WO2020089912A1 - Anti-biofilm compositions - Google Patents

Abstract

The present invention is directed to anti-biofilm D-enantiomeric polypeptides and compositions comprising the same. Further provided are methods for inhibiting or reducing the formation of biofilm on or within an article, or for treating or inhibiting an infectious disease in a subject in need thereof.

Description

ANTI-BIOFILM COMPOSITIONS

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62/754,024 titled "ANTI-BIOFILM COMPOSITIONS", filed November 1, 2018, the contents of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

[002] The present invention is in the field of microbiology.

BACKGROUND

[003] Curli fibrils, secreted by Escherichia coli and Salmonella typhimurium, play an essential role in cells adhesion, biofilm formation and architecture, host colonization, immune activation and cell invasion. Yet, the curli fibrils are not a consequence of aberrant protein misfolding, but of a highly controlled cellular process, regulated via an array of accessory proteins. In microbes, amyloids often serve as virulence determinants involved in aggressive infections. Atomic resolution molecular structures of amyloids are necessary to rationalize and describe their functions, and to devise means of controlling their formation.

[004] With respect to curli fibril, the two main proteins involved in its formation are the major curli subunit cell surface glycopeptide A (CsgA) and minor subunit CsgB. CsgA, is a main component of Enterobacteriaceae biofilm that self-aggregate into large b-rich fibrils and provides an extreme resistant to chemical or proteolytic degradation. CsgA consists of five imperfect sequence repeats (R1-R5), defined by regularly spaced Serine (Ser), Glutamine (Gln) and Asparagine (Asn) residues. The first and the last repeats (Rl and R5) form amyloid fibrils, while the other repeats (R2-R4) contain 'gatekeeper' residues that halt aggregation propensity. CsgB nucleates CsgA fibrillation in vivo through interactions with soluble and unstructured CsgA molecules secreted to the outer membrane. SUMMARY

[005] The present invention, in some embodiments thereof, is directed to anti-biofilm compositions comprising one or more peptides targeting CsgA, devices comprising the same, and methods of using the same, such as for reducing or inhibiting biofilm formation.

[006] According to a first aspect, there is provided a method for reducing or inhibiting biofilm formation on or within a surface, the method comprising: contacting a surface with at least one D-enantiomeric polypeptide selected from the group consisting of the amino acid sequences: RKRIRLVTKKKR (SEQ ID NO: 1); RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2); and RPRTRLHTHRNR (SEQ ID NO: 3), thereby reducing or inhibiting biofilm formation on or within the surface.

[007] According to another aspect, there is provided a composition comprising at least one D-enantiomeric polypeptide selected from the group consisting of the amino acid sequences: RKRIRLVTKKKR (SEQ ID NO: 1);

RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2); and RPRTRLHTHRNR (SEQ ID NO: 3) and an acceptable carrier, for use in inhibiting or reducing the formation of biofilm.

[008] In some embodiments, the surface is on or within an article or a subject.

[009] In some embodiments, the at last one D-enantiomeric polypeptide is RKRIRLVTKKKR (SEQ ID NO: 1).

[010] In some embodiments, the at last one D-enantiomeric polypeptide is RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2).

[01 1 ] In some embodiments, the at last one D-enantiomeric polypeptide is RPRTRLHTHRNR (SEQ ID NO: 3).

[012] In some embodiments, the D-enantiomeric polypeptide comprises 100% D- enantiomeric amino acid residues.

[013] In some embodiments, the biofilm is produced by a Gram-negative bacterium.

[014] In some embodiments, the Gram-negative bacterium belongs to a genus selected from the group consisting of: Salmonella, Pseudomonas, and Escherichia. [015] In some embodiments, the at least one D-enantiomeric polypeptide reduces the level of biofilm formation by at least 10%.

[016] In some embodiments, the at least one D-enantiomeric polypeptide has a half maximal inhibitory concentration (IC50) of 5 to 50 mM.

[017] In some embodiments, the at least one polypeptide is in a pharmaceutical composition.

[018] In some embodiments, the pharmaceutical composition comprises the at least one D-enantiomeric polypeptide at a concentration of 0.1-100 pM.

[019] In some embodiments, the composition is a pharmaceutical composition comprising the at least one D-enantiomeric polypeptide and a pharmaceutically acceptable carrier.

[020] Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[021 ] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE FIGURES

[022] Figs. 1A-1D are micrographs of fibrillated CsgA spine segments fibrils of (1A) ₄₅LNIYQY_5O (SEQ ID NO: 4) (imperfect repeat 1 (Rl)), (IB) ₄₇lYQYGG₅₂(SEQ ID NO: 5) (Rl), (1C) ₁₃₇VTQVGF₁₄₂ (SEQ ID NO: 6) (R5), and (ID) ₁₂₉TASNSS₁₃₄ (SEQ ID NO: 7) (R4-R5 loop) visualized and documented using transmitting electron microscopy (TEM). Scale bars = 100 pm (1A) and 0.2 pm (1B-1D). [023] Figs. 2A-2D are graphs showing concentration-dependent thioflavin T (ThT) fibrillation kinetics of the cell surface glycoprotein A (CsgA) segments. The graphs represent averaged fluorescence reading of ThT triplicated measurements of (2A) 45LNIYQY50 (SEQ ID NO: 4) (Rl), (2B) 47IYQYGG52 (SEQ ID NO: 5) (Rl), (2C) i37VTQVGFi42 (SEQ ID NO: 6) (R5), and (2D) 129TASNSS134 (SEQ ID NO: 7) (R4-R5 loop) segments at concentrations of 100 mM, 150 pM and 200 pM. (¨) 100 pM, (■) 150 pM, (A) 200 pM. Error bars of average are shown.

[024] Figs. 3A-3D are images of crystal structures of CsgA spine segments. High- resolution crystal structures of the CsgA segments show classical steric zipper cross-b fibrils of tightly mated b-sheets, with individual subunits (peptides) situated perpendicular to the fibril axis. In each structure, the left view is down the fibril axis with residues shown as sticks, and the right view is perpendicular to the fibril axis, with b-strands, shown as ribbons, run horizontally. Eight layers of b-strands are shown while actual fibrils contain thousands of layers. The carbons of each b-sheet are colored either light gray or dark gray. Water molecules are shown as small spheres. (3A) 45LNIYQY50 (SEQ ID NO: 4), (3B) 47IYQYGG52 (SEQ ID NO: 5), and (3C) 137VTQVGF142 (SEQ ID NO: 6) segments formed the classical amyloid-like class 1 steric zipper structure of tightly mated parallel b-sheets. Two possible dry interfaces in the crystal packing are shown. (3D) 129TASNSS134 (SEQ ID NO: 7) segment formed extended b-strands stack in an anti-parallel manner, which reassembled class 8 steric zippers, yet with only a small contact area between two facing b-sheets and no dry interface.

[025] Figs. 4A-4B are graphs showing that specific D-enantiomeric peptides Dpepl (SEQ ID NO: 1), Dpep2 (SEQ ID NO: 2), and Dpep3 (SEQ ID NO: 3) inhibit fibrillation of CsgA but not phenol soluble modulin a3 (PSMa3). The graphs show mean fluorescence readings of triplicate ThT measurements of (4A) CsgA and (4B) PSMa3, without or with the D-enantiomeric peptides at 1:5 mole ratio. Error bars represent standard error of the mean. CsgA and PSMa3 displayed fast fibrillation kinetics, with a short lag time followed by rapid aggregation. Dpepl and Dpep2 delayed fibril formation of CsgA and reduced the fluorescence signal (4A), while showing no effect on PSMa3 fibrillation (4B). Dpep3 had only a minor effect on CsgA fibrillation (4A).

[026] Figs. 5A-5B are graphs showing ThT fibrillation kinetics of CsgA in the presence of the D-enantiomeric peptide, described herein. The graphs show mean fluorescence readings of triplicate ThT measurements of CsgA with or without (5A) Dpepl and (5B) Dpep2. Error bars represent standard error of the mean. Dpepl and Dpep2 delayed fibril formation of CsgA and reduced the fluorescence signal in a dose- dependent manner (5A and 5B).

[027] Figs. 6A-6C are micrographs of CsgA without (6A) or with (6B and 6C) the presence of the D-enantiomeric peptide, described herein. Electron micrographs of CsgA incubated overnight showing the formation of fibrils. In the presence of (6B) Dpepl or (6C) Dpep2, only amorphous aggregates are observed. Scale bars = 100 pm (6A) and 200 pm (6B and 6C).

[028] Figs. 7A-7B are graphs showing that Dpepl delays secondary structure transitions of CsgA. Time-dependent circular dichroism (CD) spectra of CsgA incubated (7A) alone or (7B) in the presence of Dpep 1. Changes in the per-residue molar ellipticity at l=280 nm were measured along a wavelength range of 186-285 nm. Shape and color codes are indicated for each time measurement.

[029] Fig. 8 is a graph showing size exclusion chromatography with multi-angle static light scattering (SEC-MALS) analysis of freshly purified CsgA. SEC-MALS chromatogram of CsgA presents two main populations with different molecular weights. The major peak corresponds to the monomeric CsgA (-19 kDa), while the minor peak corresponds to CsgA hexamers (-109 kDa).

[030] Fig. 9 is an image of a western blot (WB) analysis showing the D-enantiomeric peptides increase CsgA SDS solubility. Migration of soluble CsgA was detected by standard WB analysis of 15% SDS-PAGE. Incubated CsgA formed insoluble fibrils and did not migrate on the gel (second lane from the left) compared to freshly purified CsgA that was still soluble (first lane from the left). CsgA incubated with Dpepl (first lane from the right) and Dpep2 (second lane from the right) showed increased solubility, indicating that D-enantiomeric peptides inhibit the formation of insoluble fibrils.

[031 ] Figs. 10A-10E are a vertical bar graph and micrographs showing that D- enantiomeric peptides reduce biofilm biomass of Salmonella typhimurium. (10A) is a vertical bar graph showing quantification of a static biofilm assay using crystal-violet analysis. The graph represents the biofilm biomass measurement (ODeoo) after the exposure to the D-enantiomeric peptides at various concentrations (0, 5, 10, 20, 37.5 and 150 mM). Statistical significance was analyzed by the Mann-Whitney non-parametric test. Error bars represent standard deviation. Asterisk (*) is p<0.05 compared to the relevant control (no inhibitor added). (10B-10E) are representative static biofilm assays of the D-enantiomeric peptides Dpepl (10C), Dpep2 (10D), and Dpep3 (10E) at 10 mM which compared to control (10B) and documented using confocal microscopy.

[032] Fig. 11 is a vertical bar graph showing the levels of measured congo red residual stain on agar following the removal of a biofilm colony of the S. typhimurium cellulose deficient mutant (MAE150 strain).

[033] Fig. 12 is a graph showing ThT fibrillation kinetics of FapC (functional amyloid in Pseudomonas) in the presence of a D-enantiomeric peptide, described herein. The graph shows mean fluorescence readings of ThT measurements of FapC with (1:5 ratio) or without Dpepl. Error bars represent standard error of the mean. Dpepl delayed fibril formation of FapC.

DETAILED DESCRIPTION

[034] In some embodiments, the present invention is directed to antimicrobial polypeptides, compositions, and devices comprising same, and methods of use thereof including, but not limited to reduction or inhibition of biofilm formation.

[035] The invention is based, in part, on the finding of polypeptides capable of significantly inhibiting fibrillation of a bacterial amyloid (CsgA) and thereby reduce biofilm biomass.

Polypeptides

[036] In some embodiments, a polypeptide comprising one or more D-enantiomeric amino acids, as described herein below, having antimicrobial activity, is provided.

[037] In some embodiments, a polypeptide of the invention comprises or consists of the amino acid sequence: RKRIRLVTKKKR (SEQ ID NO: 1).

[038] In some embodiments, a polypeptide of the invention comprises or consists of the amino acid sequence: RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2).

[039] In some embodiments, a polypeptide of the invention comprises or consists of the amino acid sequence: RPRTRLHTHRNR (SEQ ID NO: 3).

[040] In some embodiments, the polypeptide has increased binding affinity to a bacterial amyloid fiber, compared to a control. [041 ] As used herein, the term "amyloid" refers to long unbranched fibers that are characterized by a cross-beta sheet quaternary structure, in which antiparallel chains of b-stranded peptides are arranged in an orientation perpendicular to the axis of the fiber.

[042] In some embodiments, the polypeptide of the invention inhibits secondary- structure transition of a protein into an amyloid. In some embodiments, the polypeptide stabilizes an amyloid-forming protein in a random coiled structure. In some embodiments, the polypeptide inhibits an amyloid-forming protein from forming into beta sheet structures. Methods of analyzing a protein's secondary structures would be apparent to one of ordinary skill in the art. A non-limiting example of a method for protein secondary structure analysis includes but is not limited to circular dichroism (CD) spectra, such as described in the example section hereinbelow.

[043] In some embodiments, the polypeptide of the invention, or a composition comprising the same, inhibits oligomerization of microorganismal amyloids. In some embodiments, the polypeptide of the invention, or a composition comprising the same, increases solubility of microorganismal amyloids. In some embodiments, the polypeptide of the invention, or a composition comprising the same, reduces microorganismal amyloid fibrillation or polymerization. In some embodiments, the polypeptide of the invention, or a composition comprising the same, reduces microorganismal amyloid stability. In some embodiments, the polypeptide of the invention, or a composition comprising the same, reduces a microorganismal amyloid coverage %. In some embodiments, the polypeptide of the invention, or a composition comprising the same, reduces the area covered by a microorganismal amyloid. In some embodiments, the polypeptide of the invention, or a composition comprising the same, reduces biofilm formation. In some embodiments, the polypeptide of the invention, or a composition comprising the same, reduces biofilm stability. In some embodiments, the polypeptide of the invention, or a composition comprising the same, reduces the area covered by biofilm. In one embodiment, the area covered by biofilm is presented as coverage % and is calculated as follows: (the area covered by biofilm divided by the total surface area) xlOO.

[044] In some embodiments, the invention encompasses a polypeptide comprising one or more D-isomer forms of the amino acids. In some embodiments, the polypeptide comprises a combination of D-amino acids and L-enantiomeric amino acids. In some embodiments, the polypeptide comprises at least 4%, at least 8%, at least 16%, at least 25%, at least 33%, at least 40%, at least 50%, at least 60%, at least 66%, at least 75%, at least 83%, at least 91%, or comprises or consists of 100% D-amino acids, or any range and value therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the polypeptide comprises 4-8%, 8-16%, 16-25%, 25- 33%, 33-40%, 40-50%, 50-60%, 60-66%, 66-75%, 75-83%, 83-91%, or 91-100% D- amino acids. In some embodiments, the polypeptide of the invention comprises 50% or more D-amino acids. In some embodiments, the polypeptide comprises 50% or less L- amino acids.

[045] Production of retro-inverso D-amino acid peptides where at least one amino acid and perhaps all amino acids are D-amino acids is well known in the art. When all of the amino acids in the peptide are D-amino acids, and the N- and C-terminals of the molecule are reversed, the result is a molecule having the same structural groups being at the same positions as in the L-amino acid form of the molecule. The term "diastereomeric peptide" as used herein refers to a peptide comprising both L-amino acid residues and D-amino acid residues. The number and position of D-amino acid residues in a diastereomeric polypeptide of the invention may be variable so long as the peptide is capable of displaying the function of the disclosed invention, e.g., reducing or inhibiting the formation of biofilm. As used herein, the term "D-enantiomeric polypeptide" encompasses any polypeptide as described herein comprising one or more D-amino acids.

[046] In some embodiments, the polypeptide has bacterial amyloid fiber binding affinity with a dissociation constant (KD) of 0.1-1 mM, 0.5-5mM, 1-10 mM, 5-15 mM, 10-20 mM, 15-30 mM, 20-40 mM, 35-50 mM, 45-60 mM, 55-70 mM, 65-80 mM, 75- 90 mM, 85-95 mM, 90-120 mM, 100-500 nM, 250-750 nM, 0.7-1.5 mM, 1-5 pM, 4- lOpM, 8-20 pM, 15-40 pM. Each possibility represents a separate embodiment of the invention. In some embodiments, the polypeptide has bacterial amyloid fiber binding affinity with KD of 0.15 mM at most, 0.5 mM at most, 5 mM at most, 10 mM at most, 20 mM at most, 30 mM at most, 40 mM at most, 50 mM at most, 60 mM at most, 70 mM at most, 80 mM at most, 90 mM at most, 100 mM at most, 110 mM at most, 150 mM at most, 250 mM at most, 500 mM at most, 750 mM at most, 1,500 mM at most, 1 pM at most, 5 pM at most, 10 pM at most, 15 pM at most, 20 pM at most, or 30 pM at most, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. [047] Methods for determining protein-protein binding affinity would be apparent to one of ordinary skill in the art. Non-limiting examples include but are not limited to surface plasmon resonance (SPR), affinity electrophoresis, bio-layer interferometry, dual polarization interferometry, static light scattering, dynamic light scattering, flow- induced dispersion analysis, and others.

[048] In some embodiments, increase is at least 5% more, at least 10% more, at least 20% more, at least 50% more, at least 100% more, at least 250% more, at least 350% more, at least 500% more, at least 750% more, or at least 1,000% more, compared to control, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, increase is 1-15%, 10-50%, 40- 120%, 150-350%, 250-400%, 300-500%, 450-750%, or 650-1,000%. Each possibility represents a separate embodiment of the invention. In some embodiments, increase is at least 2-fold more, at least 5-fold more, at least 8-fold more, at least lO-fold more, at least 20-fold more, at least 30-fold more, at least 50-fold more, at least 75-fold more, or at least lOO-fold more compared to control, or any value and range therebetween. Each possibility represents a separate embodiment of the present invention.

[049] As used herein, "control" refers to any baseline of reference.

[050] In some embodiments, a control comprises a baseline to which the binding affinity of the polypeptide to a bacterial amyloid fiber is compared to. In some embodiments, a control is a protein having no bacterial amyloid fiber binding affinity. In some embodiments, a control is a protein having low bacterial amyloid fiber binding affinity. In some embodiments, a control is a polypeptide comprising 100% L- enantiomeric amino acid residues comprising an amino acid sequence selected from: SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, a control is a polypeptide of the invention which is initially inactivated, such as by antibody neutralization, enzymatic digestion, denaturation, or other methodologies known in the art of protein inactivation, prior to incubation in an environment comprising bacterial amyloid fiber, in vitro or in vivo.

[051] In another embodiment, a control comprises a substrate, or an article, or a surface having no polypeptide of the invention incorporating or coating thereto and/or thereon. In another embodiment, a control comprises a sample of a tissue of a healthy subject. [052] In some embodiments, the polypeptide of the invention can be chemically modified so as to include, any one of but not limited to, terminal-NH₂ acylation, acetylation, or thioglycolic acid amidation, terminal-carboxlyamidation, e.g., with ammonia, methylamine, and the like. The polypeptide can be either linear, cyclic or branched and the like, which conformations can be achieved using methods well known in the art.

[053] Included within the scope of the invention are peptide conjugates (interchangeably "fusion proteins") comprising the polypeptide of the invention joined at their amino or carboxy-terminus or at one of the side chains, such as via a peptide bond, to an amino acid sequence corresponding to or derived from a different protein.

[054] Non-limiting examples of fusion proteins of the invention exhibit a longer serum half-life while maintaining therapeutic effect.

[055] In some embodiments, the term "serum half-life" refers to the time it takes for a substance to lose half of its pharmacologic, physiologic, or radiologic activity following introduction of an amount of the substance into the serum or circulation of an organism. In another embodiment, serum half-life refers to the time it takes for a substance to be reduced to half of a starting amount introduced into the serum of an organism, following such introduction. In some embodiments, a polypeptide of the invention having one or more D-amino acids has a substantially increased serum half- life, e.g., from minutes to several days. Biological stability (or serum half-life) can be measured by a variety of in vitro or in vivo means. For example, differences in half-life can be compared by using a radiolabeled version of each protein to be compared and measuring levels of serum radioactivity as a function of time in the same or different organism. Alternatively, serum half-life can be compared by assaying the levels a polypeptide of the invention present in serum using ELISA as a function of time in the same or different organism.

[056] In some embodiments, conjugates comprising the polypeptide of the invention and a protein can be made by protein synthesis, e.g., by use of a peptide synthesizer, or by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the conjugate by methods commonly known in the art. [057] Addition of amino acid residues may be performed at either terminus of the polypeptide of the invention for the purpose of providing a "linker" by which the polypeptide of this invention can be conveniently bound to a carrier. Such linkers are usually of at least one amino acid residue and can be of 40 or more residues, more often of 1 to 10 residues. Typical amino acid residues used for linking are tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.

Anti-microbial compositions and articles

[058] In some embodiments, the invention is directed to a composition comprising as an active ingredient an effective amount of a polypeptide, and an acceptable carrier and/or diluent. In some embodiments, the invention is directed to a composition comprising as an active ingredient an effective amount of one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3. In some embodiments, the acceptable carrier facilitates incorporation or coating of the active ingredient in and/or on a substrate.

[059] In some embodiments, the composition of the invention further comprises a substrate. In some embodiments a composition comprising one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 is incorporated in and/or on at least a portion of the substrate.

[060] In some embodiments, the invention is directed to a composition comprising a substrate having incorporated in and/or on at least a portion thereof, one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

[061] As used herein, the term "a portion thereof" refers to, for example, a surface or a portion thereof, and/or a body or a portion thereof, of solid or semi-solid substrates; or a volume or a part thereof, of liquid, gel, foams and other non-solid substrates.

[062] Substrates of widely different chemical nature can be successfully utilized for incorporating (e.g., depositing on a surface thereof) one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a composition comprising thereof, thereon, as described herein. The term "successfully utilized" refers to an outcome meaning that: (i) one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a composition comprising thereof, successfully formed a substantially uniform and homogenous coating on the substrate’s surface; and/or (ii) the resulting coating imparts long-lasting desired properties (e.g., antimicrobial or anti biofilm properties, or both) to the substrate’s surface.

[063] Substrate usable according to some embodiments of the invention can therefore be hard (rigid) or soft, solid, semi-solid, or liquid substrates, and may take a form of a foam, a solution, an emulsion, a lotion, a gel, a cream, or any mixture thereof.

[064] Substrate usable according to some embodiments of the invention can have, for example, organic or inorganic surfaces, including, but not limited to, glass surfaces; porcelain surfaces; ceramic surfaces; silicon or organosilicon surfaces, metallic surfaces (e.g., stainless steel); mica, polymeric surfaces such as, for example, plastic surfaces, rubbery surfaces, paper, wood, polymer, a metal, carbon, a biopolymer, silicon mineral (rock or glass), surfaces, wool, silk, cotton, hemp, leather, fur, feather, skin (hide, pelt or pelage) surfaces, plastic surfaces and surfaces comprising or made of polymers such as but not limited to polypropylene (PP), polycarbonate (PC), polyethylene (PET), high- density polyethylene (HDPE), low-density polyethylene (LDPE), polyester (PE), unplasticized polyvinyl chloride (PVC), and fluoropolymers including but not limited to polytetrafluoroethylene (PTFE, Teflon®); or can comprise or be made of any of the foregoing substances, or any mixture thereof.

[065] Alternatively, other portions, or the entire substrate are made of the above- mentioned materials.

[066] In some embodiments, the substrate incorporating one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a composition comprising thereof, as described herein is or forms a part of an article.

[067] According to some embodiments, there is provided an article (e.g., an article- of-manufacturing) comprising a substrate incorporating in and/or on at least a portion thereof one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a composition comprising thereof.

[068] The article can be any article which can benefit from the antimicrobial and/or anti-biofilm formation activities of the polypeptide of the invention.

[069] Non-limiting examples of articles include, but are not limited to, medical devices, organic waste processing device, fluidic device, an agricultural device, a package, a sealing article, a fuel container, a water and cooling system device and a construction element.

[070] Non-limiting examples of devices which can incorporate one or more polypeptides comprising an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, or a composition comprising thereof, as described herein, beneficially, include tubing, pumps, drain or waste pipes, screw plates, and the like.

[071 ] Non-limiting example of an article include but is not limited to an element used in water treatment systems (such as for containing and/or transporting and/or treating aqueous media or water), devices, containers, filters, tubes, solutions and gases and the likes.

[072] Non-limiting example of an article include but is not limited to an element in organic waste treatment systems (such as for containing and/or disposing and/or transporting and/or treating organic waste), devices, containers, filters, tubes, solutions and gases and the likes.

[073] The method also include incorporation of compositions the active material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance.

[074] In some embodiments, the invention is directed to a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of the polypeptide of the invention, and a pharmaceutically acceptable carrier and/or diluent. In some embodiments, the invention is directed to a pharmaceutical composition comprising as an active ingredient a therapeutically effective amount of a polypeptide comprising SEQ ID NO: 1, or a polypeptide comprising SEQ ID NO:2, or a polypeptide comprising SEQ ID NO: 3, or any combination thereof. In some embodiments, the pharmaceutically acceptable carrier facilitates administration of the polypeptide of the invention to an organism. For example, the term "pharmaceutically acceptable" can mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. [075] In some embodiments, the herein disclosed invention is directed to a composition for use in reducing or inhibiting microbial growth, microbial activity, or combination thereof. In some embodiments, microbial activity comprises any activity selected from: proliferation, antibiotic resistance, cell communication and/or quorum sensing, biofilm production, toxin production and/or secretion, or any combination thereof. Microbial activity can be assayed using any method known in the art, non limiting examples of such methods include, but are not limited to, spectrophotometry, drug resistance assays using selective substrates, bioluminescence assay, liquid chromatography, mass -spectrometry, or others, some of which are exemplified herein below, and all of which are well known to one of ordinary skill in art.

[076] As used herein, the term "anti-microbial activity" refers to the ability to inhibit, prevent, reduce or retard bacterial growth, fungal growth, biofilm formation or eradication of living bacterial cells, or their spores, or fungal cells or viruses in a suspension, on a surface or in a moist environment, or any combination thereof. In some embodiments, inhibition or reduction or retardation of biofilm formation by a microorganism positively correlates with inhibition or reduction or retardation of growth of the microorganism and/or eradication of a portion or all of an existing population of microorganisms.

[077] In some embodiments, biofilm formation comprises secretion of amyloid proteins, including, but not limited to CsgA, FapC, and the like.

[078] In some embodiments, reducing is by at least 5%, at least 15%, at least 25%, at least 40%, at least 50%, at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99%, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, reducing is by 1-5%, 7- 15%, 10-25%, 20-40%, 35-50%, 45-65%, 55-75%, 70-85%, 80-90%, 87-95%, or 92- 100%. Each possibility represents a separate embodiment of the present invention.

[079] In some embodiments, a composition as described herein comprises a polypeptide comprising SEQ ID NO: 1 and a polypeptide comprising SEQ ID NO: 2 in a mole ratio ranging from 10: 1 to 1:10 mole/mole, 9: 1 to 1:9 mole/mole, 8:1 to 1:8 mole/mole, 7: 1 to 1:7 mole/mole, 6: 1 to 1:6 mole/mole, 5: 1 to 1:5 mole/mole, 4:1 to 1:4 mole/mole, 3: 1 to 1:3 mole/mole, 2: 1 to 1:2 mole/mole, 1: 1 mole/mole. Each possibility represents a separate embodiment of the invention. [080] In some embodiments, a composition as described herein comprises a polypeptide comprising SEQ ID NO: 1 and a polypeptide comprising SEQ ID NO: 3 in a mole ratio ranging from 10: 1 to 1:10 mole/mole, 9: 1 to 1:9 mole/mole, 8:1 to 1:8 mole/mole, 7: 1 to 1:7 mole/mole, 6: 1 to 1:6 mole/mole, 5: 1 to 1:5 mole/mole, 4:1 to 1:4 mole/mole, 3: 1 to 1:3 mole/mole, 2: 1 to 1:2 mole/mole, 1: 1 mole/mole. Each possibility represents a separate embodiment of the invention.

[081] In some embodiments, a composition as described herein comprises a polypeptide comprising SEQ ID NO: 2 and a polypeptide comprising SEQ ID NO: 3 in a mole ratio ranging from 10: 1 to 1:10 mole/mole, 9: 1 to 1:9 mole/mole, 8:1 to 1:8 mole/mole, 7: 1 to 1:7 mole/mole, 6: 1 to 1:6 mole/mole, 5: 1 to 1:5 mole/mole, 4:1 to 1:4 mole/mole, 3: 1 to 1:3 mole/mole, 2: 1 to 1:2 mole/mole, 1: 1 mole/mole. Each possibility represents a separate embodiment of the invention.

[082] In some embodiments, the composition comprises a polypeptide of the invention in a concentration of at least 50 nM, at least 100 nM, at least 0.5 mM, at least

1 mM, at least 2 pM, at least 5 pM, at least 10 pM, at least 15 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 75 pM, at least 100 pM, at least 150 pM, at least 200 pM, at least 225 pM, or at least 350 pM, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the composition comprises a polypeptide of the invention in a concentration of 50-250 nM, 500-1,500 nM, 1-10 pM, 5-20 pM, 10-30 pM, 20-40 pM, 25-50 pM, 30-60 pM, 40-70 pM, 50-80 pM, 65-90 pM, 70-100 pM, 80-110 pM, 90- 120 pM, 110-160 pM, 150-275 pM, or 250-500 pM . Each possibility represents a separate embodiment of the invention.

[083] In some embodiments, the composition comprises each of the polypeptides of the invention in a concentration of at least 50 nM, at least 100 nM, at least 1 pM, at least

2 pM, at least 5 pM, at least 10 pM, at least 15 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 75 pM, at least 100 pM, at least 150 pM, at least 200 pM, at least 225 pM, or at least 350 pM, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, the composition comprises each of the polypeptides of the invention in a concentration of 10-50 nM, 40-100 nM, 75-500 nM, 450-900 nM, 0.75-1.5 pM, 1-10 pM, 5-20 pM, 10-30 pM, 20-40 pM, 25-50 pM, 30-60 pM, 40-70 pM, 50-80 pM, 65-90 pM, 70-100 mM, 80-110 mM, 90-120 mM, 110-160 mM, 150-275 mM, or 250-500 mM. Each possibility represents a separate embodiment of the invention.

[084] As defined herein, the term "half maximal inhibitory concentration (IC50)" refers to a measure of the potency of a substance to inhibit a specific biological or biochemical function. In some embodiments, the composition has an IC50 at the micromolar level. In some embodiments, micromolar level comprises 1,000 mM at most, 900 mM at most, 800 mM at most, 700 mM at most, 600 mM at most, 500 mM at most, 400 mM at most, 300 mM at most, 200 mM at most, 100 mM at most, 75 mM at most, 50 mM at most, 35 mM at most, 20 mM at most, 15 mM at most, 10 mM at most, 5 mM at most, or 1 mM at most, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, micromolar level comprises 1-10 mM, 5-20 mM, 15-30 mM, 25-500 mM, 40-75 mM, 70-120 mM, 100- 200 mM, 150-300 mM, 250-400 mM, 375-500 mM, 400-650 mM, 600-850 mM, or 800- 1,000 mM. Each possibility represents a separate embodiment of the invention.

[085] As used herein, the term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active ingredient is administered. Such carriers can be sterile liquids, such as water-based and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents.

[086] Water may be used as a carrier such as when the active ingredient is comprised by a pharmaceutical composition being administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned. The carrier may comprise, in total, from about 0.1% to about 99.99999% by weight of the compositions presented herein. [087] An embodiment of the invention relates to polypeptides of the present invention, presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. In one embodiment, the unit dosage form is in the form of an ampoule, vial or pre-filled syringe.

[088] In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the nature of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in-vitro or in-vivo animal model test bioassays or systems.

[089] In one embodiment, the composition of the invention is administered in the form of a pharmaceutical composition comprising at least one of the active ingredients of this invention (e.g., polypeptides comprising SEQ ID Nos.: 1-3) together with a pharmaceutically acceptable carrier or diluent. In another embodiment, the composition of the invention can be administered either individually or together in any conventional oral, parenteral or transdermal dosage form.

[090] As used herein, the terms "administering", "administration", and like terms refer to any method which, in sound medical practice, delivers a composition containing an active agent to a subject in such a manner as to provide a therapeutic effect.

[091] Depending on the location of the tissue of interest, the polypeptide of the present invention can be administered in any manner suitable for the provision of the polypeptide to the tissue of interest. Thus, for example, a composition containing the polypeptide of the present invention can be introduced, for example, into the systemic circulation, which will distribute the polypeptide to the tissue of interest.

[092] In some embodiments, a pharmaceutical composition comprising the polypeptide is administered via transdermal, subcutaneous, intramuscular, intraperitoneal or intravenous routes of administration. The route of administration of the pharmaceutical composition will depend on the disease or condition to be treated. Suitable routes of administration include, but are not limited to, parenteral injections, e.g., intradermal, intravenous, intramuscular, intralesional, subcutaneous, intrathecal, and any other mode of injection as known in the art. Although the bioavailability of a polypeptide administered by other routes can be lower than when administered via parenteral injection, by using appropriate formulations it is envisaged that it will be possible to administer compositions comprising the polypeptide of the invention via transdermal, oral, rectal, vaginal, topical, nasal, inhalation and ocular modes of treatment. In addition, it may be desirable to introduce pharmaceutical compositions comprising the polypeptide of the invention by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir.

[093] For purposes of parenteral administration, solutions in sesame or peanut oil or in aqueous propylene glycol can be employed, as well as sterile aqueous solutions of the corresponding water-soluble salts. Such aqueous solutions may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal injection purposes.

[094] According to some embodiments, the polypeptide of the invention can be delivered in a controlled release system. In another embodiment, an infusion pump can be used to administer the polypeptide such as the one that is used, for example, for delivering insulin or chemotherapy to specific organs or tumors. In another embodiment, the polypeptide is administered in combination with a biodegradable, biocompatible polymeric implant, which releases the polypeptide over a controlled period of time at a selected site. Examples of preferred polymeric materials include, but are not limited to, polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla., the contents of which are hereby incorporated by reference in their entirety). In yet another embodiment, a controlled release system can be placed in proximity to a therapeutic target, thus requiring only a fraction of the systemic dose.

[095] The presently described polypeptide may also be contained in artificially created structures such as liposomes, ISCOMS, slow-releasing particles, and other vehicles which increase the half-life of the polypeptide in serum. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. Liposomes for use with the presently described polypeptides are formed from standard vesicle-forming lipids which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally determined by considerations such as liposome size and stability in the blood. A variety of methods are available for preparing liposomes as reviewed, for example, by Coligan, J. E. et al, Current Protocols in Protein Science, 1999, John Wiley & Sons, Inc., New York, and see also U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[096] In one embodiment, the present invention is directed to combined preparations. In one embodiment, "a combined preparation" defines especially a "kit of parts" in the sense that the combination partners as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners i.e., simultaneously, concurrently, separately or sequentially. In some embodiments, the parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners, in some embodiments, can be administered in the combined preparation. In one embodiment, the combined preparation can be varied, e.g., in order to cope with the needs of a patient subpopulation to be treated or the needs of the single patient which different needs can be due to a particular disease, severity of a disease, age, sex, or body weight as can be readily made by a person skilled in the art.

[097] In one embodiment, it will be appreciated that the polypeptide of the invention can be provided to the individual with additional active agents to achieve an improved therapeutic effect as compared to treatment with each agent by itself. In another embodiment, measures (e.g., dosing and selection of the complementary agent) are taken to adverse side effects which are associated with combination therapies.

[098] In one embodiment, depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is affected or diminution of the disease state is achieved.

[099] In some embodiments, composition comprising the polypeptide of the invention is administered in a therapeutically safe and effective amount. As used herein, the term "safe and effective amount" refers to the quantity of a component which is sufficient to yield a desired therapeutic response without undue adverse side effects (such as toxicity, irritation, or allergic response) commensurate with a reasonable benefit/risk ratio when used in the presently described manner. In another embodiment, a therapeutically effective amount of the polypeptide of the invention, is the amount of the mentioned herein polypeptide necessary for the in vivo measurable expected biological effect. The actual amount administered, and the rate and time-course of administration, will depend on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington: The Science and Practice of Pharmacy, 2lst Ed., Lippincott Williams & Wilkins, Philadelphia, Pa., (2005). In some embodiments, preparation of effective amount or dose can be estimated initially from in vitro assays. In one embodiment, a dose can be formulated in animal models and such information can be used to more accurately determine useful doses in humans.

[ 100] In one embodiment, toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. In one embodiment, the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. In one embodiment, the dosages vary depending upon the dosage form employed and the route of administration utilized. In one embodiment, the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. [See e.g., Fingl, et ah, (1975) "The Pharmacological Basis of Therapeutics", Ch. 1 p.l]

[0101 ] Pharmaceutical compositions containing the presently described polypeptide as the active ingredient can be prepared according to conventional pharmaceutical compounding techniques. See, for example, Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, Pa. (1990). See also, Remington: The Science and Practice of Pharmacy, 21 st Ed., Lippincott Williams & Wilkins, Philadelphia, Pa. (2005).

[0102] In one embodiment, a composition comprising the polypeptide of the invention formulated in a compatible pharmaceutical carrier is prepared, placed in an appropriate container, and labeled for treatment of an indicated condition. [0103] In one embodiment, a composition comprising the polypeptide of the invention is presented in a pack or dispenser device, such as an FDA approved kit, which contains, one or more unit dosages forms containing the active ingredient. In one embodiment, the pack, for example, comprises metal or plastic foil, such as a blister pack. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, the pack or dispenser is accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, in one embodiment, is labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert.

Methods of use

[0104] According to some embodiments, there present invention is directed to a method for reducing or inhibiting a biofilm formation on or within a surface.

[0105] In some embodiments, the surface is on or within a subject or an article. In some embodiments, the surface is the interphase. In some embodiments, the interphase is between two regions, areas, compartments, having different density. In some embodiments, the surface is the outer layer of a micelle. In some embodiments, the surface is within an emulsion. In some embodiments, the surface is on water-based droplets, oil-based droplets, or both, within an emulsion, including, but not limited to water in oil, oil in water, water in oil in water, oil in water in oil emulsions.

[0106] In some embodiments, the method is directed to treating a biofilm-related infectious disease in a subject in need thereof.

[0107] In some embodiments, treating or inhibiting of the disease encompasses the treatment or inhibition of symptoms associated therewith.

[0108] As used herein, "biofilm-related infectious disease" refers to any disease or disorder caused to a subject by an increased formation of a biofilm. In some embodiments, a biofilm-related infectious disease inducing-organism is selected from: bacteria, viruses, fungi, parasites, or a combination thereof.

[0109] Non-limiting examples for symptoms of an infectious disease include, but are not limited to, fever, diarrhea, fatigue, muscle aches, coughing, or their combination. [01 10] Non-limiting examples of infectious diseases include urinary tract infection, gastrointestinal infection, enteritis, salmonellosis, diarrhea, nontuberculous mycobacterial infections, legionnaires' disease, hospital-acquired pneumonia, skin infection, cholera, septic shock, periodontitis, infection, and sinusitis. In some embodiments, the infection induces a condition selected from: bacteremia, skin infections, neonatal infections, pneumonia, endocarditis, osteomyelitis, toxic shock syndrome, scalded skin syndrome, and food poisoning.

[011 1] The term "subject" as used herein refers to an animal, more particularly to non human mammals and human organism. Non-human animal subjects may also include prenatal forms of animals, such as, e.g., embryos or fetuses. Non-limiting examples of non-human animals include, horse, cow, camel, goat, sheep, dog, cat, non-human primate, mouse, rat, rabbit, hamster, guinea pig, and pig. In one embodiment, the subject is a human. Human subjects may also include fetuses.

[0112] As used herein, the terms "treatment" or "treating" of a disease, disorder, or condition encompasses alleviation of at least one symptom thereof, a reduction in the severity thereof, or inhibition of the progression thereof. Treatment need not mean that the disease, disorder, or condition is totally cured. To be an effective treatment, a useful composition herein needs only to reduce the severity of a disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide improvement to a patient or subject’s quality of life.

[01 13] As used herein, the term "prevention" of a disease, disorder, or condition encompasses the delay, prevention, suppression, or inhibition of the onset of a disease, disorder, or condition. As used in accordance with the presently described subject matter, the term "prevention" relates to a process of prophylaxis in which a subject is exposed to the presently described peptides prior to the induction or onset of the disease/disorder process. This could be done where an individual has a genetic pedigree indicating a predisposition toward occurrence of the disease/disorder to be prevented. For example, this might be true of an individual whose ancestors show a predisposition toward certain types of, for example, inflammatory disorders. The term "suppression" is used to describe a condition wherein the disease/disorder process has already begun but obvious symptoms of the condition have yet to be realized. Thus, the cells of an individual may have the disease/disorder, but no outside signs of the disease/disorder have yet been clinically recognized. In either case, the term prophylaxis can be applied to encompass both prevention and suppression. Conversely, the term "treatment" refers to the clinical application of active agents to combat an already existing condition whose clinical presentation has already been realized in a patient.

[0114] As used herein, the term "condition" includes anatomic and physiological deviations from the normal that constitute an impairment of the normal state of the living animal or one of its parts, that interrupts or modifies the performance of the bodily functions.

[01 15] In some embodiments, a composition comprising the polypeptide of the invention is directed to killing microorganisms in a living tissue or on or in an article or reducing the formation of microorganisms on or in an article.

[01 16] In some embodiments, the present invention is directed to a method of inhibiting or reducing a formation of biofilm on or within an article, comprising incorporating or coating a composition comprising one or more of a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 1, a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 2, and a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 3, on and/or within the article.

[01 17] According to some embodiments, the method comprises treating or ameliorating a biofilm-related infectious disease or a symptom thereof in a subject in need thereof, comprising administering to the subject one or more polypeptides comprising an amino acid sequence comprising SEQ ID NO: 1, a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 2, and a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 3, or a pharmaceutical composition comprising thereof.

[01 18] In some embodiments, there is provided a use of a composition comprising an effective amount of a polypeptide in the preparation of a medicament for the treatment, amelioration, reduction, or prevention of a biofilm-related infectious disease or a symptom thereof in a subject in need thereof. In some embodiments, the invention is directed to a use of a composition comprising an effective amount of one or more of a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 1, a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 2, and a polypeptide comprising an amino acid sequence comprising SEQ ID NO: 3 in the preparation of a medicament for the treatment of an infectious disease or a symptom thereof in a subject in need thereof.

[0119] In one embodiment, the polypeptide of the invention is provided to the subject per se. In one embodiment, one or more of the polypeptides of the invention are provided to the subject per se. In one embodiment, the polypeptide of the invention is provided to the subject as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. In one embodiment, one or more of the polypeptides of the invention are provided to the subject as part of a pharmaceutical composition where they are mixed with a pharmaceutically acceptable carrier.

[0120] As used herein, the term "biofilm" refers to a group of microorganisms adhering to one another, which are embedded within self -produced and self-secreted extracellular polymer comprising DNA, proteins, polysaccharides, or any combination thereof. In some embodiments, a biofilm adheres to a surface on a living host. In one embodiment, a biofilm adheres to a non-living surface.

[0121] In some embodiments, any one of the polypeptides, composition comprising thereof, or method of the present invention reduce biofilm production by at least 1%, at least 25%, at least 50%, at least 100%, at least 250, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1,000%, compared to control, or any value and range therebetween. Each possibility represents a separate embodiment of the invention. In some embodiments, biofilm production is reduced by 1-35%, 30-150%, 140-250%, 200-400%, 350-630%, 280-490%, 375-590%, 450-650%, 500-750%, or 650-1,000%, compared to control. Each possibility represents a separate embodiment of the invention. In some embodiments, biofilm production is reduced by at least 2-fold, at least 3 -fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7- fold, at least 8-fold, at least 9-fold, or at least lO-fold, compared to control, or any value and range therebetween. Each possibility represents a separate embodiment of the invention.

[0122] In some embodiments, any one of the polypeptides, composition comprising thereof, or method of the present invention inhibits de novo synthesis of biofilm. In some embodiments, any one of the polypeptides, composition comprising thereof, or method of the present invention reduces the area covered by biofilm. In some embodiments, any one of the polypeptides, composition comprising thereof, or method of the present invention induces degradation of previously formed biofilm. As used herein, previously formed refers to a biofilm produced prior to the application of any one of the polypeptides, composition comprising thereof, or method of the present invention. Polypeptide synthesis

[0123] The polypeptide of the invention may be synthesized or prepared by techniques well known in the art. The polypeptide can be synthesized by a solid phase peptide synthesis method of Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). Alternatively, the polypeptide can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984) or by any other method known in the art for peptide synthesis.

[0124] Recombinant technology may be used to express the polypeptide of the present invention.

[0125] In some embodiments, the invention encompasses polynucleotides encoding the polypeptides of the invention. In another embodiment, a polynucleotide sequence encoding the polypeptide is at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% homologous to the polynucleotide encoding a polypeptide comprising any one of SEQ ID Nos.: 1-3.

[0126] In some embodiments, a polynucleotide of the invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the polypeptide of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing tissue- specific expression of the polypeptide of the present invention. In some embodiments, the cis- regulatory sequence is suitable for directing inducible expression of the polypeptide of the present invention.

[0127] In some embodiments, a polynucleotide of the invention is prepared using polymerase chain reaction (PCR) techniques, or any other method or procedure known to one skilled in the art.

[0128] In one embodiment, the polynucleotide is inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of a recombinant polypeptide. In one embodiment, the expression vector includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhancers) and transcription and translation terminators (e.g., polyadenylation signals).

[0129] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the polypeptide of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the polypeptide coding sequence; yeast transformed with recombinant yeast expression vectors containing the polypeptide coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing polypeptide coding sequence.

[0130] In some embodiments, non-bacterial expression systems are used (e.g. mammalian expression systems) to express the polypeptide of the present invention. In one embodiment, the expression vector is used to express the polynucleotide of the present invention in mammalian cells.

[0131 ] In some embodiments, in bacterial systems, a number of expression vectors can be advantageously selected depending upon the use intended for the polypeptide expressed. In one embodiment, large quantities of polypeptide are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the polypeptide. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et ah, Methods in Enzymol. 185:60-89 (1990)].

[0132] In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. No. 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.

[0133] In one embodiment, the expression vector may further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES).

[0134] In some embodiments, mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3.l (±), pGL3, pZeoSV2(±), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.l, pSinRep5, DH26S, DHBB, pNMTl, pNMT4l, pNMT8l, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

[0135] In some embodiments, expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses are used by the present invention. SV40 vectors include pSVT7 and pMT2. In some embodiments, vectors derived from bovine papilloma virus include pBV-lMTHA, and vectors derived from Epstein Bar virus include pHEBO, and p205. Other exemplary vectors include pMSG, pAV009/A+, rMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.

[0136] In some embodiments, recombinant viral vectors, which offer advantages such as lateral infection and targeting specificity, are used for in vivo expression of the polypeptide of the invention. In one embodiment, lateral infection is inherent in the life cycle of, for example, retrovirus and is the process by which a single infected cell produces many progeny virions that bud off and infect neighboring cells. In one embodiment, the result is that a large area becomes rapidly infected, most of which was not initially infected by the original viral particles. In one embodiment, viral vectors are produced that are unable to spread laterally. In one embodiment, this characteristic can be useful if the desired purpose is to introduce a specified gene into only a localized number of targeted cells. [0137] Various methods can be used to introduce an expression vector into cells. Such methods are generally described in Sambrook et ah, Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et ah, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et ah, Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et ah, Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[0138] In one embodiment, plant expression vectors are used. In one embodiment, the expression of a polypeptide coding sequence is driven by a number of promoters. In some embodiments, viral promoters such as the 35S RNA and 19S RNA promoters of CaMV [Brisson et ah, Nature 310:511-514 (1984)], or the coat protein promoter to TMV [Takamatsu et ah, EMBO J. 6:307-311 (1987)] are used. In another embodiment, plant promoters are used such as, for example, the small subunit of RUBISCO [Coruzzi et ah, EMBO J. 3: 1671-1680 (1984); and Brogli et ah, Science 224:838-843 (1984)] or heat shock promoters, e.g., soybean hspl7.5-E or hspl7.3-B [Gurley et ah, Mol. Cell. Biol. 6:559-565 (1986)]. In one embodiment, constructs are introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach [Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463 (1988)]. Other expression systems such as insects and mammalian host cell systems, which are well known in the art, can also be used by the present invention.

[0139] It will be appreciated that other than containing the necessary elements for the transcription and translation of the inserted coding sequence (encoding the polypeptide), the expression construct can also include sequences engineered to optimize stability, production, purification, yield or activity of the expressed polypeptide.

[0140] In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of a recombinant polypeptide. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce a recombinant polypeptide of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, the cells can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.

[0141] In some embodiments, depending on the vector and host system used for production, resultant polypeptide of the present invention either remains within the recombinant cell, secreted into the fermentation medium, secreted into a space between two cellular membranes, such as the periplasmic space in E. coli; or retained on the outer surface of a cell or viral membrane. In one embodiment, following a predetermined time in culture, recovery of the recombinant polypeptide is affected.

[0142] In one embodiment, the phrase "recovering the recombinant polypeptide" used herein refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification.

[0143] In one embodiment, a polypeptide of the invention is purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

[0144] In one embodiment, to facilitate recovery, the expressed coding sequence can be engineered to encode the polypeptide of the present invention and a fused cleavable moiety. In one embodiment, a fusion protein can be designed so that the polypeptide can be readily isolated by affinity chromatography, e.g., by immobilization on a column specific for the cleavable moiety. In one embodiment, a cleavage site is engineered between the polypeptide and the cleavable moiety, and the polypeptide can be released from the chromatographic column by treatment with an appropriate enzyme or agent that specifically cleaves the fusion protein at this site [e.g., see Booth et ah, Immunol. Lett. 19:65-70 (1988); and Gardella et al., J. Biol. Chem. 265: 15854-15859 (1990)].

[0145] In one embodiment, the polypeptide of the invention is retrieved in "substantially pure" form that allows for the effective use of the protein in the applications described herein.

[0146] As used herein, the term "substantially pure" describes a peptide/polypeptide or other material which has been separated from its native contaminants. Typically, a monomeric peptide is substantially pure when at least about 60 to 75% of a sample exhibits a single peptide backbone. Minor variants or chemical modifications typically share the same peptide sequence. A substantially pure peptide can comprise over about 85 to 90% of a peptide sample, and can be over 95% pure, over 97% pure, or over about 99% pure. Purity can be measured on a polyacrylamide gel, with homogeneity determined by staining. Alternatively, for certain purposes high resolution may be necessary and HPLC or a similar means for purification can be used. For most purposes, a simple chromatography column or polyacrylamide gel can be used to determine purity.

[0147] The term "purified" does not require the material to be present in a form exhibiting absolute purity, exclusive of the presence of other compounds. Rather, it is a relative definition. A peptide is in the "purified" state after purification of the starting material or of the natural material by at least one order of magnitude, 2 or 3, or 4 or 5 orders of magnitude.

[0148] In one embodiment, the polypeptide of the invention is substantially free of naturally-associated host cell components. The term "substantially free of naturally- associated host cell components" describes a peptide or other material which is separated from the native contaminants which accompany it in its natural host cell state. Thus, a peptide which is chemically synthesized or synthesized in a cellular system different from the host cell from which it naturally originates will be free from its naturally- associated host cell components.

[0149] In one embodiment, the polypeptide of the invention can also be synthesized using in vitro expression systems. In one embodiment, in vitro synthesis methods are well known in the art and the components of the system are commercially available. Non-limited example for in vitro system includes, but is not limited to, in vitro translation.

[0150] Any concentration ranges, percentage range, or ratio range recited herein are to be understood to include concentrations, percentages or ratios of any integer within that range and fractions thereof, such as one tenth and one hundredth of an integer, unless otherwise indicated.

[0151 ] Any number range recited herein relating to any physical feature, is to be understood to include any integer within the recited range, unless otherwise indicated.

[0152] In the discussion unless otherwise stated, adjectives such as "substantially" and "about" modifying a condition or relationship characteristic of a feature or features of an embodiment of the invention, are understood to mean that the condition or characteristic is defined to within tolerances that are acceptable for operation of the embodiment for an application for which it is intended. Unless otherwise indicated, the word "or" in the specification and claims is considered to be the inclusive "or" rather than the exclusive or, and indicates at least one of, or any combination of items it conjoins.

[0153] It should be understood that the terms "a" and "an" as used above and elsewhere herein refer to "one or more" of the enumerated components. It will be clear to one of ordinary skill in the art that the use of the singular includes the plural unless specifically stated otherwise. Therefore, the terms "a," "an" and "at least one" are used interchangeably in this application.

[0154] For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0155] In the description and claims of the present application, each of the verbs, "comprise," "include" and "have" and conjugates thereof, are used to indicate that the object or objects of the verb are not necessarily a complete listing of components, elements or parts of the subject or subjects of the verb.

[0156] Other terms as used herein are meant to be defined by their well-known meanings in the art.

[0157] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

[0158] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub -combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

EXAMPLES

[0159] Generally, the nomenclature used herein, and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et ah, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et ah, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et ah, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds.) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

Materials and methods

Peptides and reagents

[0160] Segments of the CsgA peptide (custom synthesis) at >98% purity were purchased from GL Biochem (Shanghai) Ltd. The peptides were synthesized with capped termini (acetylated in the N-terminus and amidated in the C-terminus) for fibrillation assays, or with unmodified termini for crystallography. All D-peptides consisted of D-enantiomeric amino acids, C-terminally amidated, > 95% pure and were purchased from either GL Biochem (Shanghai) Ltd, peptides&elephants (Potsdam, Germany), or JPT (Berlin, Germany). The sequences were as follows: Dpepl (ANK6; SEQ ID NO: 1): RKRIRLVTKKKR-NH₂, Dpep2 (DB3DB3; SEQ ID NO: 2): RPITRLRTHQNRRPITRLRTHQNR-NHi and Dpep3 (D3; SEQ ID NO: 3): RPRTRLHTHRNR-NHi.

[0161] Thioflavin T (ThT) was purchased from Sigma- Aldrich. Dimethyl- sulfoxide (DMSO) was purchased from Merck.

Computational prediction of amyloid spine segments

[0162] Fibril-formation propensities of segments of amyloid proteins were predicted using combined information from different servers, including ZipperDB, Tango, Waltz and Zyggregator.

CsgA plasmid construction and purification

[0163] CsgA was cloned into pETl ld vector with C-terminal Hisx6-tagged. The pETl ld-CsgA vector was then transformed to competent E. coli XL-l Blue cells for amplification, and further purified by PureLink Quick Plasmid Miniprep Kit (Invitrogen).

CsgA expression and purification

[0164] E. coli BL-21 cells, transformed with a pETlld vector containing C-terminal Hisx6-tagged CsgA sequence, were grown overnight in 10 ml LB culture supplemented with 50 mg/ml ampicillin, and then further diluted into 1 L of the same medium. The culture was grown at 37 °C with 230 rpm shaking until ODeoo = 0.8-0.9 was reached. CsgA expression was induced with 0.5 mM IPTG and incubation was continued for 1 h. Cell pellets were collected by centrifuge at 4,500 rpm for 25 min and stored at -80 °C. Thawed cell pellets, collected from the 1 L culture, were resuspended in 25 ml of lysis buffer (8 M Guanidinium HC1, 50 mM potassium phosphate, pH 7.3). Lysed cells were incubated at room temperature (RT) on Intelli-mixer for 24 h. The supernatant was then separated using centrifugation at 10,000 g for 25 min, and incubated with 1.6 ml of fresh HisPur™ cobalt resin beads slurry (Thermo scientific) at RT for 1 hr. Mixture of cobalt resin beads bounded to His-tagged CsgA was loaded on a disposable polypropylene column at 4 °C. To remove GdnHCl from the sample, the column was washed with 10 ml of 50 mM potassium phosphate buffer pH 7.3, followed by another column wash with 3 ml of the same buffer, supplemented with 12.5 mM of Imidazole, to release unspecific bound proteins. The elution of CsgA was achieved with 125 mM imidazole in 50 mM potassium phosphate buffer, pH 7.3. Freshly purified CsgA protein was filtered using a 30 kDa cut-off column at 4 °C (Amicon® Ultra-4, Sigma- Aldrich) to remove insoluble protein aggregates and seeds. Imidazole was removed by desalting the protein solution at 4 °C using Zeba spin 7K desalting column (ThermoFisher Scientific) into 50 mM potassium phosphate buffer with the same pH. Protein concentration was determined via Nano-drop spectrophotometer by measuring absorption at 280 nm with molar extinction coefficient of 11,460 M ¹ cm ¹.

Multi angle light scattering size exclusion chromatography (SEC-MALS)

[0165] SEC-MALS analysis of freshly purified CsgA was performed in order to determine the accurate molecular mass and the oligomerization state of soluble CsgA. CsgA protein was concentrated using a 3 KDa cut-off spin column at 4 °C (Amicon® Ultra-4, Sigma- Aldrich) to 1.8 mg/ml concentration and analyzed via multi angle light scattering (MALS) detector, followed by size exclusion chromatography on a Superdex 75 10/300 gel filtration column (AKTA Avant).

Fibrillation assays - Thioflavin T kinetic assays of CsgA

[0166] Thioflavin T (ThT) is an amyloid indicator dye that induces fluorescence upon binding to amyloid fibrils and is used to monitor the kinetics of fibril formation. Fibrillation of CsgA, with and without D-peptide inhibitors, was monitored using this approach. All D-peptides were dissolved in pure water to 50 mM concentration and the stocks were kept at -80 °C. Freshly purified CsgA, dissolved in 50 mM potassium phosphate pH 7.3 and kept on ice, was distributed to different wells in a Greiner bio-one black 96 well flat-bottom plate. Fresh ThT reaction solution was added by diluting filtered 160 mM ThT stock, made in 50 mM potassium phosphate pH 7.3 buffer, into each CsgA containing well, as well as to control wells. D-peptides stocks were freshly diluted on ice into pure water to 2 mM concentration, and further diluted into the appropriate wells in the reaction plate. Final concentrations in the wells were 150/75/30/15/0 pM of D-peptides, 20 pM ThT and 15 pM of CsgA in a final volume of 100 pl per well. The control wells included all the components except the D-peptides that were replaced with pure water at the same volume. The plate was immediately covered with a silicone sealing film (ThermalSeal RTS, Excel Scientific), and incubated in a plate reader (CLARIOstar, BMG Labtech) overnight at 25 °C with orbital shaking at 300 rpm for 30 sec before each measurement. ThT fluorescence was recorded every two minutes using an excitation of 438 ± 20 nm and an emission of 490 ± 20 nm. The measurements were conducted in triplicates, and the entire experiment was repeated at least three times.

ThT kinetic assay of amyloid spine segments

[0167] CsgA six-residue segments with capped termini (N-Terminal Acetylation and C-Terminal Amidation) were dissolved in DMSO to 10 mM stocks and kept at -20 °C. Before each ThT experiment, peptides were thawed and diluted directly into the reaction buffer, made of 50 mM potassium phosphate, pH 7.3, directly in the reaction plate. Fresh ThT reagent dye was prepared and added for each experiment as described for CsgA. The final concentrations were 100/150/200 pM of spine peptide and 20 pM ThT in a final volume of 100 pl per well. The reaction was conducted as described above. The measurements were performed in triplicates, and the entire experiment was repeated at least three times.

Transmission Electron Microscopy (TEM) of CsgA fibrils

[0168] TEM was performed to visualize CsgA fibrils, and to test the effect of different D-peptide inhibitors. CsgA was purified as described above and incubated on the bench at RT for a few days before fixation. Alternatively, samples for TEM were taken at the end of a ThT kinetics experiment for direct visualization of the fibrillation experiment. For this experiments, equivalent wells lacking the ThT were prepared, as the dye might affect the visualization. CsgA solution was incubated in a Greiner bio-one black 96 well flat-bottom plate in a plate reader (CLARIOstar) at 25 °C with 300 rpm shaking. Fluorescence was monitored as described above until maximum ThT fluorescence enhancement was achieved. For testing the effect of D-peptides, a final concentration of 30 mM CsgA and 300 mM D-peptides were used. TEM grids were prepared by applying 5 pi samples on 400 mesh copper grids with support films of Formvar/Carbon (Ted Pella), that were charged by high-voltage, alternating current glow-discharge, immediately before use. Samples were allowed to adhere for 2 min followed by negative staining with 2% uranyl acetate for 30 sec. Specimens were examined at the Ilse Katz Institute for Nano-Science and Technology at Ben-Gurion University of the Negev, Israel, with FEI Tecnai T12 G2 transmission electron microscope, at an accelerating voltage of 120 kV.

TEM of amyloid spine segments

[0169] Capped CsgA segments were dissolved to 1 mM in DMSO and incubated at 37 °C with 300 rpm shaking for a several days. TEM grids were prepared and visualized as described above.

CsgA SDS-PAGE solubility assay for testing D-peptide inhibitory effect

[0170] Soluble CsgA protein transforms during overnight incubation to large SDS insoluble amyloid fibrils unable to migrate into Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fibrillation inhibitors should maintain a soluble form of CsgA, therefore successful inhibition may be observed by monitoring the ability of CsgA to migrate on SDS-PAGE. Freshly purified CsgA protein was diluted in 50 mM potassium phosphate buffer, pH 7.3, and mixed with freshly diluted D-peptides into 100 pl tubes to 20:200 mM CsgA to D-peptide final concentrations. Samples were then incubated for 18 hr at 25 °C with 300 rpm shaking. After incubation, samples were centrifuged at 16,100 xg for 20 min, and supernatant fractions were transferred into clean tubes. Twenty (20) pl supernatant aliquots were mixed with 10 pl 3x SDS sample buffer and incubated at 95 °C for 10 min. Samples were loaded on a 15% SDS-PAGE for protein migration. Then, proteins were transferred from the gel onto a nitrocellulose membrane using eBlot Protein Transfer System (GenScript). Ponceau red staining was performed to confirm protein transfer efficiency. After washing the membrane with doubly distilled water (DDW) to remove the Ponceau stain, the membrane was incubated with 5% milk powder solution for one hour at room temperature, followed by another one-hour incubation in 2% milk powder solution containing 1: 10,000 diluted horse radish peroxidase (HRP)-conjugated anti-His antibody (Genscript). The membrane was then washed three times, for five minutes each, with PBSTxl, and incubated in ECL solution (Western Blot Substrate) for 2 minutes. Imaging was performed by Image Quant LAS 4000 machine.

Circular Dichroism ( CD)

[0171 ] CsgA fibrillation involves conformational changes from an unstructured into a b-rich conformation. CD was used in order to monitor the secondary structure transitions of CsgA in the presence of Pepl. Immediately prior to the CD experiment, freshly purified CsgA (in 50 mM potassium phosphate buffer, pH 7.3) was dialyzed at 4 °C using Zeba spin 7K desalting column (ThermoFisher Scientific) to 2 mM potassium phosphate buffer, pH 7.3, to reduce background signal during CD measurements. CsgA protein was directly diluted into the CD cuvette to a final concentration of 10 mM. At the same time, 10 pM CsgA was mixed in a second cuvette with 20 pM Pepl, diluted from 50 mM stock made in ultra-pure water. The signal from blank solutions of either 2 mM potassium phosphate buffer pH 7.3 alone, or of the same buffer, containing 20 pM of Pepl, were taken at the beginning of each experiment, just before the addition of CsgA into the appropriate cuvette. CD measurements were performed few times across six hours, with cuvettes being incubated at RT between measurements, and mixed thoroughly before each measurement.

[ 172] CD spectra were recorded on the Applied Photophysics Chirascan qCD, using a 1 mm path-length fused quartz cell. The measurements are an average of four scans for each time point, or two scans for blanks, captured at a scan rate of 1 sec, bandwidth and step size of 1 nm, over a wavelength range of 180-300 nm. High-voltage (HV) and absorbance measurements were taken simultaneously, and CD values with corresponding HV or absorbance values above the recommended limit were omitted from the spectra. Due to CsgA highly aggregative nature, the soluble protein concentration for each measurement was evaluated at real time using UV absorbance, measured simultaneously to CD spectra by the same CD device. The soluble CsgA concentration was calculated by the Beer-Lambert law at 280 nm: A₂so = £280 x l x C. The pathlength (1) was 0.1 cm. A₂₈o was calculated by averaging the absorbance at 280 nm from the four blank corrected scans. Sequence-specific extinction coefficient (£₂₈o) of monomeric CsgA is 11,460 M ¹ em ¹, as determined via the Expasy server (http://web.expasy.org/cgi-bin/protparam/protparam). The molar ellipticity per residue (Q in mdeg cm² dmol ¹ residue ¹) was determined from the raw data (d, in mdeg.), using the following formula: G = 5 / (L x C x N), where L is the path length of the cuvette, C is the real-time molar concentration of CsgA, calculated as described above, and N is the number of residues in CsgA (i.e., 138).

Crystallization of CsgA segments

[0173] Peptides were dissolved as follows: IYQYGG (SEQ ID NO: 5) at 10 mM was dissolved in water. VTQVGF (SEQ ID NO: 6) at 10 mM was dissolved in 100% DMSO, LNIYQY (SEQ ID NO: 4) crystals grown in a mixture of 10 mM LNIYQY and 10 mM TAIVVQ (a segment from CsgB; SEQ ID NO: 8) were dissolved in 82% DMSO. TASNSS crystals grown in a mixture of 30 mM TASNSS and 10 mM TAIVVQ) were dissolved in 82% DMSO.

[0174] Crystallization experiments were set up using the hanging drop method in 96- well plates with 100 pl solution in each well. The drop volumes were 150-300 nl. Crystallization drops were formed by mixing 1: 1, 2: 1 and 1:2 volume ratio of crystallization condition and protein solution. All 96-well plates were set by a crystallization robot (Mosquito HTS of TTP Labtech) located at the Technion Center for Structural Biology (TCSB). The following commercial crystallization screens were used: Index (Hampton Research), Classics and PEGs suites (Qiagen), and JCSG (Molecular Dimensions). All plates were incubated in a Rock imager 1000 robot (Formulatrix) at 293 °K.

Structure determination and refinement

[0175] Crystals were mounted on glass capillaries glued into brass pins; a methodology developed for the study of amyloid crystals that contain a small amount of solvent. No cryo-protection was used. X-ray diffraction data was collected at the following micro-focal beamlines: ID23 at the European Synchrotron Radiation facility (ESRF), Grenoble, France; EMBL P14 at the high brilliance 3^rd Generation Synchrotron Radiation Source at DESY: PETRA III, Hamburg, Germany; beamline 24-ID-C of the Advanced Photon Source (APS), Argonne National Laboratory. Data were collected at 100 °K using 5° oscillation. Data indexing, integration and scaling were performed using XDS/XSCALE. Molecular replacement solutions for all segments were obtained using Phaser, with search model consisted of geometrically idealized b-strands. Crystallographic refinements were performed with the program Refmac5. Model building was performed with Coot and illustrated with Chimera. There were no residues detected in the disallowed region of the Ramachandran plot.

Bacterial strains and growth conditions

[0176] Salmonella typhimurium bacterial strains MAE-52 (UMR1 PagfDl strain) and MAE- 150 (A bscAC) were grown on Luria-Bertani (LB) agar plates without NaCl at 30 °C. For liquid media growth a single bacterial colony was picked from an agar plate and dipped in a non-agarose LB media (lacking NaCl) followed by incubation at 30 °C with vigorous shaking. All peptides were solubilized in ultra-pure water (BI, Connecticut, USA), their concentrations were calculated by spectrophotometer Nanodrop 2000c instrument (Thermo) using the 205 nm, and a specific extinction coefficient for each peptide was calculated by the ‘protein parameter calculator’ (http ://nickanthis com/tools/a205.html) .

Static biofilm analysis

[0177] Briefly, biofilm growth was conducted in a 96-well plate (nunclon, Roskilde, Denmark). Bacterial suspension with absorbance measurement at 600 nm (ODeoo) of between 0.05 to 0.13 were incubated in a defined LB media for S. typhimurium for 48 hours at 30 °C. The bio film was subjected to two washes with saline 0.9%. Biofilm located at the bottom of the micro-wells (surface-attached biofilm) was analyzed using Zeiss confocal laser scanning microscopy (CLSM) with lOx lenses and 458/522 and 543/682 nm excitation/emission filters. Signals were produced by a propidium iodide dye aimed to stain bacterial DNA thus localizing dead bacterial cells and thioflavin T staining dye aimed to stain bacterial curli fibers. All signals were recorded using Zeiss 710 microscopy (Jena, Germany). The biofilm formed as pellicle biofilm attached on the sides of the micro-wells at the liquid-air interface was additionally analyzed in the total biofilm measurement done by standard crystal violet assay.

[0178] The bacteria were cultured in conditions mention above, and 200 pl of the bacterial suspension were placed in each well of a 96-micro- well plate and were exposed to different D-enantiomeric peptide concentrations (0, 5, 10, 20 and 40 mM). The suspension was incubated for 48 hours at 30 °C and subsequently washed twice with 0.9% saline solution. In each well 100 pl of 0.1% of Crystal violet staining solution was added and incubated for 15 min followed by removal of the stained suspension and additional washing with 0.9% saline. Two hundred (200) mΐ of 95% ethanol solution were added to each micro-well and incubated for 30 min after which 100 mΐ from each well were transferred to a new 96-micro-well plate and recorded at absorbance of ODeoo. Statistical analysis

[0179] All variables calculated had small sample size (N < 12), and Mann- Whitney non-parametric test was applied for comparing two independent groups. The comparison of three independent groups was done using non-parametric Kruskal-Wallis test, the Mann-Whitney test for pairwise comparison, and the Bonferroni correction of the significance level. The data were not normally distributed according to the Shapiro Wilk test. All tests applied were two-tailed, and a p-value of 5% or less was considered statistically significant. Analyses were performed using SPSS software (version 24). Congo red ( CR ) modified depletion assay

[0180] The assay was based on the knowledge that the CR, which stains curli production in bacteria, can be removed along with the curli fibers (and bacteria). After removal it leaves a depleted area from the underlying agar plate. However, in S. thyphimurium the CR also stains produced cellulose, thus only a cellulose mutant strain (MAE- 150) was used in the assay.

[0181] Additionally, the inventors modified the assay in order to measure the curli production effect in the underlying agar while measuring the depletion of the CR from the agar. Briefly, the MAE- 150 strain was allowed to grow overnight in an LB media, this suspension was diluted down to OD₆oo of 0.05-0.13. The diluted suspension was exposed to different D-enantiomeric peptide concentrations (0, 10, 20, 40 or 80 mM) and incubated for 5-10 min. thereafter, 3 mΐ of the suspension was spotted on a CR agar plate comprising LB media supplemented with 20 pg/ml of CR stain (Sigma). The bacterial biofilm colonies were allowed to grow for 48 h at 30 °C, after which the colony was immersed in 0.4% paraformaldehyde followed by removal of the colony with double distilled water washes. The red colored agar was made soluble somewhat similar to the ‘b-Gal modified plate assay’. Briefly, the area underneath the colony was cut and melted with 1 ml of binding buffer BD solution (Hylabs, Rehovot, Israel) and incubated at 55 °C for 10 min. The suspension was then transferred to a cuvette and measured at a nanodrop 2000c device (Thermo) with absorbance of OD510. EXAMPLE 1

CsgA spine segments form canonical steric-zipper structure of pathological amyloids

[0182] To investigate structural features of CsgA, the inventors identified potential amyloid-forming segments that can function as structured spines of CsgA insoluble fibrils. Predictions were based on integrated information form several computational methods evaluating amyloidogenic propensities. The inventors focused on four segments: 45LNIYQY50 and 47IYQYGG52 from the Rl repeat, 129TASNSS134 from the R4-R5 loop, and ₁₃₇ VTQVGF₁₄₂ from the R5 repeat (sequence positions are indicated in subscript). TEM micrographs showed the formation of fibrils by all four segments (Fig. 1), in accordance with the prediction of their amyloidogenic propensity. Yet, while 45LNIYQY50 (Rl), 47IYQYGG52 (Rl), and 137VTQVGF142 (R5) formed amyloid-like unbranched and elongated fibrils (Figs. 1A-1C, respectively), the ₁₂₉TASNSS₁₃₄ (R4-R5 loop) segment formed wide and atypical fibrous structures (Fig. ID). The ₄₅LNIYQY₅₀ (Rl), 47IYQYGG52 (Rl) and 137VTQVGF142 (R5) segments bound the amyloid indicator dye ThT and showed dose-dependent amyloid fibrillation curve with short lag times (Fig. 2). The ₄₅LNIYQY₅₀ showed the shortest lag time and the highest fluorescence readings (Fig. 2A). The ₁₂₉TASNSS₁₃₄ segment from the R4-R5 loop did not bind ThT at the examined concentrations and conditions (Fig. 2D). The inventors solved the crystal structure of the four segments (Fig. 3). Of note, the ₄₅LNIYQY₅₀ (Fig. 3A) and ₁₂₉TASNSS ₁₃₄ (Fig. 3D) segments formed well diffracting crystals only when incubated in the presence of the TAIVVQ segment from the R5 repeat of the nucleator protein CsgB. It is possible that this related to the in-vivo nucleating effect of CsgB. A previous comprehensive Alanine mutagenesis analysis showed that positions Gln49, Asn54, Glnl39 and Asnl44 in CsgA were essential for curli assembly. Each of the ₄₅LNIYQY₅₀, ₄₇IYQYGG₅₂ and ₁₃₇VTQVGF₁₄₂ segments contained one of these essential residues (marked here in bold). ₄₅LNIYQY₅₀ and ₄₇IYQYGG₅₂ are overlapping segments from the Rl repeat, which indeed displayed a very similar structure. Both segments, as well as ₁₃₇VTQVGF₁₄₂ (R5), formed the classical amyloid steric-zipper structure with two possible dry interfaces between paired b-sheets (Fig. 3). The b-strands were oriented parallel to each other along the b-sheets. These three structures belong to class 1 steric zipper, classified by Sawaya and Eisenberg (2007) according to the organization of the b-strands and b-sheets. Interestingly, each of the three steric zippers showed two possible interfaces between mating b-sheets. In each of these interfaces, the chemical properties governing fibril stability, i.e., buried surface area and shape complementarity between sheets, resembled those of eukaryotic steric-zipper structures. Correspondingly, the three segments formed fibrils that bound the amyloid-indicator dye ThT (Figs. 1 and 2).

[0183] In contrast to the above three segments (from the Rl and R5 repeats), the 129TASNSS 134 segment was selected as a control sequence, as it was predicted by the computational methods to be amyloidogenic but was located in a region not implicated in fibrillation (the R4-R5 loop). Moreover, residue Serl33 within this sequence was unessential for fibrillation. Unlike the three spine segments that formed a tightly packed steric zipper structures, the 129TASNSS134 segment formed anti-parallel b-sheets, yet with no complementary and dry interface between mated sheets. The packing of the b- sheets mostly reassembled class 8 steric zippers, yet the small interface between the two facing b-sheets deterred specific classification. In agreement with the atypical crystal structure of 129TASNSS134, it formed atypical fibrils, which were exceptionally flat and wide (Fig. 1) and did not bind ThT (Fig. 2D).

EXAMPLE 2

CsgA shares fibrillation inhibitors with the Alzheimer’s-associated Amyloid-b

[0184] The similar structural characteristics of the CsgA cross-b steric-zipper spine segments to disease-associated amyloids set forth the idea that inhibitors designed against human amyloids could also affect fibrillation of CsgA. Therefore, the inventors tested D-enantiomeric peptides (referred here as D-peptides) developed against the aggregation of amyloid-b associated with Alzheimer's disease. Two of the D-peptides tested, named Dpepl (i.e., ANK6) and Dpep2 (i.e., DB3DB3), showed inhibitory effect on CsgA fibrillation at a dose-dependent manner. Freshly purified CsgA showed the characteristic amyloid-fibrillation curve with a very short lag time followed by rapid aggregation, while the presence of the D-peptides resulted in lower fluorescence signal and a longer lag-time, indicating on delayed fibril formation (Figs. 4A and 5). A significant effect was already observed at 1: 1 mole ratio with CsgA (Fig. 5). At 1:5 mole ratio between CsgA and the inhibitors, respectively, Dpepl managed to provide longer delay in fibrillation compared to Dpep2, yet eventually they both ended with similar fibrillation intensity, which was significantly lower compared to CsgA without the D- peptides, suggesting an effect on either fibril morphology or ThT binding. Both Dpepl and Dpep2 were more potent compared to Dpep3 (Fig. 4A), a prototype D-peptide shown to remove amyloid deposits, and to reduce inflammation and improve cognition in transgenic mouse models for Alzheimer’s disease (Van Goren et ah, (2012)). TEM micrographs correspondingly showed mostly amorphous aggregates or co-precipitates in the presence of Dpepl (Fig. 6B) and Dpep2 (Fig. 6C), compared to fibrils of CsgA alone (Fig. 6A). In contrast to CsgA, the D-peptides did not affect the fibrillation of the PSMa3 peptide from S. aureus, which forms cross-a amyloid-like fibril (Fig. 4B), suggesting that inhibition was dependent on the architecture of the amyloid.

[0185] The effect of the Dpepl inhibitor on the secondary- structure transition of CsgA during fibrillation was assessed via time-dependent circular dichroism (CD) analysis (Fig. 7). The CD spectra of freshly purified CsgA protein displayed typical random coil configuration with a detected minimum at around 200 nm. During six hours of incubation, the spectra of CsgA indicated a transition into a well-ordered b-sheet structure with distinctive maximum near 198 nm and minimum around 217 nm (Fig. 7A). The CD spectra of CsgA incubated with Dpepl showed a slower transition and remained as random coil configuration during five hours of measurements, indicating on inhibition of CsgA fibrillation (Fig. 7B).

[0186] CsgA formed insoluble fibrils that were resistant to sodium dodecyl sulfate (SDS) treatment. Therefore, the inventors sought to examine the effect of the D-peptide inhibitors on CsgA shift between soluble and insoluble states using its migration via SDS-PAGE followed by western blot (WB) analysis (Fig. 9). The inventors first assessed the oligomeric state of freshly purified CsgA using multi angle light scattering size exclusion chromatography (SEC-MALS; Fig. 8). The analysis showed that the major part of freshly purified CsgA was at a monomeric state (-19 kDa). A minor fraction indicated the formation of hexamers (6-subunit oligomer; -109 kDa). While oligomerization is expected, considering the fast-aggregative nature of CsgA, it is still not clear whether the formation of hexamers in particular has a specific role during fibrillation. The formation of penta- or hexamers as the smallest populated assembly species has also been described for amyloid-b (Wolff et ah, (2017)). Freshly purified CsgA incubated for 24 hr was insoluble and thus did not migrate in the gel, in contrast to the fresh sample (Fig. 9). The effect of the inhibitors on the solubility of CsgA at 5- fold mole access was evident: Dpep2, and moreover Dpepl, increased solubility of CsgA (Fig. 9). This suggested a rapid and robust effect of the inhibitors on CsgA fibrillation. EXAMPLE 3

D-peptides reduce biofilm formation of Salmonella typhimurium

[0187] Dpepl and Dpep2 showed dose-dependent effect on reducing the total biofilm biomass of S. typhimurium in a crystal violet-based assay (Figs. 10). A significant effect was already observed at 10 mM for both D-peptides with Dpep2 showing a more pronounced effect on biofilm formation compared to Dpepl. Dpep3 displayed a significant effect on the biofilm starting from 20 pM and was less effective compared to the other two D-peptides. The does-dependent effect on the biofilm observed up to 75 and 37.5 pM for Dpepl and Dpep2, respectively, was not attributed to bacteriostatic or bactericidal effects, as there was no effect on bacterial growth up to that concentration. Confocal microscopy of the biofilm further validated the effect of the D-peptides, visualizing a significant reduction in the formation of an otherwise thick and robust surface-attached biofilm of S. typhimurium by the addition of 10 pM Dpepl (Fig. 10C) or Dpep2 (Fig. 10D). Dpep3 showed only a slight reduction of the biofilm at this concentration (Fig. 10E), in accordance with the crystal violet-based assay (Fig. 10A) and the in-vitro effects on CsgA fibrillation (Figs. 4-7&9).

[0188] In order to confirm that the effect of the D-peptides on biofilm biomass was related to amyloid formation, the inventors used Congo Red (CR), a dye known to stain amyloids including curliated whole cells. Accordingly, growing the S. typhimurium cells on the CR- supplemented agar resulted in reddish biofilm colonies that absorbed the dye. Since CR also stains secreted cellulose in biofilm, the inventors performed an experiment using the S. typhimurium MAE- 150 mutant that does not express cellulose, in order to emphasize the effect of the D-peptide on curli fibril formation (Fig. 11). In order to comparatively quantify S. typhimurium whole-cell curliation with and without the addition of the D-peptides, the inventors utilized previous observations that the CR dye was depleted from an agar growth plate supplemented with CR when the curli- producing bacterial cells were removed. The inventors modified this approach to quantify the remaining amount of CR in the agar, such that the more CR remained indicated less curli fibrils were formed. Dpep2 was most effective in reducing curli fibril formation at a dose-dependent manner, followed by Dpepl (Fig. 11). This was in accordance with their effect on static biofilm biomass formation (Fig. 10). Dpep3 did not show an effect on curli fibril formation at the concentrations tested using the CR depletion assay (Fig. 11). [0189] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

Claims

CLAIMS What is claimed is:

1. A method for reducing or inhibiting biofilm formation on or within a surface, the method comprising: contacting a surface with at least one D-enantiomeric polypeptide selected from the group consisting of the amino acid sequences: RKRIRLVTKKKR (SEQ ID NO: 1); RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2); and RPRTRLHTHRNR (SEQ ID NO: 3), thereby reducing or inhibiting biofilm formation on or within said surface.

2. The method of claim 1 , wherein said surface is on or within an article or a subject.

3. The method of claim 1 or 2, wherein said at last one D-enantiomeric polypeptide is RKRIRLVTKKKR (SEQ ID NO: 1).

4. The method of claim 1 or 2, wherein said at last one D-enantiomeric polypeptide is RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2).

5. The method of claim 1 or 2, wherein said at last one D-enantiomeric polypeptide is RPRTRLHTHRNR (SEQ ID NO: 3).

6. The method of any one of claims 1 to 5, wherein said D-enantiomeric polypeptide comprises 100% D-enantiomeric amino acid residues.

7. The method of any one of claims 1 to 6, wherein said biofilm is produced by a Gram-negative bacterium.

8. The method of claim 7, wherein said Gram-negative bacterium belongs to a genus selected from the group consisting of: Salmonella, Pseudomonas, and Escherichia.

9. The method of any one of claims 1 to 8, wherein said at least one D-enantiomeric polypeptide reduces the level of biofilm formation by at least 10%.

10. The method of any one of claims 1 to 9, wherein said at least one D-enantiomeric polypeptide has a half maximal inhibitory concentration (IC50) of 5 to 50 mM.

11. The method of any one of claims 1 to 10, wherein said at least one polypeptide is in a pharmaceutical composition.

12. The method of claim 11, wherein said pharmaceutical composition comprises said at least one D-enantiomeric polypeptide at a concentration of 0.1-100 mM.

13. A composition comprising at least one D-enantiomeric polypeptide selected from the group consisting of the amino acid sequences: RKRIRLVTKKKR (SEQ ID NO: 1); RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2); and RPRTRLHTHRNR (SEQ ID NO: 3) and an acceptable carrier, for use in inhibiting or reducing the formation of biofilm.

14. The composition of claim 13, wherein said at last one D-enantiomeric polypeptide is RKRIRLVTKKKR (SEQ ID NO: 1).

15. The composition of claim 13, wherein said at last one D-enantiomeric polypeptide is RPITRLRTHQNRRPITRLRTHQNR (SEQ ID NO: 2).

16. The composition of claim 13, wherein said at last one D-enantiomeric polypeptide is RPRTRLHTHRNR (SEQ ID NO: 3).

17. The composition of any one of claims 13 to 16, wherein said D-enantiomeric polypeptide comprises 100% D-enantiomeric amino acid residues.

18. The composition of any one of claims 13 to 17, comprising said at least one D- enantiomeric polypeptide at a concentration of 0.1-100 mM.

19. The composition of any one of claims 13 to 18 being a pharmaceutical composition comprising said at least one D-enantiomeric polypeptide and a pharmaceutically acceptable carrier.