WO2004016650A1

WO2004016650A1 - Antimicrobial peptides, polypeptides comprising same, genes coding for said peptides, vectors, transformed organisms and compositions containing same

Info

Publication number: WO2004016650A1
Application number: PCT/FR2003/002517
Authority: WO
Inventors: Jean-Philippe Meyer
Original assignee: Entomed
Priority date: 2002-08-13
Filing date: 2003-08-12
Publication date: 2004-02-26
Also published as: FR2843591B1; AU2003276325A1; FR2843591A1

Abstract

The invention concerns an isolated peptide characterized in that it corresponds to formula (I): Xaa-Lys-Ile-Pro-Xab-Ala-Xac-Lys-Gly-Ala-Xad-Arg-Ala-Leu-Xae-Ala-Ser-Thr-Ala-Xaf-Asp-Ile-Ala-Xag-Phe-Xah-Arg-Lys- Arg-Xai (I), wherein Xaa is -NH2 or the peptide residue Arg or Gly; Xab is the peptide residue Ile-Asn or Val-Glu; Xac is the peptide residue Ile-Lys, Ile-Arg or Leu-Lys; Xad is the peptide residue Ser-Arg-Ala-Trp, Lys-Ala-Val-Gly-His-Gly-Leu or Lys-Val-Ala-Gly-Arg-Ala-Trp; Xae is the peptide residue Asp-Leu, Asn-Ile or Asp-Leu; Xaf is the peptide residue Tyr or His; Xag is the peptide residue Ser-Ile, Ser-Ala or His-Leu; Xah is the peptide residue Asn, Asp or His; Xai is -OH or the peptide residue Glu, Asn or Lys-His, derivatives and fragments thereof. The invention also concerns a polypeptide comprising said peptide, a polynucleotide encoding said peptide, a chimeric gene, a vector containing said polynucleotide or said chimeric gene and a transformed organism. The invention further concerns an antibacterial and/or antifungal composition containing at least one peptide of the invention for use in human and animal therapy, in agriculture as well as in the agri-food and phytosanitary industry.

Description

ANTIMICROBIAL PEPTIDES, POLYPEPTIDES COMPRISING SAID PEPTIDES, GENES ENCODING SAID PEPTIDES, VECTORS, TRANSFORMED ORGANISMS AND COMPOSITIONS CONTAINING THEM.

The present invention relates to new peptides having antimicrobial properties, polypeptides comprising said peptides, polynucleotides encoding said peptides, chimeric genes, vectors containing said polynucleotides or said chimeric genes and transformed organisms. The invention also relates to antimicrobial compositions containing said peptides which can be used in human and animal therapy, in agriculture as well as in the food and plant health industry.

Both in human and animal health and in plant health, the need for new molecules to fight against infections of bacterial and fungal origin is growing. In human health, nosocomial infections of bacterial and fungal origin are serious infections that affect hospital patients. They are particularly formidable for patients whose immune system is weakened following treatments such as corticotherapy or chemotherapy, interventions such as transplants or diseases affecting the immune system such as Acquired Immunodeficiency Syndrome. The situation is of particular concern for muno-depressed patients infected with Gram-positive bacteria of the genus Enterococcus. Enterococci have been recognized for the past 2 decades as the main cause of the development of bacteremia, urinary and intra-abdominal infections in hospitalized patients (Pathogenicity of Enterococci, Lynn E. Hancock and Michael S. Gilmore, Gram-Positive Pathogens, ASM Publications 2000). Among the most pathogenic Enterococci, the species Enterococcus faecalis and Enterococcus faeci m are listed in particular. Enterococcus faecalis alone is responsible for around 80% of the nosocomial infections mentioned above. Infections caused by bacteria of the genus Enterococcus are particularly difficult to treat. Enterococci tolerate a wide variety of growing conditions. In particular, they have the capacity to grow in hypotonic, hypertonic, acidic, alkaline environments and in environments whose temperature range is between 10 and 45 ° C. Sodium azide and bile salts, which inhibit the growth or even destroy most of the microorganisms, are tolerated by Enterococci. The most commonly used antibacterial compounds today are or are derived from natural substances produced by bacteria, actinomycetes and fungi. These antibacterial compounds are grouped according to the following main classes: β-lactams including penicillins, methicillin, cephalosporins and monobactams; aminoglycosides including streptomycin, gentamicin, neomycin, tobramycin, netilmycin and amikacin; tetracyclines including minocycline and doxycycline; sulfonamides and trimethoprimes; fluoroquinolones including ciprofloxacin, norfloxacin and ofloxacin; macrolides including erythromicin, azithromycin and clarithromycin; quinolones including ciprofloxin; polymyxins; glycopeptides including vancomycin; polymyxins; lincosamides; and chloramphenicol. Enterococci have naturally developed resistance mechanisms against most conventional antibacterials and in particular against β-lactams of penicillin and cephalosporins type, against a inoglycosides of gentamycin type, against quinolones, against glycopeptides of vancomycin type, against tetracyclines , against erythromycin-like macrolides, against chloramphenicol and against ampicillin (Diversity a ong Multidrug-Resistant Enterococci, Barbara E. Murray, Emerging Infectious Diseases, Vol. 4, No. 1, January-March 1998). For example, the percentage of nosocomial infections caused in the United States by Enterococci resistant to vancomycin increased by more than a factor of 20 between 1989 and 1993, from 0.3% to 7.9% (Multiple-Drug Resistant Enterococci: The Nature of the Problem and an Agenda for the Future, Mark M. Huycke, Daniel F. Sahm and Michael S. Gilmore, Emerging Infectious Diseases, Vol.4, No.2, April-June 1998).

Peptides with antimicrobial properties are produced by a wide variety of animal and plant species, in which they participate in non-specific defense mechanisms against infections (Antimicrobial peptides of multicellular organisms, Zasloff M., Nature Vol. 415, Jan 2002, 389-395).

Insects have in particular developed an effective defense system against microorganisms. This immune response is largely based on the rapid and transient synthesis of antimicrobial peptides with a broad spectrum of activity (Antimicrobial peptides in insects; structure and function, Bulet P. and al., Dev. Comp. Immunol. 23, 1999, 329-344). Among the peptides insects which have properties against Gram-positive bacteria, there are in particular cecropins, defensins, thanatin and moricin. Cecropins are peptides isolated from Diptera and Lepidoptera which have a three-dimensional structure of the type comprising 2 amphipatic helices (Amphipatic, α-helical antimicrobial peptides, Tossi A., Sandri L., Giangaspero A., Biopolymers, Vol. 55 , 4-30 (2000); WO 89/00194). Insect defensins are peptides isolated from various orders of insects which have a three-dimensional structure of the type comprising an α helix and 2 antiparallel β strands linked by 3 disulfide bridges (EP 0349451; WO 90/13646; EP 0546213 ; FR 2695392; EP 0607080; WO 97/02286). Thanatin is a peptide isolated from the hemiptera Podisus maculiventris which has a three-dimensional structure of the β-hairpin-like type comprising two antiparallel β strands linked by a disulfide bridge (WO 99/24594). Moricin is a peptide isolated from the lepidoptera of the Bombycidae family, Bombyx ori (JP 08119995; JP 11215983). Among the peptides previously listed, none has been described as having activity against Gram-positive bacteria of the genus Enterococcus.

The technical problem posed by the present invention consists in characterizing new compounds having antifungal and / or antibacterial properties and more particularly against Gram-positive bacteria of the genus Enterococcus responsible for serious opportunistic infections in humans and / or animals. By “antibacterial” is meant according to the present invention, any molecule having bacteriostatic and / or bactericidal properties. By "antifungal" is meant according to the present invention, any molecule having fungistatic and / or fungicidal properties.

In the peptide sequences reported below, the amino acids are represented by their one-letter code, but they can also be represented by their three-letter code according to the nomenclature below.

To Ala alanine

C Cyst cysteine

D Asp aspartic acid E Glu glutamic acid

F Phe phenylalanine

G Gly glycine

H His histidine

I Isoleucine island K Lys lysine

L Leu leucine

M Met methionine

N Asn asparagine

P Pro proline Q Gin glutamine

R Arg arginine

S Ser Serine

T Threonine Thr

V Val valine W Trp tryptophan y Tyr tyrosine

The Applicant has now isolated, purified and characterized new peptides from the hemolymph of immune larvae of the Lepidopteran of the family Nymphalidae, Caligo illioneus (FIG. 1). Lepidoptera of the family Nymphalidae have a daytime activity and are characterized by the presence of antennae ending in club shape, bright colors with patterns and vertical wings at rest.

The peptides isolated by the Applicant from the hemolymph of immune larvae of the Lepidopteran Caligo illioneus correspond to formula (I): Xaa-Lys-Ile-Pro-Xab-Ala-Xac-Lys-Gly-Ala-Xad-Arg -Ala-Leu- Xae-Ala-Ser-Thr-Ala-Xaf-Asp-Ile-Ala-Xag-Phe-Xah-Arg-Lys- Arg-Xai (I) in which,

Xaa is - H2 or the peptide residue Arg or Gly, Xab is the peptide residue Ile-Asn or Val-Glu,

Xac is the Ile-Lys, Ile-Arg or Leu-Lys peptide residue,

Xad is the peptide residue Ser-Arg-Ala-Trp, Lys-Ala-Val-Gly-His-Gly-Leu or Lys-Val-Ala-Gly-Arg-Ala-Trp, Xae is the peptide residue Asp-Leu, Asn-Ile or

Asp-Leu,

Xaf is the peptide residue Tyr or His,

Xag is the peptide residue Ser-Ile, Ser-Ala or His-Leu, Xah is the peptide residue Asn, Asp or His,

Xai is —OH or the peptide residue Glu, Asn or Lys-His. The main subject of the present invention therefore is an isolated peptide corresponding to formula (I): Xaa-Lys-Ile-Pro-Xab-Ala-Xac-Lys-Gly-Ala-Xad-Arg-Ala-Leu- Xae-Ala -Ser-Thr-Ala-Xaf-Asp-Ile-Ala-Xag-Phe-Xah-Arg-Lys- Arg-Xai (I) in which Xaa, Xab, Xac, Xad, Xae, Xaf, Xag, Xah and Xai are as defined above, its derivatives and its fragments.

The formula (I) corresponds to the sequence SEQ ID NO: 1 in the sequence listing in the appendix.

The present invention relates more particularly to an isolated peptide corresponding to one of the following sequences:

- ETD-P1646 G K I P I N A I R K G A K A V G H G L R A L N I A S T A H D I A S A F H R K R K H (SEQ ID

NO: 2 in the sequence listing in the appendix)

- ETD-P1647 R K I P V E A I K K G A S R A W R A L D L A S T A Y D I A S I F N R K R E (SEQ ID NO: 3 in the sequence listing in the appendix) - ETD-P1648 G K I P V E A L K K G A K V A G R

A W R A L D L A S T A Y D I A H L F D R K R N (SEQ ID NO: 4 in the sequence listing in the appendix), its derivatives and fragments.

Mention may be made, as derivatives of the peptides of the invention, of the peptides which exhibit a post-translational modification and / or chemical modification, in particular glycosylation, amidation, acylation, acetylation, methylation as well as the peptides which carry a protective group. The term “protective group” according to the present invention means any group making it possible to avoid the degradation of said peptides. The derivatives of the peptides of the invention may also be those in which one or more amino acids are enantiomers, diastereoisomers, natural amino acids of conformation D, rare amino acids in particular 1 hydroxyproline, 1 hydroxylysine, allo- hydroxyl sine, 6-N-methyl sine, N-ethylglycine, N- methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, pyroglutamine, aminobutyric acid and synthetic amino acids, especially ornithine, norleucine, norvaline, cyclohexyl-alanine and omega-amino acids. The derivatives also cover retropeptides and retro-inversopeptides, as well as peptides in which the side chain of one or more of the amino acids is substituted by groups which do not modify the antimicrobial activity of the peptides of the invention.

By way of fragments of the peptides of the invention, are meant fragments of at least 5 amino acids which exhibit antimicrobial activity. The antimicrobial activity of the derivatives and fragments of the peptides of the invention can be demonstrated using the in vitro tests described below in Example 4.

The invention also relates to a polypeptide comprising a peptide of the invention. The invention more particularly contemplates a polypeptide comprising a peptide of the invention, one and / or the other of the ends of said peptide comprising one or more amino acids necessary for its expression and / or its targeting in a host organism.

The invention also relates to an isolated polynucleotide encoding a peptide or polypeptide of the invention. The term “polynucleotide” according to the present invention means a nucleic sequence of DNA or RNA type, preferably DNA, in particular double strand.

The invention also relates to isolated polynucleotides which comprise modifications at the level of one or more nucleotides resulting from the degeneration of the genetic code and which encode the same amino acid sequence of the peptides or polypeptides of the invention. The invention also covers isolated polynucleotides encoding the peptides or polypeptides of the invention and capable of hybridizing under stringent conditions to said peptides or polypeptides. The term "stringent condition" according to the present invention means the conditions taught by Sambrook et al. (Molecular cloning, 1989, Noland C. ed., New York: Cold Spring Harbor Laboratory Press).

The invention also covers the nucleotide sequences complementary to the isolated polynucleotides defined above as well as the corresponding RNAs.

The invention also relates to a chimeric gene comprising at least, operatively linked, a constitutive or inducible promoter functional in a host organism, a polynucleotide encoding a peptide or a polypeptide of the invention and a functional terminator in an organism. host. The term “operatively linked together” according to the invention means elements linked together so that the operation of one of the elements is affected by that of another. For example, a promoter is operably linked to a coding sequence when it is able to affect the expression of the latter. All the regulatory elements of transcription, the translation and maturation of peptides or polypeptides that the chimeric gene can understand is known to those skilled in the art and the latter is able to choose them according to the host organism. The chimeric gene may in particular comprise, as an additional regulatory element, sequences coding for a signal or transit peptide so as to direct the secretion of the peptide or polypeptide of the invention in the host organism. When the host organism is a yeast, these sequences are preferably the pre-BGL2 and pro sequences of MF 1 which allow the secretion of the peptide or polypeptide of the invention directly into the culture medium. The chimeric gene may further include sequences encoding an endoprotease to increase proteolytic activity in the host organism during the secretion process. When the host organism is a yeast, these sequences are preferably the sequences of the KEX2 gene coding for the endoprotease yscf which makes it possible to cleave the pro sequence of the pre-pro-peptide of the invention during the secretion process.

The invention also relates to a cloning and / or expression vector characterized in that it contains a polynucleotide or a chimeric gene according to the invention for transforming a host organism and expressing in the latter a peptide or polypeptide of the invention . The vector can be a plasmid, a cosmid, a bacteriophage or a virus, in particular a baculovirus. The vector is advantageously a vector with autonomous replication comprising elements allowing its maintenance and its replication in the host organism as an origin of replication. In addition, the vector may include elements allowing its selection in the host organism, for example a gene for resistance to a compound. antibacterial or a selection gene that provides complementation with the respective deleted gene at the level of the genome of the host organism. The vector can also be a shuttle vector comprising elements allowing its maintenance and its replication in each of the host organisms and elements allowing its selection in each of the host organisms. Such cloning and / or expression vectors are well known to those skilled in the art and widely described in the literature.

The invention also relates to a host organism transformed with a vector of the invention. By “host organism” according to the present invention is meant any single or multicellular organism, lower or higher, in which a polynucleotide or a chimeric gene of the invention is introduced for the production of a peptide or polypeptide of the invention . According to a preferred form of the invention, the host organism is a microorganism such as a yeast, a bacterium or a fungus. The transformation of such microorganisms makes it possible to produce the peptides or polypeptides of the invention on a semi-industrial or industrial scale. As bacteria, the invention more particularly contemplates the bacteria of the species Escherichia coll. As yeast, the invention more particularly contemplates a yeast of the genus Saccharomyces, preferably of the species Saccharomyces cerevisiae, of the genus Kluyveromyces, preferably of the species Kluyveromyces lactis, of the genus Hansenula, preferably of the species Hansenula polymorpha, of the genus Pichia, preferably of the species Pichia pastoris or of the genus Schizosaccharomyces, preferably of the species Schizosaccharomyces pombe. As a mushroom, the invention envisages more particularly the filamentous fungus of the species Aspergillus nidulans.

According to another form of the invention, the host organism is an animal, in particular an arthropod cell (for example a Spodop t era f ugiperda or Trichoplusia ni cell) or a mammalian cell.

According to another form of the invention, the host organism is a plant cell or a plant. By “plant cell” is meant, according to the present invention, any cell originating from a plant and which may constitute undifferentiated tissues such as calluses, differentiated tissues such as embryos, parts of plants, plants or seeds. By “plant” is meant according to the invention, any differentiated multicellular organism capable of photosynthesis, in particular monocotyledons or dicotyledons, more particularly crop plants intended or not for animal or human food.

The peptides of the invention are also useful for conferring on plants a character of resistance to bacterial and / or fungal diseases. The invention therefore also relates to a plant cell or a plant resistant to bacterial and / or fungal diseases comprising a polynucleotide or a chimeric gene of the invention and expressing a peptide or polypeptide of the invention.

The peptides or polypeptides of the invention can also be chemically synthesized according to techniques known to those skilled in the art. The peptides of the invention have been tested for their antimicrobial activities. Tables 1 and 2 below show the in vitro antibacterial activities (IC90 μg / ml) and the in vitro antifungal activities (MIC μg / ml) of the peptides of the invention, respectively.

Table 1

Table 2

ND Not determined

The peptides of the invention are preferably active against Gram-positive bacteria and more particularly against bacteria of the genus Enterococcus. The invention therefore also aims to take advantage of the antimicrobial properties of the peptides of the invention to prevent and / or treat bacterial and / or fungal infections both in humans and animals and in plants. The invention therefore advantageously relates to the use of the peptides of the invention as medicaments in human and animal therapy. It also relates to the use of the peptides of the invention for the treatment of plants against bacterial and / or fungal infections, by applying said peptides directly to the plants.

The invention therefore relates to an antibacterial composition comprising as active agent at least one peptide of the invention advantageously combined in said composition with an acceptable vehicle. The antibacterial composition may further include another antibacterial agent. According to the present invention, the antibacterial agent is preferably chosen from β-lactams and more particularly penicillins, methicillin, cephalosporins and monobactams; aminoglycosides and more particularly streptomycin, gentamicin, neomycin, tobramycin, netilmycin and amikacin; tetracyclines and more particularly minocycline and doxycycline; sulfonamides and trimethoprime; fluoroquinolones and more particularly ciprof loxacin, norfloxacin and ofloxacin; macrolides and more particularly erythromicin, azithromycin and clarithromycin; quinolones and more particularly ciprof loxine; polymyxins; glycoproteins and more particularly vancomycin and teicoplanin; lipopeptides and more particularly daptomycin; lincosamides; chloramphenicol; and oxazo l idinone s and more particularly linezolid.

The invention also relates to an antifungal composition comprising, as active agent, at least one peptide of the invention advantageously combined in said composition with an acceptable vehicle. The antifungal composition may further comprise another antifungal agent. According to the present invention, the antifungal agent is preferably chosen from polyene derivatives and, more particularly amphotericin B, nystatin and pimaricin; azole derivatives and more particularly ketoconazole, clotrimazole, miconazole, econazole, butoconazole, oxiconazole, sulconazole, tioconazole, terconazole, fluconazole and itraconazole; allyl-thiocarbamates and more particularly tolnaftate, naftifine and terbinafine; 5-f luorocytosine; echinocandins; griseof ulvine; ciclopirox; and haloprogin.

By “vehicle” is meant according to the present invention, any substance which is added to the peptide (s) of the invention to promote the transport of the peptide (s), avoid its substantial degradation in said composition and preserve its antimicrobial properties. . The vehicle is chosen according to the type of application of the composition. In particular, when the composition is applied to pharmaceutical use in human and animal health, the vehicle is a pharmaceutically acceptable vehicle suitable for administration of the peptide of the invention by topical route, per os or by injection. When the composition is applied for cosmetic use, the vehicle is a cosmetically acceptable vehicle suitable for administration to the skin or the integuments. When the composition is applied to an agrochemical use, the vehicle is an agrochemically acceptable vehicle suitable for administration on plants or near plants without degrading them.

The peptides or the composition of the invention are also of interest in the food industry. Their use makes it possible in particular to prevent contamination by bacteria, yeasts and / or fungi during the manufacture of food products and after their manufacture for their preservation.

In the phytosanitary industry, the peptides or the composition of the invention can also be used for the manufacture of phytosanitary products in place of the usual products.

Other advantages and characteristics of the peptides of the invention will appear from the following examples concerning their preparation and their antimicrobial activities and in which reference will be made to the accompanying drawings in which:

FIG. 1 represents the Lepidoptera Caligo illioneus (family of Nymphalidae; subfamily of Brassolinae) from which the peptides of the invention were isolated.

I. Example 1; Isolation of the peptides ETD-P1646, ETD-P1647 and ETD-P1648 from the hemolymph taken from immune larvae of the Lepidopteran Caligo illioneus. 1) Induction of the biological synthesis of antimicrobial substances in the hemolymph of Caligo illioneus.

The last larvae of the last stage of the Lepidopteran Caligo illioneus were immunized by injection of a PBS solution containing Gram positive bacteria (Micrococcus luteus and Staphylococcus aureus) and Gram negative bacteria (Pseudomonas aeruginosa), spores of filamentous fungi (Aspergillus fumigatus) and yeasts (Candida albicans). The bacteria are prepared from cultures carried out in Luria-Bertani medium for 12 hours at 37 ° C. The yeasts are prepared from cultures carried out in the middle of Sabouraud for 12 hours at 30 ° C. The spores of A. fumigatus are taken from a frozen stock at -80 ° C. The animals thus infected were kept for 24 hours on their host plant, in a ventilated space. Before the removal of the hemolymph, the larvae were cooled on ice.

2) Preparation of the plasma. The hemolymph of C. ill ioneus (28 ml) was collected by lateral injection at the level of the head followed by manual pressure and collected in 1.5 ml polypropylene microcentrifuge tubes cooled in ice and containing aprotinin as a protease inhibitor (20 μg / ml in final concentration) and phenylthiourea as a melanization inhibitor (final concentration of 40 μM). The hemolymph thus collected from the immunized larvae was centrifuged at 8000 rpm for 1 min at 4 ° C in order to eliminate the hemocytes. The centrifugation supernatant was then centrifuged at 12,000 rpm. The hemolymph without blood cells was stored at -80 ° C until use. 3) Acidification of the plasma.

After rapid thawing, the plasma of C. illioneus was acidified to pH 3 with a 1% trifluoroacetic acid solution (volume by volume) containing aprotinin (20 μg / ml in final concentration) and phenylthiourea (concentration 40 μM final). The extraction of the peptides under acid conditions was carried out for 30 min with light stirring in an ice-water bath. The extract obtained was then centrifuged at 4 ° C for 30 min at 10000g.

4) Purification of peptides. a) Pre-purification by solid phase extraction.

An amount of extract equivalent to 6 ml of hemolymph of C. illioneus was deposited on a support of 5 g of reverse phase as sold in the form of cartridge (Sep-Pak ™ C ₁₈ , Waters Associates), balanced with acidified water (TFA 0.05%). The hydrophilic molecules were removed by a simple washing with acidified water. The elution of the peptide was carried out with a 60% solution of acetonitrile in water containing

0.05% TFA. The fraction eluted with 60% acetonitrile was dried under vacuum in order to remove the acetonitrile and the TFA, then it was reconstituted in sterile acidified water (TFA 0.05%) before being subjected at the first stage of purification. Step 4 a) was repeated once in order to obtain the fraction eluted with 60% acetonitrile in a larger amount. b) Purification by high performance liquid chromatography (HPLC) on reverse phase column.

First step: the 60% elution from the solid phase extraction described above containing the peptide was fractionated by reverse phase high performance liquid chromatography (HPLC) on a column Interchim ™ brand semi-preparative, Modulocart Uptiprep type, size 250 x 10 mm, porosity 300 A and particle size 15 μm with C8 grafting. The fractionation was carried out with an acetonitrile gradient / 0.05% TFA against H ₂ O / 0.05% TFA from 5 to 60% in 50 minutes at a constant flow rate of 2.5 ml / min. The fractions were collected automatically (Gilson ™ collector type FC 204) in the first 80 wells (columns A to J) of a 96-well plate (Maximum capacity of each well: 2 ml). The collection is done per unit of time and the volume collected is approximately 1.6 ml. The collected fractions were dried under vacuum, reconstituted with ultrapure water and analyzed for their antimicrobial activities using the tests described below. The step was repeated once in order to obtain larger active fractions.

Second step: fractions 37 and 45 from the previous step having activity against Gram-positive bacteria in the purification steps described above, eluted with a percentage of acetonitrile equal to 30 and 35 respectively, were purified so finer on a C18 reverse phase grafting analytical column of the Modulocart Uptisphere type (Interchim ™, 250 x 4.6 mm, 300 A and 5 μm), using a linear two-phase gradient of acetonitrile / 0.05% TFA against H ₂ O / 0.05% TFA. For example, for a fraction eluted at 30% (35%) of acetonitrile, the gradient used ranges from 2 to 26% (31%) in 10 min and from 26 (31%) to 34% (39%) in 45 min, with a constant flow rate of 0.8 ml / min. The fractions were collected manually by following the change in absorbance at 225 nm. The fractions collected were dried under vacuum, reconstituted with ultrapure water and analyzed for their activities against Gram-positive bacteria in the conditions described below. The activity was found in certain sub-fractions of the fractions described above.

Third step: the fractions described above, containing the peptides, were purified to homogeneity on a reverse phase column of Interchim ™ brand of the Modulocart Uptisphere type (C18 grafting, 3 A particle size, size 150 × 2 mm) in using a linear two-phase gradient of acetonitrile / 0.05% TFA against H ₂ O / 0.05% TFA (gradient depending on the percentage of elution of acetonitrile from the sub-fraction to be purified), with a constant flow rate of 0, 2 ml / min at a controlled temperature of 30 ° C. The fractions were collected manually by following the change in absorbance at 225 nm. The collected fractions were dried under vacuum, reconstituted with filtered ultrapure water and analyzed for their activities against Gram positive bacteria.

II. Example 2; Structural characterization of the peptides ETD-P1646, ETD-P1647 and ETD-P1648.

1) Verification of purity by Maldi-TOF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time Of Flight).

The purity check was carried out on a MALDI-TOF Bruker Biflex mass spectrometer (Bremen, Germany) in positive linear mode (see section 3 below).

2) Reduction and S-pyridylethylation with a view to determining the number of cysteines. The number of cysteine residues was determined on the native peptides by reduction and S-pyridylethylation. 400 pmoles of native peptide were reduced in 40 μl of 0.5 M Tris / HCl buffer, pH 7.5 containing 2 mM EDTA and 6 M guanidinium chloride in presence of 2 μl of 2.2 M dithiothreitol. The reaction medium was placed under a nitrogen atmosphere. After 60 min of incubation in the dark, 2 μl of freshly distilled 4-vinylpyridine were added to the reaction which was then incubated for 10 min at 45 ° C in the dark and under a nitrogen atmosphere. The S-pyridylethylated peptide was then separated from the constituents of the reaction medium by reverse phase chromatography on an Aquapore RP-300 C ₈ reverse phase analytical column (Brownlee ™, 220 x 4.6 mm, 300 Å), using a linear gradient of acetonitrile / 0.05% TFA against H ₂ O / 0.05% TFA from 2 to 52% for 70 min.

3) Determination of the mass of the native peptide, of the S-pyridylethylated peptide and of the proteolysis fragments by MALDI-TOF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time Of Flight).

The mass measurements were carried out on a MALDI-TOF Bruker Biflex mass spectrometer (Bremen, Germany) in positive linear mode. The mass spectra were calibrated externally with a standard mixture of peptides of known m / z, respectively 2199.5 Da, 3046.4 Da and 4890.5 Da. The different products to be analyzed were deposited on a thin layer of α-cyano-4-hydroxycinnamic acid crystals obtained by rapid evaporation of a saturated solution in acetone. After drying under a slight vacuum, the samples were washed with a drop of 0.1% trifluoroacetic acid before being introduced into the mass spectrometer. The difference in mass observed between the S-pyridylethylated peptide and the native peptide makes it possible to determine the number of cysteine residues present in each of the peptides.

4) Sequencing by Edman degradation.

Automatic sequencing by Edman degradation of native peptides, S-pyridylethylated peptides and different fragments obtained after the various proteolytic cleavages and the detection of phenylthiohydantoine derivatives were carried out on a sequencer of the Procise HT sequencing System type, model 492 of the Applied Biosystem brand.

5) Proteolytic cleavages.

Confirmation of peptide sequences in the C-terminal region. 200 pmol of reduced peptide and S-pyridylethylated were incubated in the presence of 5 pmol of endoproteinase-Asp-N (Endoproteinase Asp-N, specific cleavage of aspartic acid residues on the N-terminal side, Takara, Otsu) according to the conditions recommended by the supplier (20 mM sodium phosphate buffer, pH 8.9, in the presence of 0.01% Tween 20) for 16 hours. After stopping the reaction with 1% TFA, the peptide fragments were separated by reverse phase HPLC on a column of Interchim ™ brand of the Modulocart Uptisphere type (C18 grafting, particle size 3 Å, size 150 × 2 mm) with a linear gradient of acetonitrile / 0.05% TFA against H ₂ O / 0.05% TFA from 2 to 60% in 80 min with a flow rate of 0.2 ml / min and a constant temperature of 37 ° C. The fragments obtained were analyzed by MALDI-TOF mass spectrometry and the peptide corresponding to the C-terminal fragment was sequenced by Edman degradation.

The primary sequence of the ETD-P1646 peptide is represented in the sequence list in the appendix under the number SEQ ID NO: 2. The molecular mass of the native ETD-P1646 peptide is 4383 Da and the molecular mass of the ETD-P1646 S peptide pyridylethylated is 4384 Da.

The primary sequence of the ETD-P1647 peptide is represented in the annexed sequence list under the number SEQ ID NO: 3. The molecular mass of the ETD-peptide Native P1647 is 4202 Da and the molecular weight of the peptide ETD-P1647 S-pyridylethylated is 4205 Da.

The primary sequence of the ETD-P1648 peptide is represented in the annexed sequence list under the number SEQ ID NO: 4. The molecular mass of the ETD-peptide

Native P1648 is 4408 Da and the molecular weight of the peptide ETD-P1648 S-pyridylethylated is 4410 Da.

III. Example 3: Production of the peptides ETD-P1646, ETD-P1647 and ETD-P1648.

The peptides ETD-P1646, ETD-P1647 and ETD-P1648 have been synthesized chemically (Altergen).

IV. Example 4: Tests of antimicrobial activity in vitro.

1) Antibacterial tests.

Antibacterial activities were assessed in vitro by determination of IC90 (Growth Inhibition ≥ 90%). The tests were carried out on the following bacterial strains:

-Gram positive bacteria: Staphylococcus aureus (strain 21; Gift of the Institute of Molecular and Cellular Biology, Strasbourg), Enterococcus faecalis (clinical strain 17; Gift of Dr. G. Prevot, Institute of Bacteriology, Strasbourg) and Enterococcus faecium (clinical strain 18; Gift of Dr. G. Prevot, Institute of Bacteriology, Strasbourg); - Gram negative bacteria: Pseudomonas aeruginosa (Gift of the Institute of Molecular and Cellular Biology, Strasbourg). All these strains are sensitive to the antibacterials commonly used in hospitals. a) Preparation of the bacterial suspensions. A 4 ml preculture in LB medium was prepared by seeding a colony of the bacterial strain of interest. The preculture was incubated at 35-37 ° C with shaking for 6 h. The concentration of the preculture was evaluated by measuring the optical density at 600-620 nm according to the relation bacterial density≈f (OD). The concentration was adjusted by dilution so as to obtain a suspension of 2.10 ⁶ / ml in final concentration. The concentration of the bacterial suspension was checked by enumeration of the Colony Forming Units (CFU). The bacterial suspension at 2.10 ⁶ / ml was diluted in cascade (10 ^_1 , 10 ^"2 , 10 ^{" 3} , ... 10 ^"6 ) then 100 μl of the dilutions 10 ^{" 6} , 10 ^"5 and 10 ^{~ 4} were spread on LB agar plates The plates were incubated at 35-37 ° C for 16 to 18 hours and then the CFUs were counted b) Determination of IC90.

The IC90s were determined by a liquid test in 96-well microplates. 100 μl of test sample were distributed in duplicates so as to obtain a range of final concentrations from 64 to 0.125 μg / ml in the wells. 100 μl of bacterial suspension at 2.10 ⁶ / ml was added to each well so as to obtain a final concentration of 10 ⁶ / ml. The microtiter plates were incubated at 30-35 ° C for 16-18 h. The optical density of the wells was measured at 600-620 nm and the results were interpreted by calculating the percentage of growth in each well according to the formula:% Sprout = (DO well - DO TS) / (DO TP - DO TS) * 100 where DO of TP = DO of growth control (bacterial suspension without sample to be tested), DO of TS = DO of sterility control (culture medium without bacteria) and DO well = DO of the well of which one wishes calculate the percentage of growth (bacterial suspension + sample to be tested). The IC90 corresponds to the well where the percentage of growth is less than or equal to 10%. The results of the antibacterial activities (IC90) are presented in Table 1 exposed in the description. 2) Antifungal tests.

The antifungal activities were evaluated in vitro by determination of the Minimum Inhibitory Concentration (MIC).

The tests were carried out on the following yeast strains: Candida albicans (IHEM 8060 strain; Gift of Dr. H. Koenig, Civil Hospital, Strasbourg) and Candida glabrata (patient strain 1; Gift of Dr. H. Koenig, Civil Hospital , Strasbourg) as well as on the following filamentous fungi: Aspergillus fumigatus (strain GASP 4707; Gift of Dr. H. Koenig, Hôpital civil, Strasbourg).

All these strains are sensitive to antifungal agents commonly used in hospitals, a) Preparation of fungal suspensions. a-1) Preparation of yeast suspensions. A yeast loop taken from a stock of yeasts suspended at 4 ° C. was spread on a Sabouraud Agar agar plate. The dish was incubated at 30 ° C for 24 to 48 hours. A few yeast colonies were collected and suspended in 10 ml of liquid Sabouraud medium. The yeast suspension obtained, which must be “milky”, has been diluted (1 ml qs 10 ml of Sabouraud medium). The concentration of the suspension was evaluated by measuring the optical density at 600 nm according to the relationship 0.1 OD at 600 nm corresponds to 2.5 × 10 ⁶ yeasts / ml. The concentration was adjusted by dilution so as to obtain a suspension of 2.5 × 10 ³ / ml of final concentration. The concentration of the yeast suspension was checked by enumeration of the Colony Forming Units (CFU). The 2.5.10 ³ / ml yeast suspension was cascaded (10 ^_1 , 10 ^"2 , 10 ^{" 3} , ... 10 ^"6 ) then 100 μl of each dilution were spread on Sabouraud agar dishes. The dishes were incubated at 30 ° C for 24 hours then the CFUs a-2) Preparation of suspensions of filamentous fungi.

The fungi were seeded on malt agar plates which were incubated 5 to 7 days at 37 ° C. The spores formed were collected and suspended in YPG medium. The concentration of the suspension was evaluated by counting an aliquot on a counting slide (Coverslide). The concentration was adjusted by dilution so as to obtain a suspension of 5.10 ³ / ml of final concentration. b) Determination of MICs.

The MICs were determined by a liquid test on 96-well microplates according to the M27-A and M38-P protocols of the "National Committee for Clinical Standard" (NCCLS) with the difference that the RPMI-1640 medium suggested by the NCCLS protocol was replaced by Sabouraud medium (Biomérieux) for the tests on yeasts and by YPG medium (1 g peptone, 1 g yeast extract, 3 g glucose per liter) for the tests on filamentous fungi. The activities of the test samples were determined for the range of final concentrations 0.125 to 64 μg / ml. b-1) Determination of MICs on yeasts. The optical density of the microplates was measured at 600 nm with a spectrophotometer for microplates after 24 and 48 hours of incubation for yeasts of the genus Candida.

The growth percentage (% growth) was calculated from the optical density (OD) values measured according to the following formula: % growth = ((DO well with sample to be tested - DO medium only) / (DO well without sample to be tested - DO medium only)) * 100.

MICs were defined according to the following scores: MIC 0:% shoots ≤ 10%; MIC 1: 10% <% sprout ≤

20%; MIC 2: 20% <% shoots <50%; MIC 3:> 50%. The MIC was determined in the score interval MIC 0 and MIC 2. b-2) Determination of MIC on filamentous fungi. The MIC is determined by reading the microplates with the naked eye after 48 hours of incubation for Aspergillus fumigatus.

The MICs were defined according to the following scores: MIC 0: no trace of fungi at the bottom of the well; CMI 1: a point at the bottom of the well; MIC 2: mushrooms on half the surface of the well; CMI 3: three quarters of the well is occupied by the fungus;

MIC 4: whole well overgrown with fungi. The MIC was determined in the score interval MIC 0 and MIC 2. The results of the antifungal activities (MIC) are presented in Table 2 set out in the description.

Claims

1) Isolated peptide characterized in that it corresponds to formula (I): Xaa-Lys-Ile-Pro-Xab-Ala-Xac-Lys-Gly-Ala-Xad-Arg-Ala-Leu- Xae-Ala- Ser-Thr-Ala-Xaf-Asp-Ile-Ala-Xag-Phe-Xah-Arg-Lys- Arg-Xai (I) (SEQ ID NO: 1 in the sequence list in the appendix) in which, Xaa is - NH2 or the peptide residue Arg or Gly,

Xab is the peptide residue Ile-Asn or Val-Glu, Xac is the peptide residue Ile-Lys, Ile-Arg or Leu-Lys,

Xad is the peptide residue Ser-Arg-Ala-Trp, Lys-Ala-Val-Gly-His-Gly-Leu or Lys-Val-Ala-Gly-Arg-Ala-Trp,

Xae is the Asp-Leu, Asn-Ile or

Asp-Leu,

Xaf is the peptide residue Tyr or His,

Xag is the peptide residue Ser-Ile, Ser-Ala or His-Leu,

Xah is the peptide residue Asn, Asp or His, Xai is —OH or the peptide residue Glu, Asn or

Lys-His, its derivatives and its fragments.

2) Isolated peptide according to claim 1, characterized in that it corresponds to one of the following sequences:

NO: 2 in the attached sequence list)

- ETD-P1647 RKIPVEAIKKGASRAWRALDL ASTAYDIASIFNRKRE (SEQ ID NO: 3 in the sequence list in the appendix) - ETD-P1648 GKIPVEALKKGAKVAGRAWRA LDLASTAYDIAHLFDRKRN (SEQ ID NO: 4 in the sequence list in the appendix), its derivatives and its fragments.

3) Polypeptide characterized in that it comprises a peptide according to any one of claims 1 or 2.

4) Polypeptide according to claim 3, characterized in that it comprises a peptide according to any one of claims 1 or 2 in which one and / or the other of the ends of said peptide comprises one or more amino acids necessary for its expression and / or its targeting in a host organism.

5) An isolated polynucleotide characterized in that it encodes a peptide according to any one of claims 1 or 2 or a polypeptide according to any one of claims 3 or 4.

6) An isolated polynucleotide characterized in that it hybridizes to a polynucleotide according to claim 5.

7) Chimeric gene characterized in that it comprises at least, operatively linked to each other: a constitutive or inducible promoter functional in a host organism,

- a polynucleotide according to any one of claims 5 or 6, and

- a functional terminator in a host organism. 8) chimeric gene according to claim 7, characterized in that it further comprises a signal or transit peptide functional in said host organism.

9) An expression and / or cloning vector characterized in that it comprises a polynucleotide according to any one of claims 5 or 6 or a chimeric gene according to any one of claims 7 or 8.

10) Expression and / or cloning vector according to claim 9, characterized in that it is a plasmid, a cosmid or a bacteriophage.

11) Expression and / or cloning vector according to claim 9, characterized in that it is a virus, in particular a baculovirus.

12) Host organism characterized in that it comprises a vector according to one of claims from 9 to 11.

13) Host organism according to claim 12, characterized in that it is a microorganism.

14) Host organism according to claim 13, characterized in that the microorganism is a yeast of the genus S accha romyc es, preferably of the species Saccharomyces cerevisiae, of the genus Kl uyveromyces, preferably of the species Kluyveromyces lactis, of the genus Hansenula, preferably of the species Hansenula polymorpha, of the genus Pichia, preferably of the species Pichia pastoris or of the genus Schizosaccharomyces, preferably of the species Schizosaccharomyces pombe. 15) Host organism according to claim 13, characterized in that the microorganism is a bacterium, preferably of the species Escherichia coli.

16) Host organism according to claim 13, characterized in that the microorganism is preferably a fungus of the species Aspergillus nidulans.

17) Host organism according to claim 12, characterized in that the host organism is an arthropod cell preferably of Spodoptera frugiperda or Trichoplusia ni.

18) Host organism according to claim 12, characterized in that the host organism is a mammalian cell.

19) Host organism according to claim 12, characterized in that the host organism is a plant cell or a plant.

20) Plant cell or plant resistant to bacterial and / or fungal diseases comprising a polynucleotide according to any one of claims 5 or 6, or a chimeric gene according to any one of claims 7 or 8, and expresses a peptide according to any of claims 1 or 2, or a polypeptide according to any of claims 3 or 4.

21) Antibacterial composition characterized in that it comprises, as active agent, at least one peptide according to any one of claims 1 or 2, advantageously combined in said composition with an acceptable vehicle. 22) Antibacterial composition according to claim 21, characterized in that it further comprises another antibacterial agent.

23) An antifungal composition characterized in that it comprises, as active agent, at least one peptide according to any one of claims 1 or 2, advantageously combined in said composition with an acceptable vehicle.

24) Antifungal composition according to claim 23, characterized in that it further comprises another antifungal agent.

25) Composition according to any one of claims 21 to 24 for use in humans or animals.

26) Composition according to any one of claims 21 to 24 for use in plants.