Antimicrobial peptide and its use thereof

Abstract

The present invention relates to a novel peptide that exhibits effective antimicrobial activity against various gram-positive and gram-negative bacteria that are involved in food-borne pathogenesis, food spoilage and other pathogenic conditions. Therefore, this peptide can be a good candidate as antibacterial in agricultural, food and beverage industry, as well as for other medical applications and societal use.

Claims

1. A thermo-stable anti-microbial peptide having the general formula: ##STR00003## wherein ##STR00004## denotes a disulphide bridge; m and n independently range from 2 to 8 amino-acid residues; X, Y is Arginine or Lysine; each B is an Amino acid selected from the group consisting of Serine, Threonine, Isoleucine, Leucine, Valine, Phenylalanine, Tyrosine, Tryptophan, Cysteine, Arginine, and Lysine, and wherein the thermo-stable anti-microbial peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20.

2. The thermo-stable anti-microbial peptide of claim 1, wherein the thermo-stable anti-microbial peptide comprises more than one disulphide bridge.

3. A therapeutic composition comprising a therapeutically effective amount of the thermo-stable anti-microbial peptide of claim 1, and a pharmaceutically acceptable carrier.

4. A disinfecting solution comprising the thermo-stable anti-microbial peptide of claim 1.

5. A food preservative comprising the thermo-stable anti-microbial peptide of claim 1.

6. A biomedical device comprising the thermo-stable anti-microbial peptide of claim 1.

7. A sanitary pad, aseptic clothing, or bandage comprising the thermo-stable anti-microbial peptide of claim 1.

8. A method of treating an infection caused by microorganism in a subject in need thereof, wherein the microorganism is selected from Listeria onocytogenes, Bacillus cereus, Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Pectobacterium carotovoumr, and Salmonella tphimurium, and wherein the method comprises administering a therapeutically effective amount of a composition to the subject, the composition comprising the thermo-stable anti-microbial peptide of claim 1, and a pharmaceutically acceptable carrier.

9. The method of claim 8, wherein the thermo-stable anti-microbial peptide is administered singly or in combination.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1. Three dimensional structure of Pep.sup.20. Region [A] represents the charged amino acids, region [B] represents the residues following the cysteines on either side which include hydrophobic amino acids and region [C] represents the trypsin inhibitory loop.

(2) FIG. 2. Scanning Electron Microscopy (SEM) Images of B. cereus treated with pep.sup.19 at 2× MIC concentration—Cell morphology and membrane integrity of B. cereus cells were observed by using SEM after treating the cells with Pep.sup.19. Membrane surface of the peptide treated cells became roughened, lost integrity and intracellular content leakage was observed. Peptide untreated control cells were bright with a smooth surface, membrane was intact and the cells were devoid of such alterations, suggesting the peptides act on the bacterial cell membrane.

(3) FIG. 3. Confocal Microscopic Images of B. cereus treated with FITC-pep.sup.19—For the determination of site of action, bacterial cells treated for 1 hour with FITC tagged Pep.sup.19 at 1× MIC, appeared as green rods with fluorescence spread all over the bacterial cell indicating the internalization of the peptide.

OBJECTIVE OF THE INVENTION

(4) The main object of the present invention is to provide an antimicrobial peptide, which is used in food preservation and related applications and medical use.

(5) Another object of the present invention is to alter, enhance or preserve the efficacy of the designed peptide by making peptide variants.

(6) Yet another object of the present invention is to inhibit the food spoiling microorganisms using the designed peptide and/or those derived based on the 2.sup.nd object of the invention. Yet another object of the present invention is the application of the peptides included in the above claims as a combination/cocktail of two or more individual peptides, derived from the 2.sup.nd object of the invention or otherwise, and their simultaneous use with other antimicrobial agents.

(7) Yet another object of the present invention is simultaneous inhibition of more than one critical microbial target (serine proteases and bacterial membrane).

SUMMARY OF THE INVENTION

(8) Accordingly, the present invention provides a novel antimicrobial peptide for diverse applications having the following general formula

(9) An anti-microbial peptide having general formula:

(10) ##STR00001##
wherein

(11) ##STR00002## m and n can independently range from 2 to 8 amino-acid residues; X, Y is Arginine or Lysine; B is an Amino acid selected from the group consisting of Serine, Threonine, Isoleucine, Leucine, Valine, Phenylalanine, Tyrosine, Tryptophan, Cysteine, Arginine, and Lysine. Optionally if B is a cysteine on either side, additional disulphide bridges are incorporated for enhanced stability.

(12) The peptide comprises of at least one disulphide bridge as illustrated in the designs. Additional disulphide bridges could be incorporated in designs derived from the parent peptide formula.

(13) The present embodiment of the invention provides an anti-microbial peptide, wherein sequence of the anti-microbial peptide is selected from the group consisting of sequences having SEQ ID No. 1 to 20 or a variant of said amino acid sequence, said variant having >80% homology with sequences having SEQ ID No. 1 to 20.

(14) In yet another embodiment the invention provides an antimicrobial peptide wherein peptide variant is mutated by a method selected from the group consisting of substitution of one or more amino acids, deletion of one or more amino acids, and insertion of one or more amino acids.

(15) In another embodiment the invention provides a mechanism for simultaneous inhibition of more than one critical microbial target (serine proteases and bacterial membrane).

(16) In another embodiment the invention provides a method of inhibiting the bacteria, using combination of two or more individual peptides.

(17) The present invention provides a design for novel antimicrobial peptides inspired by Bowman-Birk Inhibitors. Three representative peptides are provided below for proof of principle.

(18) TABLE-US-00001 Sequence ID 1 (Pep.sup.20)- RSVIFGCTKSIPPICFVGFK (Disulfide- cys 7 to cys 15) Sequence ID 2 (Pep.sup.19)- SVIFGCTKSIPPICFVGFK (Disulfide- cys 6 to cys 14) Sequence ID 3 (Pep.sup.16)- SVIGCTKSIPPICFVK (Disulfide- cys 5 to cys 13)

DETAILED DESCRIPTION OF THE INVENTION

(19) The present invention relates to a novel antimicrobial peptide useful in food preservation and other applications

(20) The peptide proposed as a part of the main object of the invention consists of the following structural features (FIG. 1): a) A trypsin inhibitory loop with a hairpin structure stabilized by a disulphide bridge. b) Amino acids sequence on both ends of, i.e. N- and C-terminal to the trypsin inhibitory loop consisting of predominantly hydrophobic amino acids. c) Basic residues towards N- and C-terminal ends. d) In context of three-dimensional structure of the peptide, the presence of disulphide-bridge causes the peptide portions at either end of the trypsin inhibitory loop to come in close proximity to form a hydrophobic patch. e) The basic residue at the termini and the lysine from the trypsin inhibitory loop maintains an overall positive charge in the peptide.

(21) In the context of the antimicrobial activity of the peptide, the aforesaid structural features have the following functions. Trypsin inhibitory loop: Serves to inhibit extracellular and intracellular serine proteases produced by bacteria for its defense and survival. Disulphide bridge: Responsible for structural stability of the peptide in a hairpin conformation. This also imparts thermostability to the peptide. Basic residues: In the three-dimensional hairpin structure of the peptide, the basic residues at the two terminals and the lysine at the trypsin inhibitory loop form an amphiphilic structure wherein the positively charged ends of the hairpin interact with the negatively charged phosphate moiety of the phospholipid bilayer, while the hydrophobic core interacts with the lipid chains. The positive charge also serves to drive the binding of the peptides specifically to bacterial membranes that have a predominantly negative charge due to 23% phosphatidylglycerol content.

(22) Twenty representative peptides are provided below.

(23) TABLE-US-00002 Sequence ID 1- RSVIFGCTKSIPPICFVGFK (Disulfide- cys 7 to cys 15) Sequence ID 2- SVIFGCTKSIPPICFVGFK (Disulfide- cys 6 to cys 14) Sequence ID 3- SVIGCTKSIPPICFVK (Disulfide- cys 5 to cys 13) Sequence ID 4- RSFIFGCTKSIPPICFVGFK (Disulfide- cys 7 to cys 15) Sequence ID 5- RSVIFGCTKSIPPICFVGTR (Disulfide- cys 7 to cys 15) Sequence ID 6- RSVIFGCTKSIPPICFVGFRR (Disulfide- cys 7 to cys 15) Sequence ID 7- RSIIFGCTKSIPPICVFGFRR (Disulfide- cys 7 to cys 15) Sequence ID 8- RRTFIGCTKSIPPICVGFR (Disulfide- cys 7 to cys 15) Sequence ID 9- RRVVFCTKSIPPICFFRR (Disulfide- cys 6 to cys 14) Sequence ID 10- RSFGCVIFGCTKSIPPICFVGFCFVR (Disulfide- cys 5 to cys 15, Disulfide- cys 10 to cys 23) Sequence ID 11- RRFICVIFGCTKSIPPICFVGFCIFRR (Disulfide- cys 5 to cys 23, Disulfide- cys 10 to cys 18) Sequence ID 12- RRVIFGCTKSIPPICFVGFRR (Disulfide- cys 7 to cys 15) Sequence ID 13- RRLIFLCTKSIPPICFVFVGFR (Disulfide- cys 7 to cys 15) Sequence ID 14- RRLFGVCTKSIPPICFLGIRR (Disulfide- cys 7 to cys 15) Sequence ID 15- RSRSVIFGCTKSIPPICFVGFSR (Disulfide- cys 9 to cys 17) Sequence ID 16- RFRFRCTKSIPPICRFRFR (Disulfide- cys 6 to cys 14) Sequence ID 17- RSRSCVGIFCTKSIPPICGFGFCRSR (Disulfide- cys 5 to cys 23, Disulfide- cys 10 to cys 18) Sequence ID 18- RSRSKFLGCTKSIPPICFFGRSR (Disulfide- cys 9 to cys 17) Sequence ID 19- RSRSKFLGCTKSIPPICFFGVRSR (Disulfide- cys 9 to cys 17) Sequence ID 20- RTRSVIFGCTKSIPPICFVGFRSR (Disulfide- cys 9 to cys 17)

EXAMPLES

(24) The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1: Design and Process of Making the Peptide

(25) For the design of antimicrobial peptide, we started out with the sequence and structure analysis of naturally available peptides. We identified structural motifs that may possess a particular activity. For example, the trypsin inhibitory motif [CTKSIPPIC] has serine protease inhibitory activity, wherein the presence of disulfides impart thermostability, and presence of hydrophobic and charged cationic residues in the peptide aids interaction with bacterial membranes. The key idea was to assemble these motifs into a single peptide, i.e distinct regions within the same peptide performing different function. For this work, we used the β-hairpin peptide of the bowman-birk inhibitor class of serine protease inhibitors. This class already has the trypsin inhibitory loop, and addition to hydrophobic and cationic residue segments allowed the incorporation of multiple properties into a single peptide. The assembly of these distinct motifs (each with specific properties) into a single peptide can be represented by the formula claimed in claim 1 and depicted in FIG. 1. This formula can act as a general guideline for the design of β-hairpin forms of cationic antimicrobial peptides.

(26) Once the peptide sequences are derived from the above formula, the peptides can be artificially synthesized or produced by recombinant DNA based methods. For our studies, all the peptides were synthesized by solid phase synthesis (Fmoc chemistry) and purified by high performance liquid chromatography using a commercial peptide synthesizing service. The synthetic peptide production and purifications methods are conventional hence detailed description is omitted.

Example 2. Antimicrobial Assays

(27) Antimicrobial assays against various bacterial cultures were done using the micro dilution broth assay according to the Clinical and Laboratory Standards Institute. Four Gram-positive bacteria; Listeria monocytogenes ATCC13932, Bacillus cereus ATCC11778, Staphylococcus aureus ATCC12900, Micrococcus luteus ATCC4698 and Five Gram-negative bacteria; Escherichia coli ATCC11775, Pectobacterium carotovorum MCC2112, Klebsiella pneumoniae MTCC4032, Pseudomonas aeruginosa MTCC4673 and Salmonella typhimurium ATCC9844 were used in this study. All ATCC strains were procured from the American Type Culture Collection (ATCC, Manassas, Va., USA). All MTCC strains were procured from Microbial Type Culture Collection (MTCC, Chandigarh, India). All MCC strains were procured from Microbial Culture collection (MCC, Pune, India). Mueller-Hinton broth was used to dilute the peptide stock and the bacterial inoculum. Inoculum was prepared from the mid logarithmic phase culture. Each well of the microtiter plates received aliquots of 100 μL of the media containing different concentrations of peptide ranging from 0.3 to 300 μg/mL. In each wells final concentration of bacteria was 5×10.sup.5 CFU/mL. Peptides were tested in duplicates. To validate the assay, untreated growth control and a positive control with a known antimicrobial were included. Microtiter plates were incubated at 37° C. for 5-6 hours with continuous shaking at 130 RPM. MIC was determined by visually observing the color change by adding Resazurin dye into each well at a final concentration of 37 μg/100 μL. Here, MIC is defined as the lowest concentration of peptide that completely inhibited the growth of the organism. MIC values for two peptides, Pep.sup.20 and Pep.sup.19 are shown in Table 1.

(28) TABLE-US-00003 TABLE 1 Pep.sup.20MIC Pep.sup.19MIC SN Micro Organisms (μg/mL) (μg/mL) 1 Listeria onocytogenes 50 >150 2 Bacillus cereus 12.5 75 3 Staphylococcus aureus 150 >150 4 Micrococcus luteus 1.25 6.25 5 Escherichia coli 150 >150 6 Pectobacterium Carotovoumr 50 150 7 Salmonella tphimurium 85 >150

Example 3: Scanning Electron Microscopy

(29) For sample preparation, B. cereus cells were grown in LB broth at 37° C. to mid log phase under continuous shaking at 180 RPM. Cells were harvested by centrifugation at 5,500 RPM for 5 min, washed thrice with 10 mM PBS (phosphate buffer saline), diluted 1×10.sup.8 CFU/mL with PBS. Cells were incubated with 2× MIC of peptide in a 500 μL reaction for 1 hour. Control cells were incubated without peptides. After incubation cells were harvested by centrifugation at 8,000 RPM for 5 min, washed thrice with PBS, fixed with 2.5% (w/v) glutaraldehyde at room temperature for 4 hours, followed by washing twice with PBS. The cells were dehydrated for 10 min with a graded ethanol series (25%, 50%, 75%, 95%, and 100%). Pellet was dissolved in 100% ethanol and dried. The samples were mounted on the specimen holder and sputter-coated with gold. Samples were transferred to electron microscope (LEO 435 VP, USA) and observed. Results showed that the bacterial plasma membrane was thoroughly disrupted by the peptide (FIG. 2).

Example 4: Membrane Permeabilization

(30) For the determination of site of action and localization of the designed peptides, B. cereus and M. luteus cells in mid-logarithmic phase were harvested by centrifugation, washed three times with 10 mM PBS, pH 7.2.×10.sup.7 CFU/mL cells were incubated with FITC labeled peptide at 1× MIC concentration at 37° C. for 1 hour. After 1 hour, cells were pelleted down and washed 3 times with PBS and spotted on a glass slide and observed under a confocal microscope (LSM700, Carl Zeiss, Germany). Fluorescent images were obtained with a 488 nm band-pass filter for excitation of FITC. It was observed that FITC-labeled peptides were internalized into the cytoplasm of the bacteria, causing cell damage (FIG. 3).

Example 5: Hemolytic and Cytotoxicity Studies

(31) This example shows that peptides are non-hemolytic and non-cytotoxic. The hemolytic activity of the peptide was evaluated using human red blood cells (hRBCs). Erythrocytes were separated from 1 mL of blood by centrifugation at 1,500 rpm for 10 min. washed hRBCs were washed 3 times with PBS, diluted to 4% (v/v) in PBS. 100 μL of the hRBCs having peptides ranging from 4 to 200 μg/mL as added into 96 wells microtiter plate. The plates were incubated for 1 h at 37° C. without agitation and centrifuged at 1,500 RPM for 5 min. Aliquots (100 μl) of the supernatant were transferred to 96-well plates and absorbance was measured at 414 nm. PBS and 1% Triton-X 100 were used as control for 0% and 100% hemolysis. At a concentration range from 4 to 200 μg/mL, peptides demonstrated a hemolytic effect at a level below 2%.

(32) Cytotoxicity was measured using WST assay. ARPE-19 (Retinal pigmented epithelial) cells, chosen to represent human cells, growing in log phase were seeded into 96 wells cell-culture plates at 4×10.sup.4 the cells were incubated at 37° C. for 24 hours under 5% CO.sub.2. Peptide is added at concentrations of 4 to 200 μg/mL in DMEM/F12 nutrient mixture media for the treatment group, whereas for the negative control group, media alone was added. The cells were incubated for 16 hours at 37° C. under 5% CO2. 10 μL of WST-1 reagent was added into each well. Plate was incubated at 37° C. for 2 hours. Color intensity was measured at 450 nm. Cell viability was higher than 80% at a peptide concentration ranging from 4 to 120 μg/mL. Even at concentrations up to 200 μg/mL, cell viability was still nearly 70%, suggesting low cytotoxicity of peptides. These data indicate that designed peptides are biocompatible.

Example 6: Trypsin Inhibition

(33) The amidase activity of trypsin and its inhibition was assayed using the chromogenic substrate BAPNA at pH 8.2 in 0.05 M Tris-HCl containing 0.02 M CaCl.sub.2 at 37° C. The assay reaction contained 50 μL of trypsin solution (40-50 μg of trypsin in 1 mM HCl), 50 μL of water and 125 μL of the substrate. The reaction was carried out at 37° C. for 10 min and stopped by addition of 0.25 mL of 30% acetic acid. Absorbance of the liberated p-nitroaniline was measured at 410 nm against an appropriate blank in which the reaction was arrested by adding 30% acetic acid prior to BAPNA addition.

(34) The trypsin solution was incubated with an aliquot of inhibitor for 10 min at 37° C. and reaction was started by the addition of 125 μL substrate and incubated at 37° C. for 10 min. The reaction was arrested by addition of 30% acetic acid and the residual trypsin activity was measured by recording the absorbance at 410 nm. All the tested peptides showed good inhibition against trypsin.

Example 7: pH and Thermostability

(35) Thermostability was measured by the determination of protease inhibition activity of the peptides after incubation for 30, 60, 90, 120, 150 and 180 minutes at 95° C. All the tested peptides retained more than 50% of their activity even after heating at 95° C. Peptides were dissolved in 50 mM buffers of pH 2.5, 5, 9 and incubated for 2 h at room temperature. Protease inhibition activity of the peptides was assayed using the BAPNA method described earlier. Peptides were found to be stable at the tested pH range.

Advantages of the Invention

(36) The designed peptide of the invention shows very less hemolytic activity and cytotoxicity. The designed peptide of the invention shows very high stability at wide range of temperatures and pH. The designed peptide of the invention is resistant to cleavage against serine proteases. The designed peptide of the invention is short and can be easily synthesized chemically or may be produced by recombinant DNA technology. The designed peptide of the invention has been tested against wide range of microorganisms and shows antimicrobial activity. It is cost effective.