and the number of colonies counted on each plate.

(8) Finally, report the results as CFUs per unit area or volume of the original sample. It will be appreciated that the above method could be used to compare the number of CFU present on a surface before and after contact of the surface with the agent.

In an aspect, the invention provides an agent comprising, or consisting of, the N- terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation, for use in medicine.

In an aspect, the invention provides an agent comprising, or consisting of, the N- terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation, for use in preventing and/or treating a disease or condition characterised by amyloid formation.

In an aspect, the invention provides a method for preventing and/or treating a disease or condition characterised by amyloid formation in a subject, comprising administering to the subject an effective amount of an agent comprising, or consisting of, the N- terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

In an aspect, the invention provides use of an agent comprising, or consisting of, the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation, in the manufacture of a medicament for preventing and/or treating a disease or condition characterised by amyloid formation.

By "a disease or condition characterised by amyloid formation", we include the meaning of any medical condition which is facilitated by the production and/or accumulation of amyloid fibrils. The amyloid fibril may be pathological amyloid fibrils, which can lead to the development of neurodegenerative disease as described herein, or functional amyloid fibrils, which can lead to biofilm formation during microbial infection.

In an embodiment, the disease or condition characterised by amyloid formation is a microbial infection that produces a biofilm in a subject.

By "a microbial infection that produces a biofilm in a subject", we include the meaning of an infection caused by a microorganism that is capable of producing a biofilm, whereby the biofilm provides a shield for the microorganisms such as bacteria or fungi, making them more resistant to antibiotics and the body's immune system. Biofilm- associated infections are often chronic and difficult to treat, leading to persistent or recurrent infections, which reduction results in an increased susceptibility of the microorganism to anti-microbial agents. The prophylactic or therapeutic reduction in the structure of a biofilm may be achieved by preventing the initial formation of biofilms or by disrupting established biofilms.

Microbial infections and disease that can be treated by the methods of this invention include those caused by any microorganism that is capable of producing a biofilm. Non-limiting examples of such microorganism include, Streptococcus agalactiae, Neisseria meningitidis, Treponema denticola, pallidum), Burkholderia cepacia, or Burkholderia pseudomallei.

The microbial infection may be one or more of Haemophilus influenzae (nontypeable), Moraxella catarrhalis, Streptococcus pneumoniae, Streptococcus pyogenes, Pseudomonas aeruginosa, Mycobacterium tuberculosis. These microbial infections may be present in the upper, mid and lower airway (otitis, sinusitis, bronchitis but also exacerbations of chronic obstructive pulmonary disease (COPD), chronic cough, complications of and/or primary cause of cystic fibrosis (CF) and community acquired pneumonia (CAP)). Infections might also occur in the oral cavity (caries, periodontitis) and may be caused by Streptococcus mutans, Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans. In this example, the agent may be formulated in mouthwashes or toothpaste.

Infections might also be localized to the skin (abscesses, 'staph' infections, impetigo, secondary infection of burns, Lyme disease) and caused by Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Borrelia burdorferi. In this example, the agent may be formulated for topical administration.

Infections of the urinary tract (UTI) can also be treated and are typically caused by Escherichia coli. Infections of the gastrointestinal tract (GI) (diarrhea, cholera, gall stones, gastric ulcers) are typically caused by Salmonella enterica serovar, Vibrio cholerae and Helicobacter pylori. Infections of the genital tract include and are typically caused by Neisseria gonorrhoeae. Infections can be of the bladder or of an indwelling device caused by Enterococcus faecalis. Infections associated with implanted prosthetic devices, such as artificial hip or knee replacements, or dental implants, or medical devices such as pumps, catheters, stents, or monitoring systems, typically caused by a variety of bacteria, can be treated by the methods of this invention. These devices can be coated or conjugated to an agent as described herein. Infections caused by Streptococcus agalactiae can also be treated by the methods of this invention and it is the major cause of bacterial septicaemia in newborns. Infections caused by Neisseria meningitidis which can cause meningitis can also be treated. Thus, by practicing the methods of this invention, these diseases and complications from these infections can also be prevented or treated.

Examples of subjects that can be treated include humans, murine (mice and rats), domesticated livestock such as bovine, porcine, equine, and avian species. The treatment of a subject can result in a reduction in the amount of the microorganism (e.g., the number of colony forming units (cfu)) in or on the body of the subject.

The reduction can be at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the subject before the administration. In this aspect, the term "effective amount" refers to an amount that is sufficient to result in the desired effect of reducing the amount of the microorganism.

In an embodiment, the methods and uses for preventing and/or treating a microbial infection that produces a biofilm in a subject further comprises administering an antimicrobial agent to the subject having a microbial infection that produces a biofilm.

Examples of anti-microbial agents include antibiotics, antiseptics, and disinfectants. Also included are enzymes such as dispersin B and DNase which degrade the extracellular polymeric substances (EPS) that hold biofilms together, weakening their structure and facilitating their removal. Antimicrobial peptides (AMPs) that exhibit antimicrobial activity against a wide range of microorganisms, including those within biofilms. Quorum sensing inhibitors (QSIs) which interfere with bacterial cell-to-cell communication mechanisms, disrupting quorum sensing and inhibiting biofilm formation. Metal ions, such as silver, copper, and zinc, have antimicrobial properties and can disrupt biofilm formation and viability. Surfactants disrupt biofilm structure by interfering with cell adhesion and biofilm matrix formation. Bacteriophages can target and lyse biofilm-associated bacteria.

Examples of antibiotics include Rifampicin, Vancomycin, Ciprofloxacin, Doxycycline, Kanamycin, Amikacin, Gentamicin, Tobramycin, Erythromycin, Clindamycin.

Methods of administering an agent of the invention to a subject are described herein. In an embodiment, the subject is administered an effective amount of the agent.

In an embodiment, the disease or condition characterised by amyloid formation is a protein misfolding disease.

By "protein misfolding disease" we include the meaning of a disorder characterised by the accumulation of misfolded proteins in a subject. Normally, proteins fold into specific three-dimensional shapes that are crucial for their proper function. However, in protein misfolding diseases, genetic mutations, environmental factors, or age- related changes can lead to proteins folding incorrectly or becoming misfolded. Misfolded proteins have a tendency to aggregate and form insoluble structures, such as amyloid fibrils or inclusion bodies which can accumulate within both eukaryotic and prokaryotic cells or in extracellular spaces thus disrupting normal cellular functions. Examples of protein misfolding diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), and prion diseases like Creutzfeldt-Jakob disease.

In an embodiment, the disease or condition characterised by amyloid formation is an amyloid disease.

By "amyloid disease" we include the meaning of a protein misfolding disease characterised by the aberrant folding of proteins into structurally abnormal conformations. In these diseases, proteins fail to adopt their native three-dimensional structures, leading to the formation of insoluble aggregates or toxic intermediates.

Fibrillar amyloid structures are linked to around 40 human diseases, notably neurodegenerative disorders such as AD and PD, as well as systemic conditions like type 2 diabetes (Sipe, J. D. et al. International Society of Amyloidosis 2016 Nomenclature Guidelines. Amyloid 23, 209-213 (2016)). In an embodiment, the protein misfolding disease is one mediated by any one of the amyloid fibril proteins and/or intracellular protein inclusions listed in Tables 1 and 2.

In an embodiment, the protein misfolding disease is selected from the group consisting of tauopathies such as Alzheimer's disease and frontotemporal dementia, Parkinsons Disease (PD) and other synucleinopathies, Huntington's disease, familial amyloidotic polyneuropathy (FAP), senile systemic amyloidosis, Amyotrophic lateral sclerosis (ALS), Prion diseases, dialysis-related amyloidosis, secondary (reactive) amyloidosis, hereditary amyloidosis (AApoAI amyloidosis), familial amyloidosis, Finnish hereditary amyloidosis, hereditary non-neuropathic systemic amyloidosis, systemic immunoglobulin light chain amyloidosis, Immunoglobulin heavy chain amyloidosis (AH) and hereditary renal amyloidosis.

Alzheimer's disease is characterized by the accumulation of misfolded amyloid-beta (AP) protein and tau protein in the brain, leading to neuronal dysfunction and cognitive decline. Alpha-synuclein is a protein abundant in neuronal cells and is associated with the formation of Lewy bodies, characteristic pathological aggregates found in Parkinson's disease and other synucleinopathies. Tau protein is predominantly found in neurons and is involved in stabilizing microtubules. In tauopathies such as Alzheimer's disease and frontotemporal dementia, tau protein undergoes abnormal phosphorylation and forms intracellular neurofibrillary tangles. Huntington's disease is caused by the expansion of CAG repeats in the huntingtin (HTT) gene, leading to the production of misfolded mutant huntingtin protein. Accumulation of mutant huntingtin protein results in neuronal dysfunction and neurodegeneration. Transthyretin (TTR) is a transport protein primarily produced by the liver and is associated with familial amyloidotic polyneuropathy (FAP) and senile systemic amyloidosis. Amyotrophic lateral sclerosis (ALS) is caused by misfolded proteins such as superoxide dismutase 1 (SOD1) or TDP-43 which accumulate in motor neurons, leading to motor neuron degeneration and muscle weakness. Prion diseases, such as Creutzfeldt-Jakob disease (CJD) and variant Creutzfeldt-Jakob disease (vCJD), involve the misfolding of normal prion protein (PrP C) into an abnormal isoform (PrP Sc), which can propagate and induce further protein misfolding. Islet amyloid polypeptide (IAPP or amylin) is cosecreted with insulin by pancreatic beta cells and is involved in the regulation of glucose homeostasis. In type 2 diabetes, IAPP aggregates into amyloid fibrils within the pancreatic islets. Prion protein (PrP) is primarily known for its role in prion diseases, such as Creutzfeldt-Jakob disease (CJD) and bovine spongiform encephalopathy (BSE or "mad cow disease"). Beta-2 microglobulin (£2M) is a component of the major histocompatibility complex (MHO) class I molecules and is associated with dialysis- related amyloidosis in patients with end-stage renal disease. Serum amyloid A (SAA) is an acute-phase protein produced in response to inflammation and is associated with secondary (reactive) amyloidosis. Apolipoprotein A-I (apoAI) is associated with hereditary amyloidosis (AApoAI amyloidosis). Gelsolin (AGel) is associated with familial amyloidosis, Finnish type (Finnish hereditary amyloidosis). Lysozyme is associated with hereditary non-neuropathic systemic amyloidosis. Systemic immunoglobulin light chain amyloidosis is a protein misfolding disease caused by the conversion of immunoglobulin light chains from their soluble functional states into highly organized amyloid fibrillar aggregates that lead to organ dysfunction. Immunoglobulin heavy chain amyloidosis (AH) is a type of amyloidosis caused by deposition of monoclonal immunoglobulin heavy chain. Fibrinogen A alpha chain (AFib) is associated with hereditary renal amyloidosis.

As used herein, by "administering," we include the meaning of the placement of a compositions or agent as disclosed herein into a subject by a method or route which results in at least partial delivery of the agent at a desired site. Pharmaceutical compositions comprising the compounds disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject.

In an embodiment, the methods and uses for preventing and/or treating a protein misfolding disease in a subject further comprise administering a further therapeutic moiety. Further therapeutic moieties include those that are already established for use in the prevention and/or treatment of a protein misfolding disease including those described herein.

It will be appreciated that the agent of the invention could be used alongside established Alzheimer's disease therapies such as cholinesterase inhibitors (e.g., donepezil, rivastigmine, galantamine) and NMDA receptor antagonists (e.g., memantine) which are aimed at symptomatic relief. Since Alzheimer's disease involves both Ap plaques and tau tangles, the agent could be part of a regimen that also includes experimental therapies like tau aggregation inhibitors or monoclonal antibodies targeting A[3 or tau. In the case of Parkinson's disease, the agent of the invention could be administered alongside dopaminergic medications (e.g., levodopa, dopamine agonists like pramipexole or ropinirole) which are standard treatments for managing motor symptoms. MAO-B inhibitors (e.g., selegiline, rasagiline) that prevent the breakdown of brain dopamine could also be part of the combination therapy. For type II diabetes, the agent of the invention could be combined with insulin or insulin analogs in patients requiring insulin therapy. Other antidiabetic medications such as metformin, sulfonylureas, GLP-1 receptor agonists (e.g., liraglutide), and SGLT2 inhibitors (e.g., canagliflozin) could also be part of the treatment regimen.

The administration of the agent and further therapeutic moiety may be sequential or simultaneous. The dosing regimens and modes of administration may be those which are normal or traditionally administered by physicians for the further therapeutic moiety.

In an embodiment, the methods and uses for preventing and/or treating a protein misfolding disease in a subject further comprise formulating the agent for delivery across the blood-brain barrier. Methods for formulating a drug so that it may cross the blood-brain barrier are known in the art, such as in Wu, D., et al. The blood-brain barrier: structure, regulation, and drug delivery. Sig Transduct Target Ther 8, 217 (2023), which is incorporated by reference in its entirety. Such methods include formulating drugs with lipid-based carriers such as liposomes, micelles, or lipid nanoparticles can improve their solubility, stability, and transport across the BBB. Encapsulating drugs within nanoparticles can protect them from degradation and facilitate their transport across the BBB. Nanoparticles can be engineered with surface modifications to target specific receptors or utilize endocytic pathways for enhanced BBB penetration. Conjugating drugs to endogenous transport substrates that are recognized by transporters expressed at the BBB can facilitate their uptake into the brain. For example, amino acid or glucose transporter systems can be exploited to transport drugs across the BBB. Utilizing receptor-mediated transcytosis involves targeting specific receptors expressed on the surface of brain endothelial cells to trigger vesicular transport of drugs across the BBB. Ligands or antibodies that bind to these receptors can be conjugated to drugs to facilitate their transport into the brain.

In an aspect, the invention provides an agent which comprises, or consists of, the N- terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

In an embodiment, the N-terminal region of CagA (CagA^N) or the portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation is isolated or recombinant.

By "isolated" in respect of polypeptides and proteins, we include the meaning of polypeptides and proteins that are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated or recombinant" means separated from constituents, cellular and otherwise, in which the cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody or fragment(s) thereof, which are normally associated in nature.

The isolated polypeptides and proteins are intended to include isolated wildtype and recombinantly produced polypeptides and proteins from prokaryotic and eukaryotic host cells, as well as muteins, analogs and fragments thereof.

In a further aspect, the protein is conjugated or linked to a detectable label. Suitable labels are known in the art.

Agents of the invention can comprise, or consist of, fusions or conjugates comprising, or consisting of, the N-terminal region of CagA (CagA^N) or the portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation and a protein having anti-amyloid activity or anti-microbial activity. Proteins having anti-amyloid activity and proteins having anti-microbial activity are known in the art and examples are provided herein.

Moreover, agents of the invention can comprise, or consist of, fusions or conjugates comprising the N-terminal region of CagA (CagA^N) or the portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation and marker sequences, such as a peptide to facilitate purification. For example, the marker amino acid sequence may be a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc.), among others, many of which are commercially available. As described in Gentz eta/., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides for convenient purification of the fusion protein. Other peptide tags useful for purification include, but are not limited to, the hemagglutinin ("HA") tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and the "FLAG" tag. The protein can be linked to a solubility tag for enhancing the solubility, including thioredoxin tag and NT* tag (Kronqvist, N. et al. Nat Commun 8, 15504 (2017)). Fusion tags for protein solubility, purification and immunogenicity in expression systems, such as bacteria are known in the art, such as those described in Costa S et al., (2014) Front. Microbiol. 5:63, which is incorporated by reference in its entirety.

In an aspect, the invention provides a polynucleotide encoding a polypeptide as described herein, e.g., a polypeptide having at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 2 and which is capable of reducing and/or preventing and/or reversing amyloid formation, and/or fusions and/or conjugates thereof as described herein.

A polynucleotide can encode a polypeptide having at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least

83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least

89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 2.

A polynucleotide encoding a polypeptide as described herein can be readily determined by one skilled in the art by reference to the standard genetic code, where different nucleotide triplets (codons) are known to encode a specific amino acid. As is readily apparent to a skilled person, the class of nucleotide sequences that encode any protein described herein is large as a result of the degeneracy of the genetic code, but it is also finite.

In an aspect, the invention provides a vector comprising the polynucleotides as described herein. A vector is a replicating polynucleotide, such as a plasmid, phage, or cosmid, to which another polynucleotide may be attached so as to bring about the replication of the attached polynucleotide. Construction of vectors containing a polynucleotide employs standard ligation techniques known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press (1989). A vector may provide for further cloning (amplification of the polynucleotide), i.e., a cloning vector, or for expression of the polynucleotide, i.e., an expression vector. By "vector" we include plasmid vectors, viral vectors, cosmid vectors, and artificial chromosome vectors. Examples of viral vectors include, for instance, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, and herpes virus vectors. Typically, a vector is capable of replication in a microbial host, for instance, a prokaryotic bacterium, such as E. coli. Preferably the vector is a plasmid. The vector may be an expression vector.

Selection of a vector depends upon a variety of desired characteristics in the resulting construct, such as a selection marker, vector replication rate, and the like. In some aspects, suitable host cells for cloning or expressing the vectors herein include eukaryotic cells, such as CHO cells, HEK cells, insect cells. Suitable host cells for cloning or expressing the vectors herein include prokaryotic cells. Suitable prokaryotic cells include eubacteria, such as gram-negative microbes, for example, E. coli.

Vectors may be introduced into a host cell using methods that are known and used routinely by the skilled person. For example, calcium phosphate precipitation, transfection, electroporation, heat shock, lipofection, microinjection, and viral- mediated nucleic acid transfer are common methods for introducing nucleic acids into host cells.

Polynucleotides can be produced in vitro or in vivo. For instance, methods for in vitro synthesis include, but are not limited to, chemical synthesis with a conventional DNA/RNA synthesizer. Commercial suppliers of synthetic polynucleotides and reagents for such synthesis are well known.

An expression vector optionally includes regulatory sequences operably linked to the coding region. The disclosure is not limited by the use of any particular promoter, and a wide variety of promoters are known. Promoters act as regulatory signals that bind RIMA polymerase in a cell to initiate transcription of a downstream (3' direction) coding region. The promoter used may be a constitutive or an inducible promoter. It may be, but need not be, heterologous with respect to the host cell.

An expression vector may optionally include a ribosome binding site and a start site (e.g., the codon ATG) to initiate translation of the transcribed message to produce the polypeptide. It may also include a termination sequence to end translation. A termination sequence is typically a codon for which there exists no corresponding aminoacetyl-tRNA, thus ending polypeptide synthesis. The polynucleotide used to transform the host cell may optionally further include a transcription termination sequence.

A vector introduced into a host cell optionally includes one or more marker sequences, which typically encode a molecule that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence may render the transformed cell resistant to an antibiotic, or it may confer compoundspecific metabolism on the transformed cell. Examples of a marker sequence are sequences that confer resistance to kanamycin, ampicillin, chloramphenicol, tetracycline, and neomycin.

In an aspect, the invention provides a combination of an agent as defined herein, and an anti-microbial agent as defined herein.

In an embodiment, the combination of agent and an anti-microbial agent are provided in separate containers and/or separate compositions. In an alternative embodiment, the combination of agent and an anti-microbial agent are formulated as a composition. In an embodiment, the anti-microbial agent is an antibiotic. Exemplary anti-microbial agent and antibiotics are described herein.

In one embodiment, a composition is formulated for use as a coating. A coating can be used to cover a surface, can be incorporated, e.g., impregnated, into a surface, or a combination thereof. A protein described herein can be combined with agents suitable for use in coating a surface, such as, but not limited to, polymers, plasticizers, pigments, colorants, glidants, stabilization agents, pore formers, and/or surfactants. Examples of polymers useful as coating agents include, but are not limited to, cellulose polymers such as cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropyl methylcellulose phthalate, and hydroxypropyl methylcellulose acetate succinate; polyvinyl acetate phthalate, polyurethane, epoxy, acrylic acid polymers and copolymers, and methacrylic resins, zein, shellac, and polysaccharides.

In one embodiment, a composition is formulated for use as a cleaning solution. Such a composition is suitable for application to a surface for cleaning and/or disinfecting the surface. A protein described herein can be formulated into a solution in a suitable solvent for administration in a spray bottle, for use as an aerosol or a foam suitable for applying onto surfaces.

In one embodiment, a composition for use as a cleaning/disinfecting solution includes, in addition to the agent described herein, an acceptable carrier and an anti- microbial agent. In one embodiment, a composition for use as a cleaning/disinfecting solution includes, in addition to the agent described herein, a cleaning agent, a disinfecting agent, or a combination thereof. An anti-microbial agent can be microbiocidal or microbiostatic. Anti-microbial agents that can be incorporated into cleaning formulations are known in the art and include Quaternary Ammonium Compounds (QACs), which are a type of chemical that is used to kill bacteria, viruses, and mold. Methods for making formulations for use as a disinfectant are known in the art.

Examples of surfaces that can be coated and/or disinfected with a composition described herein can be biological or non-biotic. Examples of biological surfaces include, but are not limited to, a surface of an animal or a plant. Examples of non- biotic surfaces include any medium, e.g., plastic, glass, or metal, used in an article, for instance, prosthetics, floors, counters, soil, dental instruments, teeth, dentures, dental retainers, dental braces including plastic braces, medical instruments, medical devices (e.g., endoscope), contact lenses and lens cases, catheters, bandages, tissue dressings, surfaces (e.g., tabletop, countertop, bathtub, tile, filters (e.g., water filter), membranes (e.g., reverse osmosis membrane, etc.), fabrics (e.g., anti-odour fabric), tubing, drains, pipes including water pipes, gas pipes, oil pipes, drilling pipes, fracking pipes, sewage pipes, drainage pipes, hoses, fish tanks, showers, children's toys, boat hulls, cooling- and heating-water systems including cooling towers.

In an aspect, the invention provides a pharmaceutical composition comprising an agent as defined herein and a pharmaceutically-acceptable carrier, diluent and/or excipient.

As used herein, the term "pharmaceutical composition" refers to the active agent in combination with a pharmaceutically acceptable carrier e.g. a carrier commonly used in the pharmaceutical industry. The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio Pharmaceutical compositions containing an agent of the invention provided herein can be prepared for storage by mixing the agent having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m- cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Compositions, such as those described herein, can also contain more than one active compound as necessary for the particular indication being treated. In certain embodiments, compositions comprise, or consist of, the agent described herein and one or more active compounds with complementary activities that do not adversely affect each other. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. For example, an agent of the invention can be combined with one or more further therapeutic moieties. Such combined therapy can be administered to the patient serially or simultaneously or in sequence.

The pharmaceutical compositions provided herein contain therapeutically effective amounts of the agent described herein. An agent of the invention is included in the pharmaceutically acceptable carrier in an effective amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. The therapeutically effective concentration can be determined empirically by testing the compounds in in vitro and in vivo systems using routine methods and then extrapolated therefrom for dosages for humans. In one embodiment, the compositions are formulated for single dosage administration. To formulate a composition, the weight fraction of compound is dissolved, suspended, dispersed or otherwise mixed in a selected carrier at an effective concentration such that the treated condition is relieved, prevented, or one or more symptoms are ameliorated. The agent can be formulated and administered in unit-dosage forms or multiple-dosage forms. Unit-dose forms as used herein refers to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose contains a predetermined quantity of the agent sufficient to produce the desired therapeutic effect, in association with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes and individually packaged tablets or capsules. Unit-dose forms can be administered in fractions or multiples thereof. A multiple-dose form is a plurality of identical unitdosage forms packaged in a single container to be administered in segregated unitdose form. Examples of multiple-dose forms include vials, bottles of tablets or capsules or bottles of pints or gallons. Hence, multiple dose form is a multiple of unit-doses which are not segregated in packaging.

In one embodiment, a therapeutically effective dosage produces a serum concentration of agent of from about 0.1 ng/ml to about 50-100 pg/ml. The pharmaceutical compositions, in another embodiment, provide a dosage of from about 0.001 mg to about 2000 mg of agent per kilogram of body weight per day. Pharmaceutical dosage unit forms can be prepared to provide from about 0.01 mg, 0.1 mg or 1 mg to about 100 mg, 500 mg, 1000 mg or 2000 mg, and in one embodiment from about 10 mg to about 500 mg of the agent and/or a combination of other optional essential ingredients per dosage unit form.

The agent can be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and can be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values can also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens can be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Dosage forms or compositions containing antibody in the range of 0.005% to 100% with the balance made up from non-toxic carrier can be prepared. Methods for preparation of these compositions are known to those skilled in the art.

Oral pharmaceutical dosage forms are either solid, gel or liquid. The solid dosage forms are tablets, capsules, granules, and bulk powders. Types of oral tablets include compressed, chewable lozenges and tablets which may be enteric-coated, sugar- coated or film-coated. Capsules can be hard or soft gelatin capsules, while granules and powders can be provided in non-effervescent or effervescent form with the combination of other ingredients known to those skilled in the art.

In certain embodiments, the formulations are solid dosage forms. In certain embodiments, the formulations are capsules or tablets. The tablets, pills, capsules, troches and the like can contain one or more of the following ingredients, or compounds of a similar nature: a binder; a lubricant; a diluent; a glidant; a disintegrating agent; a colouring agent; a sweetening agent; a flavouring agent; a wetting agent; an emetic coating; and a film coating. Examples of binders include microcrystalline cellulose, gum tragacanth, glucose solution, acacia mucilage, gelatin solution, molasses, polyinylpyrrolidine, povidone, crospovidones, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include crosscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Colouring agents include, for example, any of the approved certified water soluble FD and C dyes, mixtures thereof; and water insoluble FD and C dyes suspended on alumina hydrate. Sweetening agents include sucrose, lactose, mannitol and artificial sweetening agents such as saccharin, and any number of spray dried flavours. Flavouring agents include natural flavours extracted from plants such as fruits and synthetic blends of compounds which produce a pleasant sensation, such as, but not limited to peppermint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene laural ether. Emetic-coatings include fatty acids, fats, waxes, shellac, ammoniated shellac and cellulose acetate phthalates. Film coatings include hydroxyethylcellulose, sodium carboxymethylcellulose, polyethylene glycol 4000 and cellulose acetate phthalate.

The agents described herein can be provided in a composition that protects it/them from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition can also be formulated in combination with an antacid or other such ingredient.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as a fatty oil. In addition, dosage unit forms can contain various other materials which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, sprinkle, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colourings and flavours.

The agent can also be mixed with other active materials which do not impair the desired action, or with materials that supplement the desired action, such as antacids, H2 blockers, and diuretics. The active ingredient is an agent or pharmaceutically acceptable derivative thereof as described herein. Higher concentrations, up to about 98% by weight of the active ingredient may be included.

In all embodiments, tablets and capsules formulations can be coated as known by those of skill in the art in order to modify or sustain dissolution of the active ingredient. Thus, for example, they may be coated with a conventional enterically digestible coating, such as phenylsalicylate, waxes and cellulose acetate phthalate.

The formulations may be liquid dosage forms. Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. Emulsions are either oil-in-water or water-in-oil. Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of a sugar, for example, sucrose, and may contain a preservative.

Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifying agents and preservatives. Suspensions use pharmaceutically acceptable suspending agents and preservatives.

Pharmaceutically acceptable substances used in non-effervescent granules, to be reconstituted into a liquid oral dosage form, include diluents, sweeteners and wetting agents. Pharmaceutically acceptable substances used in effervescent granules, to be reconstituted into a liquid oral dosage form, include organic acids and a source of carbon dioxide. Colouring and flavouring agents are used in all of the above dosage forms.

Solvents include glycerin, sorbitol, ethyl alcohol and syrup. Examples of preservatives include glycerin, methyl and propylparaben, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, acacia, tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, Veegum and acacia. Sweetening agents include sucrose, syrups, glycerin and artificial sweetening agents such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colouring agents include any of the approved certified water soluble FD and C dyes, and mixtures thereof. Flavouring agents include natural flavours extracted from plants such fruits, and synthetic blends of compounds which produce a pleasant taste sensation.

For a solid dosage form, the solution or suspension, in for example propylene carbonate, vegetable oils or triglycerides, can be encapsulated in a gelatin capsule. Such solutions, and the preparation and encapsulation thereof, are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. For a liquid dosage form, the solution, e.g., for example, in a polyethylene glycol, can be diluted with a sufficient quantity of a pharmaceutically acceptable liquid carrier, e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active compound or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Patent Nos. RE28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, those containing an agent provided herein, a dialkylated mono- or poly-alkylene glycol, including, but not limited to, 1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants, such as butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholic solutions including a pharmaceutically acceptable acetal. Alcohols used in these formulations are any pharmaceutically acceptable water-miscible solvents having one or more hydroxyl groups, including, but not limited to, propylene glycol and ethanol, in a suitable concentration to maintain protein function. Acetals include, but are not limited to, di(lower alkyl) acetals of lower alkyl aldehydes such as acetaldehyde diethyl acetal.

Parenteral administration by injection, either subcutaneously, intramuscularly or intravenously is also contemplated herein. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. The injectables, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, dextrose, glycerol or ethanol, in a suitable concentration to maintain protein function. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, and other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that a constant level of dosage is maintained (see, e.g., U.S. Pat. No. 3,710,795) is also contemplated herein. Briefly, a compound provided herein is dispersed in a solid inner matrix, e.g., polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinylchloride, plasticized nylon, plasticized polyethyleneterephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylenevinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonate copolymers, hydrophilic polymers such as hydrogels of esters of acrylic and methacrylic acid, collagen, cross-linked polyvinylalcohol and cross-linked partially hydrolyzed polyvinyl acetate, that is surrounded by an outer polymeric membrane, e.g., polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinylacetate copolymers, silicone rubbers, polydimethyl siloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride, vinylchloride copolymers with vinyl acetate, vinylidene chloride, ethylene and propylene, ionomer polyethylene terephthalate, butyl rubber epichlorohydrin rubbers, ethylene/vinyl alcohol copolymer, ethylene/vinyl acetate/vinyl alcohol terpolymer, and ethylene/vinyloxyethanol copolymer, that is insoluble in body fluids. The agent diffuses through the outer polymeric membrane in a release rate controlling step. The amount of agent contained in such parenteral compositions is highly dependent on the specific nature thereof, as well as the activity of the compound and the needs of the subject.

Preparations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products, such as lyophilized powders, ready to be combined with a solvent just prior to use, including hypodermic tablets, sterile suspensions ready for injection, sterile dry insoluble products ready to be combined with a vehicle just prior to use and sterile emulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparations include aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anaesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, Ringers Injection, Isotonic Dextrose Injection, Sterile Water Injection, Dextrose and Lactated Ringers Injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to parenteral preparations packaged in multiple-dose containers which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and dextrose. Buffers include phosphate and citrate. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcelluose, hydroxypropyl methylcellulose and polyvinylpyrrolidone. Emulsifying agents include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions includes EDTA. Pharmaceutical carriers also include ethyl alcohol, polyethylene glycol and propylene glycol for water miscible vehicles; and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

The concentration of the pharmaceutically active ingredient is adjusted so that an injection provides an effective amount to produce the desired pharmacological effect. The exact dose depends on the age, weight and condition of the patient or animal as is known in the art.

The unit-dose parenteral preparations can be packaged in an ampoule, a vial or a syringe with a needle. All preparations for parenteral administration can be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterile aqueous solution containing an active compound is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing an active material injected as necessary to produce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In one embodiment, a therapeutically effective dosage is formulated to contain a concentration of at least about 0.1% w/w up to about 90% w/w or more, in certain embodiments more than 1% w/w of the active compound to the treated tissue(s).

The agent can be suspended in micronized or other suitable form. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for ameliorating the symptoms of the condition and may be empirically determined.

In other embodiments, the pharmaceutical formulations are lyophilized powders, which can be reconstituted for administration as solutions, emulsions and other mixtures. They may also be reconstituted and formulated as solids or gels. The lyophilized powder is prepared by dissolving an agent provided herein, or a pharmaceutically acceptable derivative thereof, in a suitable solvent. The lyophilized powder can be sterile. The solvent may contain an excipient which improves the stability or other pharmacological component of the powder or reconstituted solution, prepared from the powder. Excipients that may be used include, but are not limited to, dextrose, sorbitol, fructose, corn syrup, xylitol, glycerin, glucose, sucrose or other suitable agent. The solvent may also contain a buffer, such as citrate, sodium or potassium phosphate or other such buffer known to those of skill in the art at, about neutral pH. Subsequent sterile filtration of the solution followed by lyophilization under standard conditions known to those of skill in the art provides the desired formulation. The resulting solution can be apportioned into vials for lyophilization. Each vial will contain a single dosage or multiple dosages of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injection provides a formulation for use in parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The precise amount depends upon the selected compound. Such amount can be empirically determined.

Topical mixtures are prepared as described for the local and systemic administration. The resulting mixture can be a solution, suspension, emulsions or the like and can be formulated as creams, gels, ointments, emulsions, solutions, elixirs, lotions, suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays, suppositories, bandages, dermal patches or any other formulations suitable for topical administration. The antibodies of the invention can be formulated as aerosols for topical application, such as by inhalation (see, e.g., U.S. Pat. Nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of a steroid useful for treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract can be in the form of an aerosol or solution for a nebulizer, or as a microfine powder for insufflations, alone or in combination with an inert carrier such as lactose. In such a case, the particles of the formulation will, in one embodiment, have diameters of less than 50 microns, in one embodiment less than 10 microns.

The agents can be formulated for local or topical application, such as for topical application to the skin and mucous membranes, such as in the eye, in the form of gels, creams, and lotions and for application to the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal delivery and also for administration to the eyes or mucosa, or for inhalation therapies. Nasal solutions of the active compound alone or in combination with other pharmaceutically acceptable excipients can also be administered. These solutions, particularly those intended for ophthalmic use, may be formulated as 0.01%-10% isotonic solutions, pH about 5-7, with appropriate salts.

Other routes of administration, such as transdermal patches, including iontophoretic and electrophoretic devices, and rectal administration, are also contemplated herein. Transdermal patches, including iotophoretic and electrophoretic devices, are well known to those of skill in the art. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010715, 5,985,317, 5,983,134, 5,948,433, and 5,860,957.

The agents and other compositions provided herein may also be formulated to be targeted to a particular tissue, receptor, or other area of the body of the subject to be treated. Many such targeting methods are well known to those of skill in the art. All such targeting methods are contemplated herein for use in the instant compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542 and 5,709,874. In some embodiments, the agents described herein are targeted (or otherwise administered) to the brain, such as in a patient having or at risk of having Alzheimer's Disease.

In an aspect, the invention provides a combination of an agent as defined in any one of the preceding claims, and a further therapeutic moiety as defined herein.

Further therapeutic moieties include antibiotics and probiotics such as those described herein. Exemplary probiotics include Lactobacillus species, Bifidobacterium species, Saccharomyces boulardii, Streptococcus thermophilus, Enterococcus faecium, Escherichia coli Nissle 1917 (EcN). Further therapeutic moieties include molecules having anti-amyloid activity, such as the BRICHOS domain protein (SEQ ID NO: 4 or 5).

By "molecules having anti-amyloid activity" we include the meaning of compounds or substances that exhibit the ability to inhibit or modulate the formation, aggregation, or toxicity of amyloid proteins. Molecules with anti-amyloid activity can target various stages of amyloid formation and aggregation, for example by preventing misfolding by stabilizing the native, functional conformation of proteins, reducing the likelihood of misfolding into amyloid structures; by inhibiting aggregation by interfering with the aggregation process, either by preventing the association of monomers into oligomers or by disrupting the growth of fibrils; by promoting disaggregation by facilitating the breakdown of existing amyloid aggregates into smaller, less toxic species or promote their clearance by cellular mechanisms; and/or by reducing toxicity by mitigating the toxic effects associated with amyloid aggregates, such as oxidative stress, inflammation, and neuronal dysfunction.

In an embodiment, the combination of agent and a further therapeutic moiety are in separate containers and/or separate compositions. In an alternative embodiment, the combination of agent and a further therapeutic moiety are formulated as a composition.

Separate compositions can be administered separately or simultaneously. Separate administration refers to the two compositions being administered at different times, e.g. at least 10, 20, 30, or 10-60 minutes apart, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12 hours apart. One can also administer compositions at 24 hours apart, or even longer apart. Alternatively, two or more compositions can be administered simultaneously, e.g. less than 10 or less than 5 minutes apart. Compositions administered simultaneously can, in some aspects, be administered as a mixture, with or without similar or different time release mechanism for each of the components.

The agents described herein can be conjugated or recombinantly fused to a further therapeutic moiety (or one or more therapeutic moieties). The agent may be conjugated or recombinantly fused to a therapeutic moiety, such as another protein having anti-amyloid activity, such as BRICHOS domain (SEQ ID NO: 4 or 5).

Fusion proteins are hybrid proteins created by combining the coding sequences of two or more genes, typically from different sources, to produce a single polypeptide chain with properties derived from each component. Methods for generating fusion proteins are known in the art and include recombinant DNA technology involving the manipulation of DNA sequences in vitro to create a fusion gene encoding the desired fusion protein. The DNA sequences encoding the individual proteins of interest are ligated together in-frame, allowing for the production of a single mRNA transcript and a fusion protein with both domains. Techniques such as PCR amplification, restriction enzyme digestion, and DNA ligation are commonly used in recombinant DNA technology to construct fusion genes. Gene synthesis technologies enable the custom synthesis of DNA sequences encoding fusion proteins without the need for pre-existing DNA templates. Researchers can design the desired fusion gene sequence, including appropriate linkers or spacers between the individual protein domains, and have it synthesized by commercial gene synthesis companies. Overlap extension PCR, also known as splicing by overlap extension (SOE) PCR or PCR stitching, is a technique used to amplify DNA fragments with overlapping ends that can be annealed together to form a fused product. Primers are designed to amplify overlapping regions of the two or more DNA fragments encoding the protein domains of interest. In subsequent PCR cycles, the overlapping regions anneal together, resulting in the generation of a fused DNA fragment encoding the fusion protein. Fusion proteins can be generated in vivo using genetic recombination techniques. For example, in yeast two-hybrid assays or other genetic screening systems, fusion proteins can be generated by fusing the coding sequences of two proteins to different regions of a reporter gene. Interaction between the two proteins of interest results in the production of a functional reporter protein. Ribosomal frameshifting is a natural mechanism that can be exploited to generate fusion proteins. By introducing a programmed ribosomal frameshift site between the coding sequences of two proteins, the ribosome can shift reading frames during translation, leading to the synthesis of a single polypeptide containing both protein domains.

In an aspect, the invention provides an agent for use, an agent, a pharmaceutical composition, a use, or a method, substantially as described in the accompanying description, claims and examples.

Embodiments of the invention will now be described, by way of example only, with reference to the accompanying figures, in which:

Figure 1. Architecture of H. pylori CagA and the recombinant protein preparation, (a) H. pylori colonizes the human stomach lining and has been associated with various human diseases, including gastrointestinal disorders, cardiovascular diseases, as well as neurodegenerative disorders like AD and PD. (b) The CagA protein is a multifaceted molecule encompassing both a N-terminal and a C- terminal region. The C-terminal region is disordered, and the N-terminal region can be further subdivided into three distinct sections: Domain I (DI), Domain II (D2), and Domain III (D3) (PDB ID 4DVY). Domain I is comprised of 10 o-helices, Domain II features a substantial anti-parallel 0-sheet, including a subdomain with five o-helices and two short 0-sheets, while Domain III adopts a four-helix bundle structure, (c and d) The N-terminal region, CagA^N, was expressed in E. coli, purified using IMAC, IEC and SEC. Circular dichroism (CD) was used to measure the secondary structure, revealing an o-helix dominant conformation. SDS-PAGE analysis indicated a high purity.

Figure 2. CagA^N prevents biofilm formation and functional amyloid assembling, (a) Schematic presentation of bacterial functional amyloid and biofilm formation, where functional amyloid is the structure scaffold of biofilm, (b) CagA^N inhibits pseudomonas biofilm formation. Pseudomonas sp. UK4 (wildtype and pFap species) were incubated with and without recombinant CagA^N proteins, and the biofilm formation were evaluated by Gram's crystal violet staining. For both wildtype (WT) and pFap species, CagA^N showed dose-dependent effects on the reducing biofilm formation, (c and d) Pseudomonas secrets FapC protein that assembles into amyloid fibrils. The inhibition effects of CagA^N on FapC fibril formation was studied by ThT assay with 15 pM FapC and different concentrations of CagA^N from 0 to 1280 nM. (e) The half time (T1/2) and maximum rate (rmax) were extracted by sigmoidal fitting of the dataset in (e). (f and g) E. coli secrets CsgA protein that assembles into amyloid fibrils. The inhibition effects of CagA^N on CsgA fibril formation was studied by ThT assay with 15 pM FapC and different concentrations of CagA^N from 0 to 1280 nM. (h) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (h).

Figure 3. CagA^N prevents AD associated peptides and protein from forming amyloid, (a) Schematic presentation of pathogenic amyloid formation and some of the relevant human diseases, (b) Schematic presentation of the amyloid formation and the related peptides and protein in AD. (c) The inhibition effects of CagA^N on A[342 fibril formation was tested by ThT assay with 3 pM A[342 and different concentrations of CagA^N from 0 to 50 nM. (d) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (c). (e and f) The inhibition effects of CagA^N on A[340 fibril formation was tested by ThT assay with 5 pM A[340 and different concentrations of CagA^N from 0 to 1 nM. (f) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (e). (g and h) The inhibition effects of CagA^N on tau fibril formation was tested by ThT assay with 15 pM tau and different concentrations of CagA^N from 0 to 500 nM. (h) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (g).

Figure 4. CagA^N supresses PD and T2D related protein and peptide amyloid formation (a) Schematic presentation of the amyloid formation and the related peptides and protein in PD. (b) The inhibition effects of CagA^N on 10 pM o-synuclein fibril formation was tested by ThT assay, while the concentrations of CagA^N were ranging from 0 to 3 pM. (c) The inhibition effects of CagA^N on 30 pM o-synuclein fibril formation was tested by ThT assay with CagA^N concentration from 0 to 0.5 pM. (d) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (c). (e) The inhibition effects of CagA^N on 30 pM o-synuclein fibril formation at pH 5.5 was tested by ThT assay with CagA^N concentration from 0 to 1 pM. (f) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (e). (g) Schematic presentation of the amyloid formation and the related peptide IAPP in T2D. (h) The inhibition effects of CagA^N on 10 pM IAPP fibril formation was tested by ThT assay with CagA^N concentration from 0 to 0.1 pM. (i) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (h).

Figure 5. CagA^N interferes with different microscopic events of the fibril formation of various amyloid peptides or proteins. The kinetics of amyloid aggregation involves distinct stages²⁹: in the primary nucleation phase, monomers come together to create a nucleus (primary nucleation, kn), from which a fibril can start to elongate (elongation, k+); simultaneously, during secondary nucleation (k2), monomers adhere to the fibril's surface, catalyzing the development of a new nucleus and facilitating exponential fibril growth. Refer to the schematic representation in (a) and (b) for a visual depiction of this process, (a) CagA^N supresses different bacterial functional amyloid proteins from forming amyloid, i.e., CsgA from E. coli and FapC from Pseudomonas. For these amyloidogenic proteins, CagA^N predominantly obstructs the elongation process, as indicated by the cross, (b) CagA^N impedes the formation of amyloid fibrils by various human pathogenic amyloid peptides or proteins through diverse mechanisms. In the case of AD-associated A[342 and A[340 peptides, primarily, primary nucleation is blocked. Conversely, for the AD-relevant protein tau, both secondary nucleation and elongation are affected by CagA^N. Regarding the PD-related protein o-synuclein, CagA^N inhibits the elongation process. The IAPP peptide, associated with T2D, has its fibrillization inhibited by CagA^N, affecting both secondary nucleation and elongation processes. The details of global fitting for each dataset are included in Figures 7-9.

Figure 6. The interaction of CagA^N with amyloidogenic substrates, (a) NMR spectrum of 15N-labelled o-synuclein with (red spectrum) and without CagA^N (blue spectrum), (b) The relative intensity of each amino acid of o-synuclein with and without CagA^N. Residues marked with an asterisk (*) was excluded from the analysis due to too low signal-to-noise ratio, (c and d) SPR analysis of CagA^N and o-synuclein oligomers or fibrils, (e) SPR analysis of CagA^N and A[342 monomers, (f) ZDOCK analysis of the interaction between CagA and A[342 monomers, (g) Cartoon of the D2 domain with hydrophobic amino acid residues in stick mode, (h) The inhibition effects of CagA^N D2 domain on 3 pM A[342 fibril formation was tested by ThT assay with CagA^N concentration from 0 to 100 nM. (i) The T1/2 and rmax were extracted by sigmoidal fitting of the dataset in (h). (j) CagA^N D2 domain impedes the formation of amyloid fibrils of AP42 peptides by inhibiting primary nucleation.

Figure 7. Aggregation kinetics of FapC and CsgA in the presence of different CagA^N concentrations. Aggregation kinetics of 15 pM FapC (a) or CsgA (b) in the presence of different CagA^N concentrations as denoted by the labels on the right of the subfigure. The global fits (solid lines) of the aggregation traces (dots) were constrained such that only one single rate constant, i.e., kn, k2 or k+, is the sole free fitting parameter. The tick symbolizes a satisfactory fit, while a cross signifies an unsatisfactory fit.

Figure 8. Aggregation kinetics of A(342, A(34O and tau in the presence of different CagA^N concentrations. Aggregation kinetics of 3 pM A[342 (a), 5 pM A[340 (b) or 15 pM tau (c) in the presence of different CagA^N concentrations as denoted by the labels on the right of the subfigures. The global fits (solid lines) of the aggregation traces (dots) were constrained such that only one single rate constant, i.e., kn, k2 or k+, is the sole free fitting parameter. The tick symbolizes a satisfactory fit, while a cross signifies an unsatisfactory fit.

Figure 9. Aggregation kinetics of o-synuclein and IAPP in the presence of different CagA^N concentrations. Aggregation kinetics of 30 pM o-synuclein (o-syn, (a), or 10 pM IAPP (b) in the presence of different CagA^N concentrations as denoted by the labels on the right of the subfigure. The global fits (solid lines) of the aggregation traces (dots) were constrained such that only one single rate constant, i.e., kn, k2 or k+, is the sole free fitting parameter. The tick symbolizes a satisfactory fit, while a cross signifies an unsatisfactory fit.

Figure 10. Interaction measurement of o-synuclein monomer and CagA^N by FIDA. The o-synuclein monomers were labelled with ALC480 and used as indicator, while the CagA^N was the analyte, (a) ALC480 labelled o-synuclein monomers was analysed, giving a hydrodynamic radius (Rh) of 2.9 nm, suggesting an intrinsically disordered nature, (b) ALC480 labelled o-synuclein monomers were analysed with and without a very large excess of CagA^N and the difference of rh values was small, suggesting no strong interaction between o-synuclein and protein CagA^N.

Figure 11. Coincubation of A(342 and CagA^N. (a) 3 pM A[342 incubated with and without different concentrations of CagA^N were analysed by SDS-PAGE. (b) 5 pM A[342 incubated with and without 5 pM CagA^N were analysed by native-PAGE. (c) The masses of A[342 after incubation with CagA^N were analyzed by mass spectrometer.

Figure 12. Preparation of recombinant CagA^N D2 domain, (a) SDS-PAGE analysis of purified recombinant CagA^N D2 domain, (b) CD measurement of the recombinant CagA^N D2 domain. Figure 13. Aggregation kinetics of o-synuclein and A(342 in the presence of different CagA^N D2 concentrations. Aggregation kinetics of 30 pM o-synuclein (o- syn, (a), or 3 pM A[342 (b) in the presence of different CagA^N concentrations as denoted by the labels on the right of the subfigure. The global fits (solid lines) of the aggregation traces (dots) were constrained such that only one single rate constant, i.e., kn, k2 or k+, is the sole free fitting parameter. The tick symbolizes a satisfactory fit, while a cross signifies an unsatisfactory fit.

EXAMPLES

Example 1 - Helicobacter pylori releases strong anti-amyloid protein molecule

Bacteria, the smallest and most abundant life forms on Earth, have been a source of profound insights and strategies that have had a significant impact on human science and technology. From the development of essential antibiotics to pioneering gene editing techniques, bacteria have proven to be a treasure trove of inspiration. Here, we show a discovery that bacteria can release super efficient molecules to counteract functional and pathogenic amyloid fibril formation, which are fibrillar protein assemblies relevant to bacterial biofilm formation and various human diseases, respectively. The broad anti-amyloid substrate spectrum of CagA (cytotoxin-associated gene A) protein secreted by Helicobacter pylori unveils a mechanism whereby these bacteria interfere with other bacteria and human, holding promise for pharmaceutical interventions aimed at combating bacterial infections and human protein misfolding diseases. Our discoveries emphasize the profound source of inspiration that the microbial world offers for numerous domains of human innovation and scientific exploration.

Introduction

Bacteria, often underestimated due to their microscopic scale, offer a vast reservoir of ingenious strategies spanning a myriad of scientific and technological domains. From the development of life-saving antibiotics rooted in microbial warfare¹ to pioneering gene editing techniques, inspired by the precision of bacterial defense systems², and even the creation of specialized protein syringes that mimic bacterial appendages³, these diminutive life forms provide a rich tapestry of innovation. They offer insights into genetic manipulation with potential applications in health, agriculture, and personalized medicine. This microbial realm constantly reveals its resourcefulness, fostering a profound source of inspiration for diverse fields of human innovation and discovery. Amyloid is a kind of highly ordered protein fibrillar assembly (nanofibrils), which can be functional or pathogenic⁴. Extensive characterization, in recent decades, has unraveled the highly organized nature of amyloid nanofibrils with approximately 10 nm in diameter, characterized by unbranched structures and well-defined 0-sheets oriented perpendicular to the fibril axis⁵. Natural amyloids exhibit non-pathological functionality and possess clearly defined physiological roles⁶, prevalent across various natural species, encompassing both human (e.g., p-mel and certain polypeptide hormones⁴) and invertebrates (e.g., insects⁷ and spiders⁸-⁹). Notably, bacteria also contribute to this functional amyloid spectrum by secreting amyloids, including Escherichia coli (curli) and various Pseudomonas species (FapC) that are common inhabitants of the human gut microbiome¹⁰. The bacterial amyloid fibrils play a key role in maintaining the structural integrity of biofilms that enables bacteria to adhere to surfaces and to other bacterial cells, retain nutrients and water, and also act as a protective barrier. Pathogenic microbes associated with biofilms are protected from antimicrobial agents and host immune system attacks, making them more infectious and difficult to treat efficiently, and in fact, 80% of all chronic infections are related to bacterial biofilms¹¹. When amyloid-forming proteins are mutated or the amyloid formation is inhibited, the biofilms are disrupted and bacteria become more vulnerable to environmental factors¹¹. On the flip side, pathogenic amyloids represent a class of pathogenic protein aggregates characterized by their distinct fibrillar structure and insolubility, intricately linked to a broad spectrum of incurable debilitating disorders known as amyloidosis, including Alzheimer's disease (AD), Parkinson's disease (PD), and type II diabetes (T2D)⁵-¹². Each disorder is characterized by the deposition of specific amyloidogenic peptides or proteins, showcasing the diverse pathogenicity of amyloid aggregates and the associated clinical manifestations⁴. Inspired by the antibiotic-producing abilities of some bacteria and the importance of amyloid formation in bacterial biology, it is conceivable that certain bacterial species might have evolved mechanisms to hinder amyloid formation, ultimately affecting the growth of neighboring bacterial populations.

Helicobacter pylori, a prevalent Gram-negative bacterium, establishes residence within the human stomach lining, impacting around 50% of the global population¹³. This microorganism has captured substantial attention from the realms of medicine and science due to its pivotal role in provoking an array of gastrointestinal ailments including gastric ulcer. Moreover, it has raised intriguing prospects of connections to heightened susceptibility to cardiovascular diseases and neurodegenerative disorders¹⁴, including PD and AD, albeit the precise mechanisms remain elusive. H. pylori is also recognized for its ability to modify the microbiota in both the gastric and intestinal regions, leading to a wide array of diseases, and impact the production and physiological regulation of gut metabolic hormones¹⁵-¹⁶. H. pylori strains can be categorized into cagA (cytotoxin-associated gene A)-positive and cagA-negative types depending on the presence or absence of the cagA gene. Interestingly, CagA-positive strains of H. pylori can induce changes in the diversity and abundance of gut microbiota¹⁵'¹⁷'¹⁸. CagA also interferes with the production of antimicrobial peptides in the gut¹⁹ and activates host immune responses¹⁷, both of which can further alter gut microbiota composition and diversity. CagA expression within Drosophila intestinal stem cells is enough to alter host microbiota²⁰. Despite the increasing body of research on the effects of CagA protein on the gut microbiota, the precise mechanism by which it alters the gut microbiota remains incompletely understood.

The above observations together have prompted the hypothesis that bacteria in natural environments may have evolved mechanisms to regulate amyloid formation, potentially influencing the growth as well as behaviour of other bacteria residing within biofilms and/or associated with human protein misfolding disease. In this study, surprisingly, we found that H. pylori CagA is a super-efficient anti-amyloid molecule with a broad substrate spectrum, which unveils a mechanism whereby H. pylori interferes with other bacteria and human, holding pharmaceutic potentials in interfering with bacterial infections and human protein misfolding diseases.

CagA prevents biofilm formation and function amyloid assembling

The CagA protein is a virulence factor secreted by H. pylori, delivered into the cytoplasm of the host cells via the complex type IV secretion system, and this protein has been detected in the blood of patient after infection with a micromolar level concentration²¹. CagA consists of multiple domains/regions: the N-terminal structured region and a disordered C-terminal tail for perturbing host signal transduction (Fig. la and b)²²'²³. The CagA protein's size ranges from 130 to 145 kDa due to structural polymorphism in the C-terminal region²². Notably, the N-terminal region is suggested to function as an inhibitory domain for the effects of the C-terminal region, and the region spanning amino acids 800-1216, when expressed independently, induces the highest degree of epithelial cell elongation²⁴. Additionally, CagA undergoes protease cleavage in human cells, resulting in two fragments of approximately 100-105 kDa and 35-40 kDa, and the latter corresponds to the region from amino acid 800-1216 that contains the EPIYA segmengments²⁵. Therefore, this study focuses on the N- terminal region spanning from position 1 to 884, designated as CagA^N, which can be divided into Domain I, Domain II, and Domain III. Domain I comprises 10 o-helices, Domain II features a substantial anti-parallel 0-sheet, encompassing a subdomain with five o-helices and two short 0-sheets, and Domain III adopts a four-helix bundle structure with the C-terminal region from a long o-helix connecting Domain II and III (Fig. lb)^{23 26}. The expression of CagA^N was carried out in E. coli, followed by purification using immobilized metal affinity chromatography (IMAC), ion exchange chromatography (IEC), and size exclusion chromatography (SEC). The resulting recombinant CagA^N exhibited an o-helix dominant conformation with high purity (Fig. 1c and d), aligning well with the crystal structure of the N-terminal part (PDB ID 4DVY) (Fig. lb).

Bacteria, such as E. coli (curli) and several Pseudomonas species (FapC), commonly found in the human gut microbiome, have the capacity to generate amyloids¹⁰. These functional amyloid fibrils are vital for upholding the structural stability of biofilms— densely packed microbial communities— and establishing a protective shield (Fig. 2a)²⁷. This shield possess challenges in combating pathogenic microbes associated with biofilms, rendering them less susceptible to antimicrobial agents and complicating effective microbial treatment¹¹. Disruption of amyloid-forming proteins by mutations or inhibition of amyloid formation results in the breakdown of biofilms, rendering bacteria more susceptible to environmental influences. To assess the potential of CagA^N in preventing biofilm formation, we examined its effects on Pseudomonas species, encompassing both wildtype and pFapC proteins variants (Fig. 2b). Surprisingly, CagA^N demonstrated efficient concentration-dependent inhibition effects on the biofilm formation of both wildtype Pseudomonas and the pFapC species (Fig. 2b). This observation raises intriguing possibilities regarding the mechanisms underlying the ability of CagA-positive strains of H. pylori to induce changes in the diversity and abundance of gut microbiota, suggesting that H. pylori may possess a capacity to inhibit biofilm formation across different bacterial species.

For further insights into whether the anti-biofilm effects are raised from the inhibition of functional amyloid formation, considering that amyloid is essential for stabilizing the biofilm architecture, we evaluated the effectiveness of CagA^N against two common proteinaceous components, FapC from Pseudomonas and CsgA from E. coli (Fig. 2c, d, f and g), both prevalent in the human gut microbiome¹⁰, with regards to amyloid fibril formation. This assessment was carried out using a Thioflavin T (ThT) fluorescence assay, where ThT is a dye specifically designed to bind to amyloid structures with a resulting increase of the fluorescence intensity²⁸. Surprisingly, CagA^N exhibited exceptional efficacy against amyloid formation, with strikingly inhibition effects at nanomolar concentrations when tested against 15 pM of either FapC or CsgA. Quantified through parameters such as half time (n/2) and maximum rate (r_max) (Fig. 2d, e, g, and h), the concentration-dependent impact of recombinant CagA^N on both FapC and CsgA was evident. With using 1.3 pM CagA^N against 15 pM amyloidogenic substrates, the n/2 of FapC was extended from approximately 3 hours to 16 hours, while for CsgA, it increased from around 2 hours to 32 hours (Fig. 2e and h), suggesting H. pylori CagA is a highly efficient candidate for inhibiting functional bacterial amyloid fibril formation. These results reveal a potential mechanism where H. pylori alters the growth of neighboring bacterial populations by changing the biofilm integrity.

CagA efficiently inhibits pathogenic amyloid formation

The impressive effectiveness of CagA in inhibiting functional amyloid formation led our investigation towards the potential extension of CagA's anti-amyloid properties to encompass pathogenic amyloid-forming peptides or proteins. These pathogenic amyloid species are intricately associated with human diseases such as AD, PD, and T2D (Fig. 3a), i.e., amyloid-0 (A0, encompassing the isoforms A042 and A04O) and tau, o-synuclein, and islet amyloid polypeptide (IAPP), respectively⁵-¹². Initially, we evaluated the effectiveness of CagA^N against the pathogenic amyloid-forming peptides and proteins associated with AD— specifically A042, A04O, and tau (Fig. 3b), which were produced and purified from E. coli— using the ThT fluorescence assay. To our surprise, CagA^N displayed exceptional efficacy, revealing outstanding inhibition effects at nanomolar concentrations when tested against 3 pM A042 fibril formation (Fig. 3c). Quantitative analysis underscored the concentration-dependent impact of recombinant CagA^N on A042 (Fig. 3d). The n/2 was extended from approximately 1.5 hours to 21 hours when using only 50 nM CagA^N (to prevent the formation of fibrils by 3 pM A042. Notably, the r_max exhibited no significant changes (Fig. 3d). Interestingly, the efficacy against A042 fibril formation extended to A04O peptide as well (Fig. 3e and f), and 1 nM CagA^N was enough for delaying fibril formation from around 14 hours to 19 hours for 5 pM A04O, with no significant changes in max (Fig. 3e and f), suggesting CagA is a powerful inhibitor of A0 fibril formation. Expanding our inquiry beyond the shorter A0 peptides, we explored CagA^N's activity against tau protein, a larger positively charged amyloidogenic protein associated with AD. Recombinant tau proteins, purified from E. coli, underwent scrutiny for fibril formation using the ThT fluorescence assay (Fig. 3g and h). Similar to its effects on A0 peptides, CagA^N's impact on tau amyloid fibril formation exhibited a concentration dependent delay of the fibrillization, with significant effects evident at 10 nM when applied against 15 pM tau (Fig. 3g and h). Noteworthy was the extension of n/2 from approximately 38 hours to 81 hours, achieved with 500 nM CagA^N to impede fibril formation by 15 pM tau. These results suggest CagA^N is an efficient amyloid forming inhibitor with different sized and charged substrates.

To evaluate the extensive anti-amyloid capabilities of CagA^N, we further investigated its effectiveness against o-synuclein, a protein known to form fibrils and aggregates into Lewy bodies— a crucial histological finding in PD (Fig. 4a). Using o-synuclein produced and purified from E. coli, we employed the ThT fluorescence assay to assess CagA^N's efficacy against this PD-associated amyloid-forming protein. In line with the effect against amyloid formation for other protein systems (Fig. 2 and 3), CagA^N demonstrated exceptional efficacy also for o-synuclein, revealing complete inhibition effects at nanomolar concentrations when tested against 10 pM of o-synuclein (Fig. 4b). For instance, 100 nM CagA^N completely prevented the formation of fibrils of 10 pM of o-synuclein. Subsequently, we increased the concentration of o-synuclein to 30 pM and employed varying concentrations of CagA^N (Fig. 4c), and then the concentration-dependent impact of recombinant CagA^N on o-synuclein became apparent. Quantitative analysis, including parameters /2 and r_max (Fig. 4d), highlighted the concentration-dependent effect of CagA^N on o-synuclein. Using 500 nM CagA^N extended n/2 from approximately 20 hours to 65 hours, effectively inhibiting the formation of fibrils by 30 pM of o-synuclein (Fig. 4d). Notably, CagA^N's anti-amyloid capacity against o-synuclein persisted even under acidic conditions. At pH 5.5, CagA^N exhibited concentration-dependent effects, preventing the formation of fibrils by 30 pM of o-synuclein (Fig. 4e). Using 1 pM CagA^N extended n/2 from approximately 10 hours to 35 hours, which shows its remarkable efficacy in inhibiting the formation of fibrils by 30 pM of o-synuclein at acidic pH (Fig. 4f), suggesting CagA can tolerate different pHs without impairment of its anti-amyloid activity.

Continuing our exploration of CagA^N's effectiveness against amyloid formation, we examined another human disease, T2D, where the peptide hormone IAPP produced by pancreatic [3-cells can form amyloid fibrils (Fig. 4g). Utilizing IAPP produced and purified from E. coli, we applied the ThT fluorescence assay to evaluate how CagA^N performed against this amyloid-forming peptide. Remarkably, CagA^N also exhibited exceptional efficacy for IAPP, showcasing noticeable inhibition effects at nanomolar concentrations during testing against 10 pM of IAPP (Fig. 4h). To further illustrate the efficacy, 100 nM CagA^N hindered the formation of fibrils by 10 pM of IAPP, reducing the half time n/2 from approximately 0.3 hours to 1.7 hours while the r_max decreased (Fig. 4i). Examination of the parameters n/2 and r_max (Fig. 4i) provided insight into the concentration-dependent impact of recombinant CagA^N on IAPP. This indicates the potency of CagA^N in impeding the formation of fibrils by IAPP, further emphasizing its potential as a therapeutic candidate for addressing T2D-related amyloid pathology.

Taken together, these findings indicate that the CagA protein secreted by H. pylori possesses outstanding efficacy as an anti-amyloid molecule with a wide substrate spectrum associated with different biological functions. This observation suggests that the anti-amyloid property of H. pylori secreted CagA has evolved with specificity, distinct from general chaperone functions.

The diverse mechanisms of CagA in inhibiting amyloid formation

The kinetics of amyloid aggregation involves several microscopic processes²⁹-³⁰: in the primary nucleation phase, monomers come together to form a nucleus (primary nucleation,_n), from which a fibril eventually can evolve and start to elongate (elongation, +); simultaneously, during secondary nucleation (/o), monomers adhere to the fibril's surface, catalyzing the development of a new nucleus and facilitating exponential fibril growth. Various proteins, such as molecular chaperones³¹, can influence distinct microscopic events. Furthermore, even a single mutation in a given protein can alter its impact on nucleation events. This underscores the intricate nature of the interplay between protein dynamics and the generation of amyloid structures³²-³³. To assess the impact of CagA^N on the formation of amyloid fibrils across diverse amyloidogenic substrates and to determine its effects on specific microscopic processes, we conducted comprehensive analyses through global fitting of the kinetic datasets. The experiments involved maintaining a constant concentration of amyloid peptide or protein while varying the concentration of CagA^N. In this global fitting process, utilizing an integrated rate law, we imposed constraints such that only a singular rate constant— namely k_n, ki, or k+— was allowed to vary as the sole fitting parameter. This approach allowed us to isolate and scrutinize the individual contributions of these microscopic processes, shedding light on the interplay between CagA^N and amyloid fibril formation dynamics.

The kinetics analysis revealed that CagA^N exerts a multifaceted influence on the formation of amyloid fibrils in both bacterial functional amyloid proteins and human pathogenic amyloid peptides/proteins. In the case of bacterial functional amyloid proteins, i.e., CsgA from E. coli and FapC from Pseudomonas, CagA^N demonstrated a pronounced suppressive effect. It predominantly obstructed the elongation ( +) process, impeding the formation of amyloid structures (Fig. 5a, Fig. 7a and b)). Turning to human pathogenic amyloid peptides and proteins, the impact of CagA^N varied across different entities. In the context of AD, CagA^N selectively targeted specific processes. For AD-associated A[342 and A[340 peptides, primary nucleation was the primary target of inhibition (Fig. 5b, Fig. 8a and b). Conversely, in the case of the AD-relevant protein tau, CagA^N affected both secondary nucleation and elongation processes (Fig. 5b, Fig. 8c). For the PD-related protein o-synuclein, CagA^N specifically hindered the elongation process, showcasing a distinct mode of interference (Fig. 5b, Fig. 9a). Furthermore, in the case of IAPP, associated with T2D, CagA^N exhibited predominant inhibitory effects on both secondary nucleation and elongation processes (Fig. 5b, Fig. 9b). These findings collectively highlight the diverse ways in which CagA^N modulates the intricate process of amyloid fibril formation across a spectrum of amyloidogenic proteins and peptides.

To shed light on the complex molecular interactions between CagA^N and various amyloidogenic peptides and proteins, we focused on o-synuclein and A[342. We first utilized flow-induced dispersion analysis (FIDA) to investigate potential interactions between CagA^N and monomeric o-synuclein. Here, ALC480-labelled monomeric o- synuclein served as the indicator, with CagA^N as the analyte. Notably, we observed no significant interaction between monomeric o-synuclein and CagA^N. Nevertheless, a marginal increase in size was detected in the presence of a substantial excess of CagA^N (Fig. 10), suggesting a minimal interaction. Further, using nuclear magnetic resonance (NMR) spectroscopy we recorded 2D ^-^N HSQC experiments with¹⁵N-labelled o- synuclein monomers and unlabelled CagA^N. Only minor interactions between CagA^N and the o-synuclein monomers were detected, as evidenced by no crosspeak signal intensity decrease corresponding to the relative intensities of each amino acid residue nor any chemical shift changes by comparing the overlapping spectra (Fig. 6a and b). In contrast, surface plasmon resonance (SPR) analysis indicated a strong binding affinity between CagA^N and o-synuclein oligomers, with a binding affinity (KD) of approximately 95 nM (Fig. 6c). Moreover, CagA^N demonstrated a high affinity, with a KD of roughly 58 nM, for o-synuclein fibrils (Fig. 6d), suggesting it may selectively inhibit the formation of o-synuclein fibrils. With A[342 peptides, the binding dynamics diverged. CagA^N bound to the monomeric form with a nanomolar affinity KD = ~77 nM) (Fig. 6e), yet SPR showed no detectable binding to A[342 fibrils. This indicates a preferential binding of CagA^N to A[342 monomers, highlighting its potential regulatory effect on A[342 aggregation in line with the global fit analysis in Fig. 5b. Furthermore, coincubation of CagA^N with A[342 followed by analysis via SDS-PAGE and native-PAGE demonstrated a concentration-dependent modulation by CagA^N, promoting the stabilization of A[342 monomers (Fig. Ila and b). These monomers remained intact in residue sequence as confirmed by mass spectrometry (Fig. 11c). Collectively, these findings illuminate the role of CagA^N in disrupting the different stages of amyloid formation across a broad spectrum of amyloidogenic peptides and proteins, thereby hindering their aggregation into amyloid fibrils with different mechanisms.

The essential role of CagA D2 domain on supressing amyloid activity

CagA is composed of distinct domains and regions, with a structured N-terminal and a disordered C-terminal tail. The N-terminal can be further divided into three domains: Domain I, II, and III, with different structures²²-²³ (Fig. lb). The N-terminal structured domain I is interacting with tumor suppressors; Domain II and/or Domain III are important for the delivery into host cells and subsequent membrane localization²²-²³. Domain II has a charged K-Xn-R-X-R motif located in the o-helix that can bind phosphatidylserine²². This modular organization of CagA underscores its complex structural architecture, with distinct regions contributing to its overall functionality. To identify the active domain responsible for CagA's anti-amyloid activity, we employed the ZDOCK program³⁴ to simulate the interaction between CagA and A[342 monomers. Remarkably, the A[342 monomers docked into the 0-meander structure of Domain II (Fig. 6f), characterized by exposed hydrophobic patches (Fig. 6g). To further evaluate the activity of Domain II experimentally, this domain was recombinantly expressed in E. coli and subsequently purified (Fig. 12a, Fig. 6j), and adopted a correct folding as indicated by CD measurements (Fig. 12b). Intriguingly, Domain II exhibited efficient activity against A[342, preventing the formation of amyloid fibrils (Fig. 6h). With 100 nM Domain II, the n/2 extended from approximately 1.5 hours to 12.5 hours (Fig. 6i). The rmax displayed a slight linear decrease with the increasing concentration of Domain II (Fig. 6i). Kinetic analysis revealed that, similar to CagA^N, Domain II primarily inhibited the primary nucleation of A[342 fibril formation (Fig. 13), underlying the same mechanism as the CagA^N against A[342 (Fig. 5b, Fig. 8a), indicating D2 as an important domain in CagA for the anti-amyloid activity.

Discussion

In this study, we uncover that certain bacterial species have evolved to obstruct amyloid and biofilm formation with remarkable efficiency, profoundly influencing the growth of proximal bacterial colonies. This phenomenon may constitute a pivotal natural foundation, including the potential for antibiotic production and gene editing strategies. The discovery that H. pylori CagA protein acts as an anti-amyloid molecule not only sheds light on microbial regulation of amyloid dynamics, but also proposes a novel intersection between microbial interactions and human disease. The dual role of H. pylori, particularly its impact on other bacterial populations and its influence on human protein misfolding diseases, reveals a complex network of biological interactions. These insights bring new prospects for harnessing microbial products in pharmaceutical advancements, especially in the realms of bacterial infection management and protein misfolding disorder treatments. Consequently, bacteria continue to emerge as a reservoir of biological innovation, presenting strategies that could forward advancements across diverse scientific disciplines, from novel medical treatments to unraveling intricate biological interactions within the human body.

Bacterial infections, manifesting in acute and chronic forms, significantly influence human health. Notably, the pathogenic activities of H. pylori underscore the importance of understanding the pathophysiology, transmission dynamics, and clinical management of bacterial infections for global health, influencing both preventative and therapeutic strategies. The H. pylori CagA protein, demonstrated herein to have exceptional efficacy in suppressing amyloid formation, suggests a unique mechanism by which H. pylori may contribute to human pathologies.

Gut microbiota, pivotal for normal physiology and disease susceptibility, exhibits immense diversity and can be influenced by host-specific factors such as infections³⁶. These factors can lead to various health outcomes, including gastrointestinal and neurological disorders³⁷. CagA-positive strains of H. pylori have been shown to alter the diversity and abundance of gut microbiota¹⁵'¹⁷'¹⁸; however, the impacts of CagA- negative strains remain to be fully elucidated. Furthermore, the association between H. pylori infection, particularly CagA-positive strains, and neurodegenerative diseases like AD involves multiple potential mechanisms, including bacterial access to the brain via the oral-nasal-olfactory route or via infected circulating monocytes³⁸. Despite of that, different studies have shown contradictory results regarding the link between H. pylori and AD, which might be due to the prevalence of CagA-positive H. pylori infection varies, and further large-scale randomized controlled trials are necessary to clarify this relationship³⁸. For example, no significant difference in the prevalence of H. pylori infection was found between AD patients and controls in Japan, where the majority of strains are CagA-positive³⁹'⁴⁰. Interestingly, mice infected with H. pylori displayed severe gastritis and increased neuroinflammation but not brain amyloid deposition or systemic inflammation⁴¹. While the precise relationship between H. pylori infection and AD remains uncertain, it is noteworthy that the prevalence of CagA-positive H. pylori infection varies geographically. In Western countries, about 30-40% of H. pylori strains are cagA-negative, while nearly all strains isolated from East Asian countries are cagA- positive⁴⁰. The strong anti-amyloid activity of CagA provided a mechanistic explanation, that CagA can block the structural scaffold of biofilm— functional amyloid- formation that subsequently make neighbouring bacteria more susceptible to microenvironment. On the other hand, amyloid fibrils bear convincing structural similarities with both viral fusion domains and anti-microbial peptides, as well as sequence similarities with a specific family of bacterial bacteriocins⁴³. The superefficient anti-amyloid activity of CagA might explain the underlying mechanism where it binds anti-microbial peptides that share similar structure as amyloid and prevent the anti-bacteria capacity. Understanding the molecular mechanisms behind the interaction between H. pylori and the CagA protein will provide valuable insights into the pathogenesis of diseases associated with this bacterium and may pave the way for novel therapeutic strategies. Functional amyloids strengthen bioflms and are a major threat to human health, since the (chronic) infections they cause are difficult to treat due to the biofilm structural integrity and insufficient penetration of drugs, thus promoting antibiotic resistance. Targeting biofilms and their amyloid components by the super-efficient anti-amyloid molecule could be a novel approach to fight antibiotic resistance and induce the changes of diversity and abundance gut microbiota.

Proteins, essential for a myriad of biological functions, must maintain precise three- dimensional conformations to function effectively. However, there is an intrinsic propensity for proteins to deviate from their native states, forming aggregates that can manifest as non-fibrillar amorphous aggregates or fibrillar amyloid structures⁴⁴: non- fi bril la r amorphous aggregates associated with a spectrum of diseases including cancer and cataract⁴, and fibrillar amyloid structures that exhibit neurotoxicity and are linked to around 40 human diseases, notably neurodegenerative disorders such as AD and PD, as well as systemic conditions like T2D⁴⁴. Molecular chaperones play a crucial role in the proteostasis network, facilitating proper folding and preventing aberrant aggregation. However, age-related decline in proteostasis network efficiency correlates with the increased prevalence of protein misfolding diseases in the elderly⁴⁵-⁴⁶. Recent advancements in monoclonal antibody drugs targeting A|3 have shown promise in AD treatment, supporting the approach of targeting amyloid as an effective strategy for treating AD⁴⁷. Experimental data indicate that molecular chaperones can prevent protein aggregation in neurodegenerative diseases, including AD, both in vitro and in vivo⁴⁸. For example, the BRICHOS domain has demonstrated preventive and therapeutic effects against amyloid fibril formation and toxicity in AD models⁴⁹. Comprehensive interventions targeting the multifaceted aspects of AD, including tau pathology, neuro-inflammation, and oxidative stress, are increasingly recognized as necessary⁵⁰. The challenge of protein misfolding diseases, exemplified by the most common form of dementia, AD, emphasizes the need for innovative research approaches due to the high failure rate in clinical trials. The CagA protein, distinct from molecular chaperones, represents a novel bacterial protein with remarkable activity against amyloid formation with a broad substrate spectrum. Future investigations into translating this activity for treating human protein misfolding disease are warranted, in particular the subdomain.

Methods

Preparation of recombinant CaoA relevant proteins

The recombinant N-terminal region ranging from position 1-884 (designated as CagA^N) and the Domain II (D2, with fused to a NT* solubility tag⁵¹) of CagA protein from H. pylori were expressed in E. coli BL21(DE3) cells, respectively. The cells were cultured in LB medium containing 100 pg/mL ampicillin at 37°C overnight, which was then transferred into fresh LB medium containinglOO pg/mL ampicillin at 37°C with a ratio of 1 : 100 (v/v). The cells were incubated under 37°C until OD600 reached to ~0.9. The temperature was cooled down to 20°C, and the cells were incubated overnight with 0.5 mM (final concentration) Isopropyl-B-D-thiogalactopyranoside IPTG). For CagA^N, cells were harvested by centrifugation and resuspended in buffer A (20 mM Tris pH 8.0, 150 mM NaCI). Subsequently, the cells were disrupted using sonication, and the lysate was cleared by centrifugation (12,000 x g for 30 min at 4 °C). The supernatant containing CagA^N was applied to a 5 mL Ni-column (Cytiva) pre-equilibrated with buffer A. The column was washed sequentially with four column volumes of buffer A, two column volumes of buffer B (buffer A with 20 mM imidazole), and two column volumes of buffer C (buffer A with 300 mM imidazole). The eluted proteins were dialyzed against buffer D (20 mM Tris pH 8.0, 50 mM NaCI) and subsequently subjected to ion exchange chromatography using a 5 mL QTrap anion exchange column (Cytiva), with elution achieved using a linear gradient of 0-1 M NaCI. Further purification was performed via size exclusion chromatography using a Superdex 200 increase 10/300GL column (Cytiva) with buffer D. Regarding the D2 domain, cells expressing this domain were resuspended in buffer E (20 mM Tris pH 8.0, 500 mM NaCI) and then sonicated on ice. After centrifugation (12,000 x g for 30 min at 4 °C), the supernatant containing recombinant D2 domain was collected and loaded onto a 5 mL Ni-column preequilibrated with buffer E. The column was washed successively with two column volumes of buffer E, buffer F (buffer E with 20 mM imidazole) and eluted by buffer G (buffer E with 300 mM imidazole). The eluted proteins were dialyzed against buffer H (20 mM Tris pH 8.0, 500 mM NaCI), cleaved by thrombin (1: 1000) overnight, and subjected to immobilized metal affinity chromatography again using a Ni-column preequilibrated with buffer E. The CagA D2 domain was eluted from the Ni-column using buffer F and buffer I (20 mM Tris pH 8.0, IM NaCI), followed by size exclusion chromatography using a Superdex 200 increase 10/300GL column equilibrated with buffer J (20 mM sodium phosphate buffer pH 8.0, 0.2 mM EDTA, 50 mM NaCI). Preparation of the amyloidogenic proteins and peptides

Functional amyloidogenic proteins-. CsgA and FapC were expressed and purified according to the established protocols⁵². Briefly, full-length FapC (amino acids 25-250) from Pseudo- monas sp. UK4 and CsgA from E. coli (amino acids 21-151) were produced using pET28d and pETlld vectors BL21(DE3) E. coli cells. A single colony was used to inoculate 50 mL of liquid culture, which was grown overnight. This culture was then diluted 40 times to achieve a starting OD600 of 0.1 for the main growth. Protein production was initiated when the OD600 reached 0.6-0.8, using 0.5 or 1 mM IPTG for CsgA and FapC, respectively. After 12 hours for CsgA and 4 hours for FapC, the cells were collected and centrifuged (6000g for 10 minutes at 4°C). Cell lysis was performed in 50 mM Tris pH 7.5 containing 8 M guanidinium chloride. The clarified lysate was then processed through a His-Tag affinity column. For effective elution, the proteins were released in two imidazole gradients (300 mM and 500 mM in 50 mM Tris pH 7.5). Desalting into 50 mM Tris pH 7.5 was done using a PD-10 column (GE Healthcare) with Sephadex G-25 beads.

For AD associated protein or peptides’. The 42 or 40 amino acid residues of A[3 (A[342 or A£40) were fused to the NT*FIS_P solubility tag and expressed in BL21(DE3) E. coli strain. Briefly, NT*FIS_P-A[342 or NT*FIS_P-A[340 was purified using a Ni-NTA column, following the previously described protocol⁵³. The NT*FIS_P-A[342 or NT*FIS_P-A[340 fusion proteins were then cleaved with NT*Fis_P-Tev and subjected to lyophilization. The resulting lyophilized material was dissolved in 20 mM Tris (pH 8.0) containing 7 M guanidium chloride. Subsequently, the A[342 or A[340 monomers were separated using a Superdex30 26/600 column (Cytiva) in 20 mM NaPi (pH 8.0) with 0.2 mM EDTA, and then aliquoted into low-binding Eppendorf tubes (Axygene). The concentrations of AP monomers were determined using an extinction coefficient of 1424 M^-1 cm^-1 at (A280-A300). Recombinant, native, and full-length tau protein was expressed and purified following established protocol⁵⁴. In short, the full-length tau proteins were expressed in BL21(DE3) E. coli cells. For purification, the cell pellet was thawed, resuspended, and cell disruption was achieved using a homogenizer. After centrifugation and heat treatment, the heat-stable tau protein in the supernatant is subjected to cation-exchange chromatography. The tau protein was then eluted using a NaCI gradient and fractions containing tau were identified via SDS-PAGE. These fractions were pooled, desalted, and the tau protein concentration was determined. Finally, the tau protein was aliquoted, frozen, and lyophilized for long-term storage.

For PD associated protein-. Preparation of recombinant wildtype o-synuclein monomers was performed according to previously published reports⁵⁵. Briefly, o-synuclein was expressed in BL21(DE3) E. coli cells. Induced bacterial cultures were pelleted and sonicated for the lysis of the cells. After centrifugation, the supernatant was purified by anion exchange chromatography (HiPrep 16/10 Q FF, Cytiva), followed by reversephase HPLC (Jupiter 300 C4, 20 mm I.D. x 250 mm, 10 pm average bead diameter, Phenomenex) and lyophilized. The o-synuclein powder was dissolved in Tris pH 7.5, and the o-synuclein monomers were isolated by a superdex 200 26/600 column (Cytiva).

For T2D associated peptide The IAPP peptide was expressed in BL21(DE3) E. coli cells and purified according to the previous protocol⁵⁶. Briefly, cell pellets were resuspended in a solution of 8 M urea and 20 mM Tris pH 8.0, and then sonicated until the mixture became clear. The resulting lysate was filtered through a 0.22 pm filter to remove any insoluble material and then loaded onto a HisPrep FF 16/10 column (Cytiva, Sweden). The column was washed with a solution containing 15 mM imidazole, 8 M urea, and 20 mM Tris pH 8.0, and the protein was eluted with a solution of 200 mM imidazole, 8 M urea, and 20 mM Tris pH 8.0. The eluted NT*Fis_P-MetIAPP protein was then dialyzed against 20 mM Tris pH 8.0. For cleavage, NT*Fis_P-MetIAPP was treated with 100 mM CNBr in a pH 1.0 environment by adding 2 M HCI until the final concentration of HCI reached 0.1 M, and incubated at room temperature overnight. The IAPP was then pelleted, dissolved in 6 M Guanidine-HCI, and applied to a 3 mL Resource RPC column (Cytiva) pre-equilibrated with 0.1% Trifluoroacetic acid (TFA) and 2% Acetonitrile. A linear gradient of 2 to 80% Acetonitrile was used to elute the IAPP peptide as a single peak. The peak fractions were collected, pooled, lyophilized, redissolved in hexafluoroisopropanol (HFIP) at a concentration of 100 pM, and stored at -20°C until needed.

Circular Dichroism (CD) Spectroscopy

CD spectra were acquired at 25°C using a J-1500 CD spectrophotometer (JASCO) equipped with a PTC-517 Peltier thermostat cell holder. Samples of CagA^N at a concentration of 5 pM were prepared in buffer D, while samples of the CagA D2 domain were diluted in buffer J. Quartz glass cuvettes with a path length of 1 mm were used for measurements. Five consecutive scans were recorded for each sample in the wavelength range of 180 to 260 nm, with an increment of 0.5 nm. Averaged spectra were obtained after blank subtraction and converted to mean residue ellipticity (MRE, deg • cm²/ dmol).

Measurement of biofilm formation with crystal violet A single colony of Pseudomonas sp. UK4 (W and pFap) was transferred to a 50 ml tube with 30 ml LB medium and ampicillin (100 pM) to be incubated overnight at 28°C, 180 RPM. The solutions were diluted to have an ODeoo~0.5 in the presence of ampicillin (100 pM) before 270 pl hereof were added to each well of a 96-well plate (Thermo Fisher scientific 236105). Peg lids (Nunc-Tsp, Thermo Fisher scientific 445497) were inserted in the plate wells for 1 h to initiate biofilm growth on the peg lids, before being transferred to a new 96-well plate with 270 pl fresh LB medium with either 0.03, 0.3, 3 or 6 pM CagA^N. The plate was then incubated at 28°C for 48 h to initiate growth on the peg lids. Following incubation, the peg lid was then washed in milliQ water and dried for 1 h at room temperature. The peg lid was then submerged in a new 96-well plate with 270 pl Gram's crystal violet solution for 15 min, before being washed twice to remove excess stain from the dye. The bound crystal violet was then released from the peg lid by placing the peg lid in a new 96-well plate with 270 pl 33% v/v acetic acid (glacial acetic acid diluted with milliQ water) for 30 min at room temperature. The released crystal violet is then transferred to a new 96-well plate with a clear bottom before being measured on a Varioskan LUX. The absorbance intensity 590 nm was used as a measure of the amount of biofilm that had grown on the peg lid.

Thioflavin T (ThT) fluorescence Assay

To investigate the effects of CagA^N or the CagA D2 domain on A[342, A[340, tau, o- synuclein, and IAPP fibril formation, ThT assay were employed. ThT fluorescence was recorded using a microplate reader (FLUOStar Galaxy from BMG Labtech, Offenberg, Germany) equipped with a 440 nm excitation filter and a 480 nm emission filter. For all experiments, aggregation traces were normalized and averaged using 4-5 replicates, and data for one set of experiments were recorded from the same plate.

For A/342 and A[340: 20 pL solution (20 mmol/L sodium phosphate buffer pH 8.0 with 0.2 mM EDTA) containing 10 pmol/L ThT, 3 pmol/L A[342, and different concentrations of the CagA^N or the CagA D2 domain (ranging from 0 to 100 nmol/L), were added to each well of half-area 384-well black polystyrene microplates with clear bottoms and nonbinding surfaces (Corning Glass 3766, USA). The microplates were then incubated at 37°C under quiescent conditions. For monitoring the effects of CagA^N on A[340 fibrillization, a similar 20 pL solution (20 mmol/L sodium phosphate buffer pH 7.4 with 0.2 mM EDTA) containing 10 pmol/L ThT, 5 pmol/L A[340 monomer and different concentrations of the CagA^N (ranging from 0 to 1.0 nmol/L) were added to wells of halfarea 384-well microplates with a clear bottom, and the plates were incubated at 37°C. For taw. 15 pM monomeric tau protein was incubated in the absence and presence of 0-500 nM CagA^N in 20 mM sodium phosphate buffer pH 7.4 and 0.2 mM EDTA together with 50 pM ThT. The samples were distributed with six replicates to a 384-well plate together with two glass beads (1 mm diameter), and the fluorescence intensity was measured every 10 minutes at 37°C. Shaking condition was applied with orbital shaking at 300 rpm for 9 minutes in-between the measurements.

For a-synucleirr. To detect the effect of CagA^N or the CagA D2 domain on o-synuclein fibril formation, a 20 pL solution containing 10 pmol/L ThT, 30 pmol/L or 15 pmol/L o- synuclein monomer, and various concentrations of CagA^N or the D2 domain (ranging from 0 to 3 pmol/L) was prepared. The plates were incubated at 37 °C with shaking.

For IAPP-. 20 pL solution (20 mmol/L sodium phosphate buffer pH 8.0 with 0.2 mM EDTA) containing 10 pmol/L ThT, 10 pmol/L IAPP, and different concentrations of the CagA^N (ranging from 0 to 100 nmol/L), were added to each well of half-area 384-well black polystyrene microplates with clear bottoms and nonbinding surfaces (Corning Glass 3766, USA). The microplates were then incubated at 37°C under quiescent conditions.

Kinetics analysis

For extracting the aggregation half time T_1/2 and the maximal growth rate r_max, the aggregation traces of different amyloidogenic proteins or peptides with and without CagA^N or the D2 domain were fitted to a sigmoidal equation³²-⁵⁷,

F = F_o + 4/(1 + exp [r_max (r_1/2 — t)J), where A is the amplitude and Fo is the base value.

For global fit analysis, the aggregation traces of the total fibril mass concentration, M(t), is described by the following integrated rate law, where the additional coefficients are functions of A and K described by Cohen et al⁵⁸. where the intermediate coefficients are functions of A and K, and nc and ri2 are the reaction orders for primary and secondary nucleation, respectively:

The aggregations graces of amyloid-forming peptides or proteins with and without CagA^N or the D2 domain were globally fitted through AmyloFit 2.0 platform⁵⁹ (https://amylofit.com/amylofitmain/fitter/) with models of Secondary nucleation Dominated (unseeded), where the fits were partially constrained with two fitting parameters held to a constant value, resulting in that only one rate constant (kn, k-i- or k2) is the sole fitting parameter. The parameters nc and r?2 are the reaction orders for primary and secondary nucleation, respectively, and both were set to 2.

Flow-induced dispersion analysis (FIDA)

The o-synuclein monomers were labelled with ALC480, and the free dyes were removed by a desalting column. For FIDA (Fida 1 Platform, Fidabio) analysis ALC480 labelled o- synuclein monomers were used as indicator and the CagA^N was the analyte. Settings: measurement time 4-6 mins/data point, detection Fida 1-480 nm detector, tray 1 5°C, capillary 25°C, PC capillary, 400 mbar

Surface plasmon resonance (SPR)

SPR assays were conducted using a BIAcore 3000 instrument (BIAcore AB). A[342 monomers, o-synuclein monomers, oligomer, and fibrils were individually immobilized via amine coupling onto flow cells on different types of sensor chips (Cytiva). All immobilization procedures were carried out using phosphate buffer (20 mM sodium phosphate, 0.2 mM EDTA, pH 8.0) as the running buffer, with a flow rate of 20 pL/min, and following the manufacturer's instructions. Blank reference surfaces were prepared on flow cell 3 of each sensor chip using the same coupling protocol, with no protein injected. The different protein or peptide, diluted in 10 mM sodium acetate buffer at pH 4.5 to a concentration of 2.5 pM, were immobilized onto flow cell 4 of a CM5 sensor chip using the amine-coupling immobilization wizard in the BIAcore 3000 control software, to achieve an immobilization level of 300 RU. Following immobilization, the flow cells were stabilized overnight in HEPES-buffered saline without detergent (10 mM HEPES, 150 mM NaCI, 0.2 mM EDTA, pH 7.5) at a flow rate of 20 pL/min to remove nonspecifically bound proteins. For binding analysis, analytes were diluted in HEPES-buffered saline to different concentrations ranging from 0 to 100 nmol/L for CagA^N. The samples were injected individually in duplicates over the chip surfaces at 25 °C and a flow rate of 30 pL/min. All experiments were conducted with HEPES-buffered saline as the running buffer and 30 mM NaOH for regeneration of the chip surfaces. The response from the blank surface was subtracted from the immobilized surface response for each concentration of analyte. For affinity analysis, steady state affinities for CagA^N to different amyloidogenic proteins or peptides were estimated by plotting the maximum binding response. The baseline of the sensorgrams were adjusted to zero and buffer spikes were excluded for global fits to reflect the binding affinity.

Coincubation of AB42 and CaqA^N

A[342 monomer at concentrations of either 3 pmol/L or 5 pmol/L was mixed with various concentrations of CagA^N (ranging from 0 to 3.0 pmol/L) in a 150 pL solution of 20 mmol/L sodium phosphate buffer at pH 8.0 with 0.2 mM EDTA. The mixture was prepared on ice, and 90 pL of the samples were added to the wells of half-area 384- well black polystyrene microplates. The microplates were then incubated at 37°C for 25 hours. Additionally, 60 pL of the fresh samples were kept at -20 °C for further analysis. After the incubation period, the supernatant of the incubated samples was collected by centrifugation (13 000 rpm for 1 h at 4°C). Subsequently, the samples were analyzed either by SDS, Native gels or mass spectrometry.

Measurement of biofilm formation with crystal violet

A single colony of Pseudomonas sp. UK4 (WT and pFap) was transferred to a 50 ml tube with 30 ml LB medium and ampicillin (100 pM) to be incubated overnight at 28°C, 180 RPM. The solutions were diluted to have an ODeoo~0.5 in the presence of ampicillin (100 pM) before 270 pl hereof were added to each well of a 96-well plate (Thermo Fisher scientific 236105). Peg lids (Nunc-Tsp, Thermo Fisher scientific 445497) were inserted in the plate wells for 1 h to initiate biofilm growth on the peg lids, before being transferred to a new 96-well plate with 270 pl fresh LB medium with either 0.03, 0.3, 3 or 6 pM CagA^N. The plate was then incubated at 28°C for 48 h to initiate growth on the peg lids. Following incubation, the peg lid was then washed in milliQ water and dried for 1 h at room temperature. The peg lid was then submerged in a new 96-well plate with 270 pl Gram's crystal violet solution for 15 min, before being washed twice to remove excess stain from the dye. The bound crystal violet was then released from the peg lid by placing the peg lid in a new 96-well plate with 270 pl 33% v/v acetic acid (glacial acetic acid diluted with milliQ water) for 30 min at room temperature. The released crystal violet is then transferred to a new 96-well plate with a clear bottom before being measured on a Varioskan LUX. The absorbance intensity 590 nm was used as a measure of the amount of biofilm that had grown on the peg lid.

Nuclear Magnetic Resonance (NMR) spectroscopy

2D HSQC spectra of 18 |_iM o-synuclein samples in the absence and presence of 18 piM CagA^N were recorded on a 700 MHz Bruker Avance NMR spectrometer equipped with a triple cryogenic probe. The spectra were recorded at 281 K in 20 mM NaP pH 7.4, 0.2 mM EDTA + 20 mM Tris pH 8 and 50 mM NaCI (91/9 H2O/D2O), and the crosspeak assignment were based on previously published work⁶⁰. The software Topspin v.4.0.7 was used to process the data, and Poky⁶¹ was used for the data analysis. Residues with a signal-to-noise ratio of >12 were excluded from the analysis due to too low signal.

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Sequence listing table

Claims

1. Use of an agent for reducing and/or preventing and/or reversing amyloid formation, wherein the agent comprises the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

2. The use of Claim 1, wherein CagA has the sequence of amino acids 1-1186 of H. pylori strain 26695, or an orthologue thereof, optionally wherein CagA has the amino acid sequence of SEQ ID: NO: 1.

3. The use of Claim 1 or 2, wherein the N-terminal region of CagA (CagA^N) has the sequence of amino acids 1-884 of CagA from H. pylori strain 26695, or an orthologue thereof, optionally wherein CagA^N has the amino acid sequence of SEQ ID NO: 2.

4. The use of any preceding claim, wherein the portion or variant of CagA^N is between 20 and 400 amino acids in length.

5. The use of any preceding claim, wherein the variant of CagA^N has at least 35%,

36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,

50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,

64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,

78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or has 100% sequence identity to SEQ ID NO: 2.

6. The use of any preceding claim, wherein the portion of CagA^N comprises, or consists of, Domain II (D2) of CagA.

7. The use of any preceding claim, wherein the portion of CagA^N comprises, or consists of, the amino acid sequence of SEQ ID NO: 3.

8. The use of any preceding claim, wherein the agent comprises, or consists of, the amino acid sequence of SEQ ID NO: 2.

9. The use of any preceding claim, wherein the capability of the agent to reduce and/or prevent and/or reverse amyloid formation is determined using a Thioflavin T (ThT) fluorescence assay, optionally using a ThT assay as described in the accompanying Examples.

10. The use of any preceding claim, wherein the amyloid is formed from amyloid fibril proteins and/or amyloid protein inclusions; preferably selected from those recited in Tables 1 and 2.

11. The use of Claim 10, wherein the amyloid is formed from microbial amyloid fibril proteins and/or amyloid protein inclusions selected from the group comprising : curli proteins; CsgA proteins; FapC proteins, TasA proteins, Biofilm-associated protein (Bap); PSM; SuhB; CdrA; CsgA; Pl; WapA; SMU_63C; Microcin E492; RbmA; TasA; MTP; Tu Elongation factor; HpaG; BE-AM1; Rodlins; Chaplins; Het-s; HET-s- like proteins (HET-s-like), Sup35, Ure2, HIV-1 Tat protein, Hepatitis C virus (HCV) core protein, Influenza A virus (IAV) matrix protein Ml, and Polyomavirus capsid proteins.

12. Use of an agent for reducing and/or preventing and/or reversing microbial biofilm formation, wherein the agent comprises the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

13. A method for reducing and/or preventing and/or reversing microbial biofilm formation, the method comprising the step of contacting a biofilm, or a surface susceptible to biofilm formation, with an agent comprising the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

14. An agent comprising the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation, for use in medicine.

15. An agent comprising the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation, for use in preventing and/or treating a disease or condition characterised by amyloid formation.

16. Use of an agent comprising the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation, in the manufacture of a medicament for preventing and/or treating a disease or condition characterised by amyloid formation.

17. A method for preventing and/or treating a disease or condition characterised by amyloid formation in a subject, comprising administering to the subject an effective amount of an agent comprising the N-terminal region of CagA (CagA^N), or a portion or variant thereof, that is capable of reducing and/or preventing and/or reversing amyloid formation.

18. The agent for use of Claim 15, or the use of Claim 16, or the method of Claim 17, wherein the disease or condition characterised by amyloid formation is a protein misfolding disease or a microbial infection that produces a biofilm.

19. The agent for use, use or method of Claim 18, wherein the protein misfolding disease is one mediated by any one of the amyloid fibril proteins and/or intracellular protein inclusions listed in Tables 1 and 2.

20. A combination of an agent as defined in any one of the preceding claims, and an anti-microbial agent.

21. A pharmaceutical composition comprising an agent as defined in any one of the preceding claims and a pharmaceutically-acceptable carrier, diluent and/or excipient.

22. A combination of an agent as defined in any one of the preceding claims, and a further therapeutic moiety.

23. An agent for use, an agent, a pharmaceutical composition, a use, or a method, substantially as described in the accompanying description, claims and examples.