A peptide capable of inhibiting bacterial biofilm / fibril formation

Abstract

The present invention provides a peptide capable of inhibiting and disintegrating bacterial biofilm / fibril formation. Particularly, the present invention provides a retro inverso peptide which can inhibit the biofilm formation as well as disintegrates the pre-formed biofilm of Staphylococcus aureus.

Description

FIELD OF THE INVENTIONThe present invention relates to the field of peptide chemistry. Particularly, the presentinvention relates to peptide and its potential of inhibiting and disintegrating bacterial biofilmand fibril formation. More particularly, the present invention relates to retro-inverso peptidesand their inhibitory and disintegrating effect on bacterial biofilm and fibrils.BACKGROUND OF THE INVENTIONAntimicrobial resistance (AMR) is one of the top challenges being faced by the global healthindustry. Antimicrobial Resistance occurs when a pathogen such as a bacteria, virus or fungidevelops drug resistance and no longer respond to antimicrobial medicines. As a result of thedrug resistance, antimicrobial medicines such as antibiotics become ineffective and infectionsbecome difficult or impossible to treat.One of the reasons for developing drug resistance in bacteria is formation of biofilms orfibrils. The self-aggregation of proteins and peptides forms amyloids into highly orderedfibrillar structures in bacteria. Amyloid aggregates are key components of biofilms of manybacterial species. These biofilms acts as protection layer for bacterial cells and minimizesimpact of external agents such as antibiotics, chemotherapy, and disinfectants.The amyloid form of proteins and peptides are also responsible for many human degenerativepathologies like Alzheimer's, Parkinson's, and Huntington's diseases. The highthermodynamic stability of such protein and peptide aggregates resists disruption of amyloidsby proteases and detergents.Biofilm formation is exhibited by both the free-floating microorganisms as well asmicroorganisms which grow on surface such as on medical devices. Medical device associated biofilms also pose a serious threat to the life and health ofpatients. Microorganisms can adhere to almost all medical devices. Formation of biofilmsaround these microorganisms develops resistance to antimicrobial agents thereby leading tomedical device-associated biofilm infections in patients.Among the various microorganisms exhibiting drug resistance, Staphylococcus aureus isposing a serious challenge. Staphylococcus aureus (S. aureus), a gram-positive bacterium, isfrequently found in the nose, respiratory tract, and on the skin, and causes infections such asendocarditis, necrotizing pneumonia, and septic shock. S. aureus is also one of the mostcommon microorganisms for medical device-associated infections. S. aureus triggeredinfections posing challenge in treatment options are primarily due to the formation of biofilmaround the bacterial surface. Biofilms provide a protective safeguard shielding bacterial cellsfrom host immune responses, antibiotics and disinfectants.In Staphylococcus aureus biofilms, the amyloid fibrils are formed primarily by phenol-soluble modulins (PSMs), small amphipathic peptides that are classified into α-type (20-25amino acids) and β-type (44 amino acids). PSMs contribute to biofilm formation and exhibitcytolytic activity, with PSMα3, a 22-residue α-type peptide, exhibiting the highestcytotoxicity.Disrupting biofilm integrity by targeting amyloid fibrils involves several strategies. Smallmolecule inhibitors, such as Congo Red, curcumin, and EGCG, can destabilize amyloidfibrils and reduce biofilm stability. Peptide-based approaches, including anti-amyloid orfibril-binding peptides, can block or destabilize amyloid fibrils, preventing biofilm formation.Enzymatic degradation using amyloid-degrading enzymes like proteinase K can also cleavefibrils and disrupt the biofilm matrix. Nanomaterials such as silver nanoparticles and carbonnanotubes interact with amyloid fibrils, destabilizing the biofilm structure. Physical methodslike ultrasound (sonication) and electroporation can mechanically disrupt biofilms, includingtheir amyloid components.Despite their potential, challenges such as ensuring specificity (to target bacterial amyloidswithout affecting human proteins), the high resistance of the biofilm matrix to antimicrobialsand immune responses, and the need for effective in vivo translation remain significantobstacles for these strategies. Also, none of the existing methods have been investigated todisrupt the structural integrity of biofilms by targeting the unique cross-α amyloid fibrilswithin the biofilm matrix.The present invention addresses these shortcomings of the prior art and provides a newpeptide which facilitates S. aureus biofilm dissolution by preventing both cross-α fibrilformation and disassembling preformed fibrils from PSM-α3 peptide.OBJECTIVE OF THE INVENTIONAn important objective of the present invention is to provide a retro inverso phenol solublemodulins alpha-3 (RI-PSMα3) peptide.SUMMARY OF THE INVENTIONThe present invention provides a peptide of SEQ ID No. 1 or an analogue thereof, said peptideis capable of inhibiting and disintegrating bacterial biofilm / fibril formation, wherein thepeptide is a retro-inverso peptide. Particularly, the present invention relates to a retro inversophenol soluble modulins alpha-3 (RI-PSMα3) peptide and its effect on the cross-α amyloidassembly of native PSMα3, facilitating Staphylococcus aureus biofilm dissolution. Morespecifically, RI-PSMα3 peptide acts on the biofilm of S. aureus and stops or disintegrate thebiofilm formation.BRIEF DESCRIPTION OF THE DRAWINGSThe accompanying drawings, which are incorporated in and constitute a part of thisdisclosure, illustrate exemplary embodiments and together with the description, serve toexplain the disclosed invention.Figure 1: (A) represents the topological relationship shown for a model peptide and its fullyretro-inverso analogue. (B) Represent the sequence of PSMα3 and RI-PSMα3 peptides. (C)Shows the aggregation kinetics of L-PSMα3 and RI-PSMα3 were monitored by ThTfluorescence assay. The concentration of each peptide was 25 μM. (D) Shows theaggregation kinetics at an increased concentration (25 μM, 50 μM, 100 μM and 200 μM) of theRI-PSMα3 peptide as monitored by the same ThT fluorescence assay. (E) Shows therepresentative SEM images of the a) L-PSMα3, and b) RI- PSMα3 after 14 hours ofincubation at 25 μM concentration. c) The same was taken for the RI-PSMα3 peptide at aconcentration of 200 μM.Figure 2: Shows analytical RP-HPLC profile (λ = 214 nm) together with ESI-MS data(inset) of the crude and purified peptides. A linear gradient of 15%-75% buffer B in bufferA (buffer A = 0.1% TFA in water; buffer B = 0.08% TFA in acetonitrile) at 40 °C over 10minutes including 4 min equilibration time using Agilent Zorbax SB-C3, 5 μm, 4.6 x 150mm, LC column with 0.9 mL / min flow rate was used for the chromatographic separation.Figure 3: (A) shows the kinetics of PSMα3 amyloid formation in the presence of the RIPSMα3, monitored by ThT fluorescence. Keeping the proportion of the L-PSMα3 and RIPSMα3 peptides same, the concentration of each peptide varied from 12.5μM to 50μM. (B)Shows the representative SEM images of PSMα3 fibrils after 14 hours of incubation in thepresence of an equimolecular amount of the RI-PSMα3 peptide. (C) Represent the dose-dependent Kinetics of PSMα3 amyloid formation in the presence of the RI-PSMα3,monitored by ThT fluorescence. The molar equivalent of the RI-PSMα3 peptide varied from0.05 to 1.00 with respect to the L-PSMα3 peptide. (D) Shows the zoomed-in version ofFigure C visualizes the complete suppression of the L-PSMα3 ThT intensity when theequivalent amount of the RI-PSMα3 peptide was 0.25 and above. (E) Shows therepresentative SEM images of PSMα3 fibrils after 24 hours of incubation in the absence orpresence of the different equivalent amountsof RI-PSMα3 peptideFigure 4: (A) illustrates Dose-dependent ThT kinetics for the disaggregation of preformedfibrils of PSMα3 in the presence of RI-PSMα3. The molar equivalent of the RI- PSMα3peptide varied from 0.25 to 2.00 with respect to the L-PSMα3 peptide's amount. (B) ShowsDose-dependent disaggregation of the preformed PSMα3 fibrils from end-point fluorescencedata taken after 15 hours. (C) Shows the representative SEM images of pre-formed PSMα3fibrils treated with different equivalents of RI-PSMα3 peptides.Figure 5: (A) Depicts the sequence of the chemically synthesized PSMα3 peptide and itsanalogues. (B) shows the schematic representation of two-component dynamiccombinatorial disulphide formation of peptides. In the figure, the orange color bar is the Atype peptide and the blue color bar is the B-type peptide as shown in Table C. Thedisulphide-bonded complex can be detected after the guanidine-mediated denaturation andRP-HPLC ESI-MS analysis. (C) represent in the table, the A-type peptide represents the LPSMα3 peptide having C-terminal cysteine residue while the B-type peptide represents anyof the two other peptides having Cys-residue either at the N- terminus or the C-terminus(RI-PSMα3(Cys) and (Cys)RI-PSMα3). (D) Represent that an equal equivalent amount ofpeptide-A was mixed with peptide-B in the folding condition in the presence of redoxreagent (20 mM phosphate, 1 mM GSH, 0.2 mM GSSG, pH 8.0). For experiments 1-3, theindividual peptide was the only component whereas for experiments 4-5, the mixture of twopeptides has been used. The column chart represents the quantity of individual dimer(percentage) obtained when the reaction reached equilibrium. The percentage of theproducts was quantified after the guanidine-mediated denaturation by the intensity of thepeak (area under the curve) detected at 214 nm wavelength in analytical RP-HPLC. (E)Shows the far-UV CD spectrum (190nm-260nm) was obtained by measuring the ellipticityof the sample in the aqueous phosphate buffer (20 mM phosphate, pH 7.4). All the peptidesshowed characteristic alpha-helical signatures with L-peptides showing characteristicnegative minima at 208 nm and 222 nm while the RI-peptides showed positive maxima atthe same two wavelengths. (F) Shows the DLS data of L-PSMα3 aggregating species weretaken at different time points and represented as color traces. (G) Shows the DLS data of RIPSMα3 peptide and buffer alone. Only the data point after 1 hour of the incubation of RIPSMα3 is shown here. (H) Shows the DLS data of L-PSMα3 aggregating species taken after1-hour incubation and the addition of the equivalent amount of the RI-PSMα3 peptide. Theaggregating species at different time points are represented as color traces.Figure 6: (A) Shows percentage MRSA biofilm inhibition by RI-PSMα3. Chloramphenicolwas used as a positive control. All experiments were carried out in triplicate, and datarepresent the mean standard error (one-way ANOVA P < 0.0001). (B) Shows the micrographsof disrupted matured biofilms of the tested MRSA strain (ATCC 43300) formed on glasssurfaces by the RI-PSMα3 at different concentrations by scanning electron microscopy (CarlZeiss, Evo 18). (a, d) Growth control; (b, e) RI-PSMα3 (100 μg / ml); and (c, f) RI-PSMα3 (150μg / ml).Other embodiments, features and advantages of the present invention will become apparentfrom the following detailed description. It should be understood, however, that the detaileddescription and specific examples, while indicating preferred embodiments of the invention,are given by way of illustration only. Since various changes and modifications within thespirit and scope of the invention will become apparent to those skilled in the art from thisdetailed description such changes and modifications are covered within the scope of thepresent invention.DETAILED DESCRIPTION OF THE INVENTIONThe details of one or more embodiments of the invention are set forth in the accompanyingdescription below including specific details of the best mode contemplated by the inventorsfor carrying out the invention, by way of example. It will be apparent to one skilled in the artthat the present invention may be practiced without limitation to these specific details.The use of "comprise", "comprises", "comprising", "contain", "contains", "containing","include", "includes", and "including" are not intended to be limiting. Particularly, the word"comprising" is intended to be used to cover within its ambit other possible ingredients orsteps or features of an aspect or embodiment of the invention which are apparent to a skilledperson after reading the present disclosure. The word "comprising" is intended to mean"including" but not necessarily "consisting of" or "composed of." It is to be understood thatboth the foregoing general description and this detailed description are exemplary andexplanatory only and are not restrictive.It is noted that the examples given in the description below are intended to clarify theinvention and are not intended to limit the invention to those examples per se. Except in theoperating and comparative examples, or where otherwise explicitly indicated, all numbers inthis description and claims indicating amounts of material or conditions of reaction, physicalproperties of materials and / or use are to be understood as modified by the word "about".Numerical ranges expressed in the format "from x to y" are understood to include x and y.When for a specific feature multiple preferred ranges are described in the format "from x toy", it is understood that all ranges combining the different endpoints are also contemplated.Unless otherwise defined, scientific and technical terms used herein shall have the meaningsthat are commonly understood by those of ordinary skill in the art. Further, unless otherwiserequired by context, singular terms shall include pluralities and plural terms shall include thesingular. Generally, nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology described herein are those well-known and commonly usedin the art.The present invention provides a peptide capable of dissolving and disintegrating the bacterialbiofilm. The peptide of the invention has inhibitory potential on the amyloid formation of thenative peptide, which is one of the components of the bacterial biofilm. Particularly, thepresent inventors identified peptide-based inhibitors which can impede the development ofthe extracellular polymeric substance (EPS) matrix and, more importantly, disassemble thefully formed EPS matrix within Staphylococcus aureus biofilms. These and other aspects,features and advantages of the invention will become apparent to those of ordinary skill in theart from a reading of the following detailed description and the appended claims.The present invention provides a retro inverso peptide capable of inhibiting the formation ofor disintegrating the formation of bacterial biofilm. Particularly, the invention provides aretro-inverso phenol soluble modulins alpha-3 (RI-PSMα3) peptide and explores the effect ofthe RI-PSMα3 on the cross-α amyloid assembly of PSMα3, facilitating Staphylococcus aureusbiofilm dissolution.The bacterial strain used for the present invention MRSA (Methicillin-resistant S. aureusATCC 43300), was procured from the American Type Culture Collection (ATCC).The team of inventors studied the property of the retro-inverso (RI) sequence of the cross-αamyloid-forming peptide PSMα3, which is among the most lytic members of the PSMfamily peptides of S. aureus. In retro-inverso peptide, the amino acid sequence is reversed(retro) and each amino acid is replaced withits enantiomer (inverse) (Figure 1A scheme). Theinventors designed a peptide structure, which despite the reversed sequence and inversedchirality, can still interact with the same receptor as the parent peptide, as the side chaintopology of the retro-inverso sequence is so designed that it mimics the original peptide'sconformation. Additionally, retro-inverso peptide of the invention has the advantage ofincreased resistance to proteolysis due to the presence of D-amino acids.The sequence of the said retro-inverso peptide is represented by SEQ ID No. 1. Accordingly,in an embodiment, the present invention provides a retro inverso phenol soluble modulinsalpha-3 (RI-PSMα3). The RI-PSMα3 peptide of the invention is capable of inhibiting ordisintegrating the biofilm formation in bacteria. The invention thus provides a newtherapeutic approach for biofilm-associated infections.In a specific embodiment, the invention provides a peptide of SEQ ID No. 1 or an analoguethereof or a sequence >60% similar thereof, said peptide is capable of inhibiting anddisintegrating bacterial biofilm / fibril formation, wherein the peptide is a retro-inversopeptide.In a preferred embodiment, the present invention provides that the peptide is retro inversophenol soluble modulins alpha-3 (RI-PSMα3).In a preferred embodiment, the present invention provides that the sequence of the peptideconsists of non-natural amino acids.In a preferred embodiment, the present invention provides that the peptide acts on the biofilmof Staphylococcus aureus and stops the biofilm formation and disintegrates pre-formedmatured biofilms. In yet another preferred embodiment, the peptide disintegrates the biofilmby targeting the amyloid fibrils within the biofilm matrix.In a preferred embodiment, the peptide selectively acts on the cross-α amyloid formation ofPSMα3 peptide.In yet another preferred embodiment, the peptide binds selectively to the native PSMα3 in anantiparallel fashion.In a preferred embodiment, the present invention provides a composition comprising the saidpeptide along with pharmaceutically acceptable additives and carriers.In yet another embodiment, the present invention provides a surface disinfectant comprisingthe said peptide along with pharmaceutically acceptable additives, carriers and / or otherdisinfectant agents.Yet another embodiment of the invention provides that a method of synthesizing the saidpeptide comprising the step of:i. coupling of amino acid Cysteine (0.25M) for 2-10 min at room temperaturefollowed by 5-15 min at 45-65 °C on resin under an N2 atmosphere with vortexmixing using using coupling reagent and additives;ii. coupling of all other amino-acids in the SEQ ID No 1 (0.25M) for 5-10 min at 60-70 °C under an N2 atmosphere with vortex mixing using coupling reagent andadditives;iii. after every coupling cycle, carrying out Fmoc deprotection by 20% piperidine inDMF at 50 °C;iv. after completion of the coupling of all residues, using a cocktail of TFA, phenol,water, DODT , and TIPS to cleave peptides from the resin;v. evaporating TFA to reduce the solution volume;vi. precipitating the cleaved synthesized peptide with diethyl ether, and lyophilizing.In another embodiment, the cocktail used for cleave peptides from the resin contains 85 %TFA, 5% phenol, 5%water, 2.5% DODT and 2.5% TIPS.In yet another embodiment the resin, coupling reagent and additives used namely are Rinkamide aminomethyl, DIC and oxyma with DIEA respectively.The following examples are given by way of illustration and for better understanding of thepresent invention and should not be construed to limit the scope of present invention.EXAMPLES:Example 1: The method of synthesis of the RI-PSMα3 peptideThe present inventors prepared RI-PSMα3 peptides as well as the Cysteine-containinganalogs using Fmoc chemistry solid phase peptide synthesis. The crude peptides werepurified using reverse-phase HPLC (RP- HPLC). The purified peptide was then subjected toa pre-treatment protocol with a TFA-HFIP (1:1) mixture. This pretreatment procedure wasdesigned to effectively dissect all aggregates present in the sample of interest.Chemicals used for the synthesis of the RI-PSMα3 peptide:N,N-diisopropylethylamine (DIEA), Ethyl cyanohydroxyiminoacetate (Oxyma) and all theL- and D-amino acids for Fmoc-SPPS was obtained from Chem-Impex International, USAand Gyros Protein Technologies. The side-chain protecting groups used were, Glu(OtBu),Asp(OtBu), Asn(Trt), Lys(OtBu), Ser(tBu) and Cys(Trt). Dichloromethane (DCM), diethylether, N,N'-dimethylformamide (DMF), HPLC grade N,N'-diisopropylcarbodiimide (DIC),1,1,1,3,3,3-Hexafluoroisopropanol (HFIP), 2,2,2 - Trifluoroethanol (TFE), phenol, 2,2 -(Ethylenedioxy)diethanethiol (DODT), Triisopropylsilane (TIPS) and trifluoroacetic acid(TFA) were purchased from SRL chemicals India. The HPLC grade acetonitrile (CH3CN) forthe peptide purification was purchased from Thermofisher Scientific, India. Piperidine wasobtained from AVRA chemicals, India. Thioflavin T (stain for amyloid grade) was obtainedfrom Sigma-Aldrich. The Rink amide aminomethyl resin (polystyrene resin with 1% crosslinked with divinylbenzene) was obtained from Supra Science Private Limited, India.a) Peptide Synthesis:Fmoc-SPPS was carried out using amino acids (AA) (0.25 M), DIC (0.25 M) as a couplingreagent, and oxyma (0.25 M) with DIEA (0.025 M) as additives. Peptides were synthesizedon Rink amide aminomethyl resin with a loading capacity of 0.5-0.6 mmol / g. Cysteine wascoupled for 5 min at room temperature followed by 10 min at 50 °C and all other amino-acidcoupling was performed for 7 min at 65 °C under an N2 atmosphere with vortex mixing.Fmoc deprotection after every coupling cycle was carried out by 20% piperidine in DMF at50 °C. After synthesis, a cocktail of TFA (85%), phenol (5%), water (5%), DODT (2.5%),and TIPS (2.5%) was used to cleave peptides from the resin. TFA was evaporated to reducethe solution volume, the cleaved peptide was precipitated with diethyl ether, and lyophilized.Analytical reverse-phase high-performance liquid chromatography (RP-HPLC) wasperformed (Figure 2) to assess the purity of cleaved peptides on an Agilent HPLC instrumentusing an Agilent zorbax SB-C3 (5 μm), 4.6x150 mm reverse-phase silica column at a flowrate of 0.9 mL / min using a linear gradient of 15-75% solvent B (0.08% TFA in acetonitrile)in solvent A (0.1% TFA in H2O) in 10 minutes. The UV absorbance of the column eluentwas monitored at 214 nm wavelength. Preparative (RP-HPLC) of crude peptides wasperformed on a Waters 1525 preparative HPLC system using Agilent ZORBAX-SB C3 (5μm, 80 Å, 9.4 x 250 mm) columns at 40 °C using a linear gradient of 25-55% solvent B(0.08% TFA in acetonitrile) in solvent A (0.1% TFA in H2O) in 60 minutes at 45 °C.Fractions containing the purified target peptide were identified by ESI-MS (see Table 1). Thedeconvolution of the observed mass was carried out using Agilent Mass-Hunter QualitativeAnalysis software (version B.07.00), and the deconvoluted mass was reported with anuncertainty of ± 0.02 Da.Table 1. Sequence of the wild type PSMα3 peptide and the synthesized analogs.**The capital letters denote L-amino acids and the small letters denote D-amino acids.b) Peptide pre-treatment:The lyophilized powder of the L-PSMα3 and RI-PSMα3 peptide analogs were reconstitutedin a mixture of TFA-HFIP (1:1) to achieve a concentration of 2 mg / ml. Next, the solution wassubjected to sonication for a duration of 10 minutes at a temperature of 37°C. The solutionwas then allowed to evaporate in a chemical hood over a period of 2 days. To remove anyremaining solvent, a high vacuum apparatus was used for solvent evaporation. In cases whereimmediate testing was not performed, the treated peptides were stored at a temperature of -80°C.Example 2: Schematic representation of the structure of RI-PSMα3 peptideA schematic exemplary structure providing the stereochemical information can be depictedas bellow:Example 3: Characterization of the structure of the prepared RI-PSMα3 peptideThe secondary structure of the RI-PSMα3 peptide in the buffer (20 mM phosphate, pH 7.4)was determined by recording far-UV circular dichroism (CD) spectra. The CD spectrarevealed alpha-helical nature of peptide, wherein the RI-PSMα3 peptide showing the reverseCD signature, as expected for a D-peptide (Figure 4E). To quantify the equilibriumsecondary structure components in the buffer, they used a server-based algorithm BESTsel.The result indicated that RI-PSMα3 peptide displayed an equal proportion of alpha-helicalcontent with the native one.Example 4: Amyloid formation of the individual peptidesThioflavin-T (ThT) aggregation kinetics assay was used for demonstrating the formation ofamyloid aggregates under the experimental conditions. In the ThT fluorescence assay for the LPSMα3 peptide at 25 μM concentration, a very short initial lag phase was observed, followedby a rapid growth phase, and further followed by the plateau phase, indicative of equilibriumbetween fibril formation and fibril dissociation or fragmentation (Figure 1C). However,at anequal concentration of RI-PSMα3, no change was observed in fluorescence signal in thisassay, suggesting that, unlike L-PSMα3 peptide, RI-PSMα3 does not form cross-α type amyloidfibril (Figure 1C).Moreover, the visualization of the samples using scanning electron microscopy (SEM) after14 hours of incubation revealed the presence of the amyloid formation for the L-PSMα3peptide (Figure 1E-a), but not the RI- PSMα3 peptide (Figure 1E-b). Importantly, it isobserved that RI-PSMα3 did not exhibit any fibril formation even at a concentration as highas 200μM (Figure 1D & 1E-c).Example 5: Inhibition of L-PSMα3 amyloid formation by RI-PSMα3The potential inhibitory effect of RI-PSMα3 on amyloid formation of the L-PSMα3 peptide,was assessed by conducting ThT assays in presence of RI-PSMα3.Solutions containing L-PSMα3 were incubated with an equal proportion of RI-PSMα3 in 10mM sodium phosphate buffer containing 150 mM NaCl at physiological pH in the presence of200 μM of ThT for 14 hours. A complete suppression of the fluorescence signal wasobserved (Figure 3A). Similar results were observed at varied peptide concentration used(12.5 μM, 25 μM, or 50μM) in the assay (Figure 3A). These ThT assay results were furthersubstantiated by SEM analysis, which visually confirmed the inhibition of amyloidformation in the presence of RI-PSMα3 (Figure 3B).Furthermore, the inventors conducted co-incubation experiments in which L-PSMα3 peptidewas combined with increasing concentration (equivalent amounts: 0.05, 0.10, 0.15, 0.25,0.37,0.50, 0.75, 1.00) of RI-PSMα3 peptide, while maintaining a constant L-PSMα3concentration of 25μM relative to the untreated L-PSMα3, considered as 100% aggregation. Itwas observed that the presence of RI-PSMα3 dramatically reduced the level of amyloid fibrils(Figure 3C). Even with a 0.25 molar equivalent of RI-PSMα3 peptide, no increase in theThT fluorescence signal was observed during the 14-hour incubation period. The inhibitionof L-PSMα3 fibril formation by RI-PSMα3 was found to be dose-dependent. Thevisualization of samples by SEM further underscores the dose-dependent reduction ofamyloid formation in the presence of RI-PSMα3 (Figure 3E a-d).These experiments comprehensively demonstrate the inhibitory efficacy of RI-PSMα3 on thecross-alpha amyloid formation of the L-PSMα3 peptide, highlighting the potential of RIPSMα3 as a valuable peptidic agent for modulating amyloid formation by L-PSMα3 peptide.Example 6: Disaggregation potential of RI-PSMα3 on pre-formed fibrilsTo assess the potential of the RI-PSMα3 peptide to disrupt pre-formed L-PSMα3 fibrils, theinventors conducted a series of in-vitro experiments to investigate its ability to disassemblethese amyloid structures. At first, the L-PSMα3 peptide were allowed to form aggregates in 10mM sodium phosphate buffer containing 150 mM NaCl at physiological pH. The progress of thefibril formation was monitored using the ThT assay. After 4.5 hours, when the ThTfluorescence intensity reached maximum (Figure 4A), varied concentrations of RI- PSMα3peptide were introduced to the matured fibrils, followed by continuous monitoring of the ThTfluorescence intensity for the subsequent 11 hours.The results revealed a striking difference in the signal behaviour. In the absence of RI-PSMα3,the ThT signal remained at the plateau, indicating the stability of the pre-formed L-PSMα3fibrils. However, upon immediate addition of RI-PSMα3, they observed a drastic reduction ofthe ThT signal, indicative of the disruption of the pre-formed fibrils and oligomers in a dose-dependent manner (Figure 4A). Notably, the most significant reduction of the ThT signal,approximately 80% disaggregation, was observed when a 1.5-fold excess of RI-PSMα3 wasintroduced (Figure 4B), which was further supported by the SEM analysis (Figure 4C). Inuntreated preformed L-PSMα3 assemblies, a fibrillar network characteristic of amyloidpresence was evident (Figure 4C-a). However, in the presence of RI-PSMα3, the fibril densitygradually diminished (Figure 4C b-d). The magnitude of this effect varied depending on themolar equivalent of the RI-PSMα3 peptide used in the study.Interestingly, in the presence of one equivalent of RI-PSMα3 no trace of fibrils wasdetectable, suggesting an apparent transformation of the preformed fibrils into smallersoluble oligomers or monomers. These smaller species were not detectable by ThT assay,further emphasizing the disaggregation potential of RI-PSMα3.Example 7: Study of dynamic combinatorial disulphide bond formation chemistryTo investigate the binding mode and affinity of the native L-PSMα3 and RI-PSMα3 peptides,the inventors used the dynamic combinatorial disulphide bond formation chemistry.Generally, the extent of disulphide bond formation between two peptides would reflect theextent of interaction between them, forming the thermodynamically stable homo- or heterodimer.For this study, the inventors designed and chemically synthesized peptides with cysteineresidue either at the C-terminus or the N-terminus, enabling the use of dynamic disulfidebond formation chemistry with LC-MS as the read-out. Since all analogs share the sameamino acid compositions, an additional (arbitrary) residue was introduced at either the N- orC-terminus of each peptide to distinguish them using mass- spectrometry (Figure 5A).A schematic of the dynamic combinatorial disulphide bond formation chemistry has beenillustrated in Figure 5B. The experiment relied on the disulphide bond formation betweentwo peptides under GSH / GSSG mediated folding conditions (20mM phosphate, 1mM GSH,0.2mM GSSG, pH 8.0) followed by denaturation and reverse-phase high-performance liquidchromatography (RP-HPLC) coupled with online electron spray mass spectrometry (ESI-MS)analysis.The first three experiments in which individual peptides (Figure 5C, Exp1-Exp3) weretreated under redox folding conditions revealed a quantitative homo-dimer product, withtraces of glutathione adduct formation (Figure 5D).In the next two experiments (Figure 5C, Exp4-Exp5), the equimolecular amounts of bothpeptides was incubated under redox folding conditions. It was observed that when the Cterminal cysteine-containing peptide L-PSMα3 (Cys) and RI-PSMα3 (Cys) were incubated, theequilibrium product distribution followed mostly statistical pattern (Figure 5D,Expt. No. 4).However, when the C-terminal cysteine-containing L-PSMα3 (Cys) peptide was incubatedwith the N-terminal cysteine-containing (Cys) RI-PSMα3 peptide, the thermodynamicequilibrium distribution shifted nearly quantitatively towards the hetero-dimeric product(Figure 5D, Expt. No. 5).With the help of the dynamic combinatorial disulphide bond formation chemistry describedabove, the inventors found that the RI-PSMα3 peptide preferentially binds with the L-PSMα3peptide, indicating a strong interaction between the two peptides when properly oriented. TheRI-PSMα3 peptide preferentially binds with the native PSMα3 peptide in an antiparallelfashion.Example 8: Dynamic light scattering experiment for trapping of transient oligomericspeciesThe inventors conducted Dynamic light scattering (DLS) experiments with L-PSMα3 and RIPSMα3. In the first experiment, L-PSMα3 and RI-PSMα3 peptides were allowed to evolveindependently to an equilibrium characterized by a maximally soluble higher oligomeric state.DLS data collection was done over the span of an hour, data being recorded at 10-minuteintervals. The inventors found that the population distribution of the L-PSMα3 peptide reachedequilibrium, with significant enrichment of species exhibiting average hydrodynamic radiiof 925.6 nm (Figure 4F). This equilibrium coexisted with minor species havinghydrodynamic radii of 3.12 nm and 126.2 nm, indicating their participation in the complexpeptide ensemble. In contrast, the population distribution of RI-PSMα3 remained skewedtoward species with very low hydrodynamic radii, residing primarily at 2.0 nm. (Figure 4G)In the subsequent experiment, the inventors observed a significant shift in the DLS profilewhen an equal proportion of RI-PSMα3 was added to the pre-equilibrated PSMα3 oligomericspecies. The dynamics of the population distribution were monitored at four different timepoints within an hour (5 minutes, 15 minutes, 30 minutes, and 60 minutes). A remarkablefinding was revealed during the experiment. The initially dominant population of species withhydrodynamic radii of 925.6nm underwent a profound shift, transitioning to significantlylower-order oligomers characterized by hydrodynamic radii of 342.3nm. At the same time,equilibrium was maintained with the remaining smaller species at 3.2nm, with bothpopulations exhibiting nearly equal intensity (Figure 4H).The dynamic light scattering (DLS) experiment of the peptide PSMα3 in the absence andpresence of the RI-PSMα3 revealed that the RI- PSMα3 peptide possesses a remarkableability to capture transient oligomeric species along the intricate cross-alpha amyloidformation pathway and drives the equilibrium towards specific lowly-populated oligomericstates, suggesting a dynamic and versatile role for RI- PSMα3 in modulating amyloidformation.Example 9: In vitro experiment showing dose- dependent MRSA biofilm inhibition byRI-PSMα3 peptideIn vitro study was performed to show the inhibition of the formation of the extracellularpolymeric substance (EPS) matrix and, dismantling the mature EPS matrix within S. aureusbiofilms.A substantial reduction in the biofilm mass in the bacterial population (MRSA strain ATCC43300) was observed in a crystal-violet staining assay when RI-PSMα3 peptide was applied toa matured biofilm-containing bacterial colony in a dose-dependent manner (Figure 6A). TheRI-PSMα3 at 150μg / ml led to ~50% inhibition in the biofilm biomass formation. Thisinvestigation serves as a compelling demonstration of the singular anti-biofilm potentialexhibited by the RI-PSMα3 peptide.Further, the SEM analysis of MRSA biofilm was conducted which revealed the inhibitoryeffect of RI-PSMα3 on S. aureus biofilm formation. Fewer bacterial cells and less extra-cellularpolymeric substance (EPS) accumulation on glass coverslips(Figure 6B) were observed whentreated with of RI- PSMα3. These results are in agreement with the results obtained from thecrystal violet spectrophotometry assay.Thus, the above in-vitro experiment established that the RI-PSMα3 peptide can disperseMRSA biofilm biomass.While the present invention has been particularly shown and described with reference toexemplary embodiments thereof, it is evident that many alternatives, modifications, andvariations will be apparent to those skilled in the art in light of the above teachings.Modification is possible. Accordingly, the spirit of the present invention should beunderstood in accordance with the claims set forth below, and all of its equivalents will fallwithin the scope of the present invention.Advantages of the invention:- The retro-inverso PSMα3 (RI-PSMα3) peptide, can be used as a therapeutic agent fortreating biofilm-associated infections, particularly those caused by Staphylococcusaureus (e.g., MRSA).- RI-PSMα3 peptide, itself does not form fibrils even at higher concentrations.Therefore the peptide can be effectively used to inhibit the amyloid fibril formation ofPSMα3 peptide uses higher doses.- RI- PSMα3 can be used in combination with conventional antibiotics or disinfectantsto increase their efficacy.- The peptide could be applied as a coating to medical devices (e.g. catheters, implants,prostheses) to prevent biofilm formation and associated infections,- RI- PSMα3, particularly useful in healthcare settings to reduce the incidence ofdevice-related infections.- RI-PSMα3 could be incorporated into wound dressings, gels, or sprays to treat andprevent biofilm-related infections in wounds, particularly in burn victims, diabetics,or those with chronic wounds.- The RI-PSMα3 peptide could be formulated into new types of disinfectants or surfacedisinfectants used in healthcare, food processing, and other industries where bacterialbiofilms pose a significant risk.- RI-PSMα3 peptide of the invention is capable of enhancing patient outcome, decreasein antimicrobial resistance, and boosting the efficacy of current antimicrobialmedicines by inhibiting the production of amyloid fibrils and dissolving pre-existingbiofilms.- Since biofilms are known to shield bacteria from standard therapies, the RI-PSMα3peptide of the invention is especially helpful in treating persistent and recurringinfections.

Claims

1. A peptide of SEQ ID No. 1 or an analogue thereof or a sequence >60% similar thereof, said pepide is capable of inhibiting and disintegrating bacterial biofilm / fibril formation, wherein the peptide is a retro-inverso peptide.

2. The peptide as claimed in claim 1, wherein the peptide is retro inverso phenol soluble modulins alpha-3 (RI-PSMα3).

3. The peptide as claimed in claim 1, wherein the sequence of the peptide consists of non-natural amino acids.

4. The peptide as claimed in claim 1, wherein the secondary structure of the peptide is alpha-helical.

5. The peptide as claimed in claim 1, wherein the peptide acts on the biofilm of Staphylococcus aureus and stops the biofilm formation and disintegrates pre-formed matured biofilms.

6. The peptide as claimed in claim 1, wherein the peptide disintegrates the biofilm by targeting the amyloid fibrils within the biofilm matrix.

7. The peptide as claimed in claim 1, wherein, the peptide selectively acts on the cross-α amyloid formation of PSMα3 peptide.

8. The peptide as claimed in claim 1, wherein the peptide binds selectively to the native PSMα3 in an antiparallel fashion.

9. A composition comprising the peptide as claimed in claim 1 along with pharmacetually acceptable additves and carriers.

10. A surface disinfectant comprising the peptide as claimed in claim 1 along with pharmacetually acceptable additves, carriers and / or other disinfectant agents.

11. A method of synthesizing the peptide as claimed in claim 1, wherein the method comprises the steps of: i. coupling of amino acid Cysteine (0.25M) for 2-10 min at room temperature followed by 5-15 min at 45-65 °C on resin under an N2 atmosphere with vortex mixing using using coupling reagent and additives; ii. coupling of all other amino-acids in the SEQ ID No 1 (0.25M) for 5-10 min at 60-70 °C under an N2 atmosphere with vortex mixing using coupling reagent and additives; iii. after every coupling cycle, carrying out Fmoc deprotection by 20% piperidine in DMF at 50 °C; iv. after completion of the coupling of all residues, using a cocktail of TFA , phenol, water, DODT , and TIPS to cleave peptides from the resin; v. evaporating TFA to reduce the solution volume; vi. precipitating the cleaved synthesized peptide with diethyl ether, and lyophilizing.

12. The method as claimed in claim 11, wherein the cocktail used for cleave peptides from the resin contains 85 % TFA, 5% phenol, 5%water, 2.5% DODT and 2.5% TIPS.

13. The method as claimed in claim 11, wherein the resin, coupling reagent and additives used are Rink amide aminomethyl, DIC and oxyma with DIEA respectively.