FR2695391A1 - Glycopeptides, their process of obtaining, and their biological applications. - Google Patents

Abstract

Glycopeptides are disclosed having antibacterial properties due to an aminoacid sequence containing at least one aminoacid substituted by a glycosyl group selected from monosaccharides, amino sugars, polysaccharides and/or polyamino sugars. These glycopeptides can be used against bacteria.

Description

GLYCOPEPTIDES, THEIR PRODUCTION PROCESS, AND THEIR
BIOLOGICAL APPLICATIONS
The subject of the invention is glycopeptides, their production and their biological applications.

 It relates more particularly to glycopeptides having in particular antibacterial properties.

 Antibacterial peptides are known to have been isolated from insects. Certain species indeed have an effective resistance against bacteria. Recent work has established that this defense is based, to a large extent, on the rapid synthesis (2 to 6 h) of several families of peptides or polypeptides. This synthesis can be induced, in particular, by a septic injury or, in general, a trauma, or by immunization, that is to say by injection of a low dose of bacteria.

Among the peptides or polypeptides produced, the following four groups can be distinguished
i) cecropins which are cationic peptides of 4 kDa forming two amphipathic a helices; (ii) cationic peptides rich in proline, of small size (2 to 4 kDa), partially characterized, such as apidaecins and abaecins; (iii) several distinct polypeptides, having a molecular weight (PM) of 8 to 27 kDa, for the most part cationic and frequently rich in glycine residues such as attacins, sarcotoxins II, diptericins and coleoptericins, and (iv) defensins , non-glycosylated, moderately cationic peptides with a pI of 8.0 to 8.5, comprising from 38 to 43 amino acids and containing a motif characteristic of 6 cysteine residues engaged in 3 intramolecular disulfide bridges.

 The antibacterial peptides mentioned above could be induced in lepidoptera, diptera, hymenoptera and beetles. However, work done to date on other species has not made it possible to detect their presence.

 Note that to date the chemical structure attributed to these peptides corresponded to a simple chain of amino acids.

 On the basis of this teaching, a person skilled in the art, seeking to develop by synthesis peptides having such properties, was therefore led only to construct a peptide sequence by linking amino acids determined according to the techniques classics.

 However, in the context of their work on the study of the molecular bases of the immune response of insects, the inventors have found that the antibacterial activity of certain inducible peptides in particular species of insects was linked to the presence of groups of substitution determined on amino acids.

 The elimination of these groups leads to a very strong reduction in antibacterial activity, or even to its complete disappearance.

 The invention therefore aims to provide new substituted peptides having a high level of activity vis-à-vis a large number of bacteria.

 It further aims to provide methods for obtaining such peptides making it possible to obtain them in large quantities.

 The invention further aims to take advantage of the biological properties conferred by substitution groups for the development of antibacterial agents usable in human and veterinary therapy and, in general, for the fight against bacteria.

 The peptides according to the invention are characterized in that they have antibacterial properties conferred by an amino acid sequence containing at least one amino acid substituted by a group chosen from oses, osamines, poly-oses and / or- osamines.

 The substituted amino acid is a hydroxylated amino acid, such as threonine, serine or tyrosine. Threonine is particularly preferred.

 The substitution group of the amino acid (s) will be designated in the following description and the claims by the term "glycosyl", the peptides of the invention also being called glycopeptides. The glycosyl group is linear or cyclic, substituted or not, in a or p form.

It advantageously contains 5 or 6 carbon atoms. The substituents optionally present are advantageously chosen from alkyl radicals of 1 to 4 carbon atoms or, in particular in the case of osamines, from acyl radicals, more especially acetyl, these radicals more particularly substituting the amine function.

 Suitable dare groups include pentoses, such as ribose, and hexoses, such as galactose and glucose. Osamine groups are advantageously constituted by a galactosamine or glucosamine radical.

 The poly-oses and / or -osamines comprise a series of ose and / or osamine units, in particular from 1 to 10 units, in particular from 1 to 5 units.

 As shown by the work of the inventors reported in the examples, these substitution groups, surprisingly, appear essential to have an appreciable level of antibacterial activity.

The glycopeptides according to the invention are further characterized in that they are of the type of glycopeptides as obtained in arthropods, and in particular in larvae or adults of insects, by a process comprising
- induction of their synthesis, in particular by injection into insects of bacteria in sufficient doses, or by septic injury or other trauma,
- the extraction in order to collect the peptides having undergone post-translational glycosylation and, if necessary,
- fractionation of the isolated extract in order to selectively separate the glycopeptides according to the level of their antibacterial activity.

 The invention relates to the corresponding extracts, mixtures of glycopeptides and fractions.

 The glycopeptides of the invention are also characterized in that they exhibit antibacterial activity against Gram-negative germs.

 According to a provision of the invention, the glycopeptides are of the type of those produced by the dipterans.

 More particularly, the glycopeptides according to the arrangement of the invention considered are characterized in that their peptide sequence responsible for their antibacterial activity, which comprises at least one amino acid substituted by a group chosen from oses, osamines, poly-oses or -osamines as defined above, is contained in a domain comprising at least approximately 30% of proline groups.

This sequence more specifically comprises a consensus motif of proline-threonine / serine O-glycosylation
Xaal-Xaa2-proline in which the Xaal and Xaa2 motifs, identical or different, are variable amino acids.

 The chain of amino acids of the biologically active sequence, that is to say having an antibacterial activity, advantageously comprises at least one hydrophobic amino acid such as threonine, substituted by a glycosyl group. The substitution group is more particularly constituted by an N-acetylhexosamine unit, optionally itself substituted by a hexose.

 Preferred glycopeptides are as inducible in brachycera.

 A group of these glycopeptides which is advantageous with regard to its antibacterial properties is such that it is inducible in Drosophila.

The chain of amino acids of the glycopeptides of this group is in particular understood or constituted by the following sequence (I)
MKFTIVFLLLACVFAMAVATPGKPRPYSPRPTSHPRPIRVRREALAIEDHLAQAAIRPP
PILPA
More particularly, said sequence is understood or constituted by the following sequence (II) of amino acids GKPRPYSPRPXSHPRPIRV
The underlined threonine units are the units substituted, in accordance with the invention, by a glycosyl group.

It is recalled that the amino acids are conventionally represented by letters or are designated by a three-letter code, according to the following correspondence
A = Ala; R = Arg; D = Asp; N = Asn; C = Cys; Q = Gln
E = Glu; G = Gly; H = His; I = Ile; L = Leu; K = Lys;
M = Met; F = Phe; P = Pro; S = Ser; T = Thr; W = Trp
Y = Tyr; V = Val.

 In another preferred group of the invention, the glycopeptides are of the type of those inducible in Phormia terranovae.

Such glycopeptides advantageously comprise or consist of a chain of amino acids, as determined according to the degradation of Edman, corresponding to the following sequence (III)
DEKPKLILPTPAPPNLPOLVGGGGGNRKDG
FGVSVDAHQKVWTSDNGGHSIGVSPGYSQH
LPGPYGNSRPDYRIGAGYSYNE
Other sequences include as Nterminal part, at least part of the following sequences (IV) and (V).

DEKPKLVLP (S) XAPPNLPQLVGGGGGNNKX
GXXVSINAAQKV (1V) or
DEK (P) KLIXPXXA (P) XNLXQLVGGGGGNNK
KXXGVXVXXAO (V)
In this sequence the threonine in position 10 and / or that in position 54 is (are) substituted (s) by a glycosyl group.

 The glycosyl group is advantageously an osamine motif itself substituted by a ose, a suitable motif for having antibacterial glycopeptides corresponds to a (N-acetylhexosamine) -hexose group.

 Yet another group includes glycopeptides such as are inducible in hymenoptera, especially in bees.

 The invention also relates to the fragments or variants of the glycopeptides defined above, since these fragments or these variants are recognized by antibodies directed against said peptides and / or have charge and / or hydrophobicity or hydrophilicity characteristics. conferring antibacterial activity against Gram-negative germs, as observed in native glycopeptides. By variants is meant glycopeptides in which one or more amino acids are deleted, substituted by a group other than a glycosyl, or replaced by another amino acid with a similar charge.

 The hydrophilicity and hydrophobicity characteristics of the amino acids are given by Kyte et al in J. Mol.

Biol., 157, 105-132, 1982.

 Antibodies formed against glycopeptides and their fragments and variants also fall within the scope of the invention.

 According to another aspect, the invention relates to the nucleotide sequences comprising the genetic information corresponding to the amino acids of the glycopeptides defined above.

 It also targets the nucleotide sequences capable of hybridizing under stringent conditions with the above sequences.

By stringent conditions is meant the conditions defined in the book "Molecular Cloning" by Sambrook,
Fritsch, Maniatis, 1989, Cold Spring Harbor Laboratory
Press.

The invention further relates to the nucleotide sequences complementary to those defined above, as well as the
Corresponding RNAs.

 The expression and / or cloning vectors comprising at least one fragment of the nucleotide sequences defined above also form part of the invention, as well as the hosts transformed by these vectors.

 By way of examples, suitable hosts for the implementation of the invention include bacteria, such as E. coli, yeasts or also arthropod, vertebrate or plant cells.

 The invention also relates to the expression products of the above vectors.

 The chemical structure of glycopeptides with antibacterial activity having been characterized as indicated above, those skilled in the art will easily choose the most suitable techniques for their development.

 By operating synthetically, there is easy access to the structures of glycopeptides as inducible in arthroprodes and their variants.

 Advantageously, one or more of the amino acids of the sequence is substituted with a glycosyl group, then the introduction of blocking groups of the functions to be protected and a determined peptide chain is formed, advantageously using of a synthesizer, the substituted amino acid (s) being introduced sequentially in order to occupy the desired position in the sequence. The blocking groups are eliminated at the end of the synthesis.

 According to a variant, these glycopeptides can be obtained according to genetic engineering techniques by introducing into an appropriate vector the nucleotide sequences capable of expressing the peptide backbone of the glycopeptide sought, and by carrying out the expression in a host capable of ensuring a post-translational glycosylation. Alternatively, when the host used cannot provide this functionalization, chemical synthesis routes are used.

 According to yet another variant, these glycopeptides are obtained by immunizing arthropods containing genes capable of coding for the desired glycopeptide (s), in particular by injection of bacteria, in an amount sufficient to cause their synthesis, or by trauma , such as a septic injury. A few hours after the immunization or the trauma, the glycopeptides are extracted and then fractionated and the fraction (s) corresponding to the glycopeptide (s) sought (s) isolated.

 The extraction is carried out under conditions making it possible to avoid any phenomenon of degradation of the glycopeptides, such as oxidation or action of proteases.

 For the purification of the products, it is advantageous to use supports which are not very retentive and which have a high separating power. Appropriate supports are chosen from the supports used in reverse phase.

 The implementation of these provisions makes it possible to isolate glycopeptides, in pure form, from animals as small as adults of Drosophila.

 It will also be noted that this extraction technique, as described in the examples, makes it possible to isolate glycopeptides whose fragility is well known.

 The antibacterial properties imparted to the peptides of the invention by the glycosyl group are used to develop antibacterial agents which can be used in human or animal therapy, or to treat a given environment.

 The invention therefore relates to the use, for developing antibacterial agents and compositions, of peptides containing at least one amino acid substituted by a group chosen from oses, osamines, poly-oses and / or -osamines, having an activity antibacterial conferred by the glycosyl substitution group (s).

 These compositions are characterized in that they contain an effective amount of the glycopeptides defined above, in combination with an inert vehicle suitable for the intended application.

 It is advantageous to incorporate excipients such as protease inhibitors, antioxidants and bacteria multiplication inhibitors.

 The compositions or agents intended for human or veterinary therapy will advantageously be in the form of injectable solutions, or in a form for external use, such as ointments, creams, powders, or solutions.

 They are particularly indicated in the case of septicemia, or for the treatment of eyes, ears, oral and dental care and in gynecology.

 It is advantageous to use the usual dosages in these areas.

 The compositions of the invention are also of great interest in agrochemicals as phytosanitary agents. They are advantageously used in the form of powders or spreading solutions.

 The use of the antibacterial compositions and agents of the invention in the food industry makes it possible to prevent contamination by Gram-negative germs, during the manufacture of products and their conservation.

 The invention also relates to the genomes of plants as modified by the presence of genes coding for the antibacterial glycopeptides defined above. These plant cells and plants are chosen from those capable of ensuring the glycosylation of the peptide sequences so as to confer on them antibacterial activity. Plants resistant to pathogens are thus available.

 By way of illustration, other examples and advantages of the invention are reported in the examples which follow.

In these examples, reference is made to FIGS. 1 and 2 which respectively represent
FIG. 1, the variation of the absorbance as a function of time of a peptide eb according to the invention, and
- Figure 2, nucleotide sequences capable of coding for a peptide of the invention.

 Example: Obtaining antibacterial glycopeptides by extraction from Drosophila.

 - Method of induction of biological synthesis.

Drosophila are anesthetized with carbon dioxide and immunized by puncture in the thoracic region near the wing insertion using a needle immersed before each inoculation in a bacterial suspension of
Microccocus luteus (Gram positive) and Escherichia coli 1106 (Gram negative). The insects are kept at 250C for 24 hours and killed by freezing in liquid nitrogen.

- Extraction, fractionation and purification of the synthesized glycopeptides:
* extraction
The legs, wings and heads of adult Drosophila are sieved, the thorax and abdomen are ground in the presence of liquid nitrogen for 30 minutes in a mortar kept cold. To the powder thus obtained are added 10 volumes of acidified water EO, 1% of trifluoroacetic acid (TFA 0.1%) 1 containing aprotinin (total of 2 mg). After 30 minutes of extraction with stirring, the extract is centrifuged at 10,000 rpm for 30 minutes at 4 "C. The supernatant thus obtained is immediately subjected to the various stages of purification.

* Fractionation of the extract on Sep-Pak cartridges
C18
After depositing the extract on a reverse phase support, as sold in the form of cartridges under the Sep-Pak brand C8B, by Waters Millipore, the molecules of hydrophilic nature are eliminated by a simple washing with 5 ml of acidified water (TFA 0.05%).

The hydrophobic molecules are eluted with solutions of 10.40 and 80% acetonitrile in acidified water (TFA 0.05%).

 The fractions collected are called "Elution 10%", "Elution 40%" and 11Elution 80% "and concentrated in vacuo. The fractions are then reconstituted with Milli Q water before analysis by HPLC.

 * Purification by HPLC of molecules with antibacterial activity.

a- First purification step
The "Elution 10%" fraction is analyzed on an Aquapore OD 300 C18 R reverse phase column with a linear acetonitrile gradient from 0 to 40% in acidified water (TFA 0.05%) in 90 minutes (i.e. increase of 0.44% acetonitrile per minute) for a flow rate of 1 ml / min.

 The "Elution 40%" fraction is analyzed in the same column as the "Elution 10%" fraction. Elution is carried out in a linear gradient of acetonitrile from 2 to 52% in acidified water (TFA 0.05%) in 90 minutes (progression of 0.55% acetonitrile per minute) at a flow rate of 1 ml / min.

 The "Elution 80 *" fraction is analyzed on an Aquapore RP 300 C8 reverse phase column. Elution is carried out in a linear gradient of acetonitrile from 10 to 60% in acidified water (TFA 0.05%) in 90 minutes (i.e. a progression of 0.55% acetonitrile per minute) for a flow rate 1 ml / min.

 The fractions are collected according to the variation in absorption at 225 nm, taking account of the shoulders. Each fraction thus collected is attributable to a peak in optical density.

 All the fractions are evaporated to dryness under vacuum and reconstituted in Milli Q water. The antibacterial activity of each fraction is detected by the technique of the growth inhibition test in liquid medium on 10 p1 aliquots: a such fraction is equivalent to an extract of 300 adults of Drosophila.

b- Final purification
Two additional steps are necessary for the final purification of the molecules with antibacterial activity. The "Elution 10%" fractions exhibiting biological activity are analyzed on an Aquapore OD 300 C18 R reverse phase column. The elution is carried out in a linear acetonitrile gradient from O to 20% in acidified water (TFA). 0.05%) in 90 minutes (i.e. an increase of 0.22% acetonitrile per minute) for a flow rate of 0.8 ml / min.

 The fractions which exhibit antibacterial activity, measured according to the method indicated below, are purified on the column previously described in a linear gradient of acetonitrile from O to 20% in acidified water (TFA 0.05%) in 90 minutes for a flow rate of 2 ml / min.

 The variation in the optical density DO at 225 nm as a function of time is reported in FIG. 1 during the final purification step of the glycopeptide. The dotted line corresponds to the acetonitrile (in%) added for the purification. The arrow in solid line (t) indicates the purified glycopeptide and that in dotted line (<)) indicates the synthetic peptide which will be discussed below. Column (() represents the antibacterial activity.

 The molar mass measured is 2564.4 daltons.

The microsequence analysis is carried out by carrying out an automated Edman degradation of the peptide and the detection of phenylthiohydantoin derivatives on an automatic pulsed liquid sequencer (Applied
Biosystems) model 473 A or 477 A.

 The amino acid analysis is carried out on a 420 A analyzer (Applied Biosystems) with HPLC analyzer and microcomputer.

The peptide is hydrolyzed at 1200C for 24 hours with
HCl 6N in gas phase (Pico-Tag, Millipore). Derivatives are formed with phenyl isothiocyanate, then amino acids are detected at 254 nm.

The isolated and purified peptide has the amino acid sequence (II) given above, namely
NH2 - Gly - Lys - Pro - Arg - Pro - Tyr - Ser - Pro - Arg
Pro - Xaa - Ser - His - Pro - Arg - Pro - Ile - Arg - Val
COOH
The measurement of the biological activity was carried out by the following method
* Preparation of strains for the antibacterial test
For each strain, an isolated colony is withdrawn and suspended in 30 ml of PB medium (LB medium devoid of yeast extract) and incubated at 30 ° C. overnight with slow agitation. The bacteria in the exponential growth phase are reduced by dilution in fresh PB medium to a concentration of 400,000
CFU (colony forming units) / ml for E.coli and 40,000
CFU / ml for M.luteus (i.e. a D0600 = 0.001).

* Antibacterial test
The test sample (10 μl) is placed in wells of microtitration plates to which 100 μl of a bacterial culture in the exponential growth phase reduced to OD600 = 0.002 are added. After 24 hours of incubation at 25 "C., the bacterial growth is measured at 600 nm. The inhibition of growth of the bacteria, which reflects the antibacterial activity, is demonstrated by the difference in OD 600 existing between the fractions tested and a control culture in which the 10 μl of sample are replaced by 10 μl of sterile water.

 The results obtained are reported in the table below and are expressed in CFU / ml x 103.

Incubation time 1 min 1 h 3 h 5 h 24 h
Glycopeptide 1088 1026 688 600 323 antibacterial
Control experience 1,220 1,164 1,200 1,250 1,088
These results clearly demonstrate the bactericidal activity of the products of the invention.

 For comparison, synthetically prepared using a peptide synthesizer such as that indicated above, a peptide having the backbone of amino acids of sequence I, but in which the threonine in position 11, unlike to the glycopeptide of the invention, is not glycosylated.

The sequence of the synthetic peptide was verified by carrying out a degradation of Edman and by determining the molar mass using a VG mass spectrometer.
Biotech Bio Q.

 A different elution of the peptide is observed in HPLC, as shown in FIG. 1.

 The determination of the molar mass gives a value of 2199.6 daltons which differs by 364.8 daltons from that of the glypeptide of Example 1.

 In addition, this synthetic peptide, devoid of a glycosyl group, exhibits a biological activity 10,000 times less than that of the glycopeptide of Example 1.

 In order to prove that the difference between the glycopeptide and this synthetic peptide results from the glycosylation on threonine in position 11, the purified glycopeptide of example 1 was subjected to the action of anhydrous HF, which specifically hydrolyzes the glycosyl bonds.

 This treatment led mainly to a compound of 2199.6 daltons, therefore of the same mass as the unsubstituted synthetic peptide and to a compound of 2403.0 daltons. On the other hand, the same treatment applied to the synthetic peptide does not alter the latter, the molar mass of which remains unchanged.

 It will be noted that the substituent of 365 daltons separated by the treatment with anhydrous HF can be broken down into 2 units, one of 203 daltons and the other of 162 daltons, by subjecting it in turn to this same treatment, which corresponds to an N-acetyl hexosamine and to a hexose. Two molecules of water are eliminated respectively when binding N-acetylhexosamine with threonine and when binding hexose with N-acetylhexosamine.

 Example: Nucleotide sequences coding for antibacterial glycopeptides.

Isolation of cDNA Clones
using a degenerate probe 5 'TAG GGN CGN GGC TTG
CC 3 'and from 30,000 phages containing the corresponding insert, a screening of a Drosophila cDNA library is carried out (the development of this library is reported below).

 30 positive hybridization clones are obtained.

Sequencing
Ten of these clones are sequenced. It can be seen that the inserts all contain an open reading frame of 65 codons starting with a methionine. Inserts 1 to 9 do not show any variation in sequence. The longest is shown in Figure 2. The insert 10 shows variations of bases which are shown in Figure 2.

The AATAAA consensus polyadenylation signal is indicated in bold. The first line of the nucleotide sequence corresponds to those of clones 1 to 9, the second line mentions the bases different from clone 10, the other bases being identical to those given on the first line are not indicated.

 The deduced amino acid sequence corresponds to a precursor of the peptide sequence of the glycopeptide of Example 1 and has the sequence I given above.

This sequence I comprises the sequence II of 19 amino acids and in the N-terminal part a potential peptide signal (motifs 1 to 19) and a Thr-Pro dipeptide (in positions 20 and 21). The C-terminal part comprises a peptide of 22 amino acids (occupying positions 43 to 64 in sequence I) separated from sequence II by a dibasic Arg-Arg cleavage site (in positions 41 and 42).

Preparation of the cDNA library
A cDNA library of selected size is prepared by subjecting Drosophila larvae to the third larval stage to a bacterial challenge. RNA enriched in poly (A) is extracted as described by Reichhart et al in
EMBO J. 11, 1469-1477 (1992). A bank> gt 22 is constructed using the Superscript lambda system marketed by Gibco BRL. The cDNAs are subcloned into Mt3mpl9. The two strands are sequenced using the Sanger dideoxy method with the sequencing kit sold under the brand Sequanase R by USB.

 Example 3: Obtaining antibacterial glycopeptides by expression from Saccharomices cerevisae.

 By operating according to conventional techniques of genetic engineering, a nucleotide sequence of a cDNA clone of Example 2 is inserted into a vector and expression is carried out in Saccharomices cerevisae.

 The glycopeptide produced is recovered and analyzed to verify its sequence.

Example: Obtaining Antibacterial Glycopeptides of the Type Induced by Synthesis
Phormia.

 A threonine is reacted with a Nacetylgalactosamine. The hydroxyl groups of galactosamine are blocked using protective groups usually used for this purpose. Benzoyl chloride is used, for example, to protect the hydroxyl groups with a benzoyl radical.

 The amino acid sequence III given above is carried out using a peptide synthesizer, as used in Example 1.

 The analysis by mass spectrometry of the product obtained and the degradation of Edman make it possible to verify that the glycopeptide sought has indeed been synthesized.

 Example 5 Formulation usable to treat septicemia.

Peptide of Example 1: 10 to 100 mg
Excipient: physiological water
The formulation is administered by injection 4 times a day, for a week.

Claims (26)

 1 / Glycopeptides, characterized in that they have antibacterial properties conferred by an amino acid sequence containing at least one amino acid substituted by a glycosyl group chosen from oses, osamines, poly-oses and / or -osamines.  2 / Glycopeptides according to claim 1, characterized in that the substituted amino acid is a hydroxylated amino acid such as threonine, serine or tyrosine.  3 / Glycopeptides according to claim 1 or 2, characterized in that the glycosyl group is linear or cyclic, substituted or not, in a or p form, advantageously comprising 5 or 6 carbon atoms, the substituents optionally present being chosen in particular from alkyl radicals of 1 to 4 carbon atoms or, in particular, for osamines, among the acyl radicals, more especially acetyl.  4 / Glycopeptides according to claim 3, characterized in that the glycosyl group is chosen from hexoses such as galactose or glucose, hexosamines such as galactosamine and glucosamine, or hexosamines substituted by hexose units such as (N -acetylhexosamine) -hexose.  5 / Glycopeptides according to one of claims 1 to 4, characterized in that they are of the type of peptides as obtained in arthropods, and in particular in larvae or adults of insects by a process comprising  - the induction of their synthesis in particular by injection of bacteria in sufficient doses or by septic injury or other trauma,  - the extraction in order to collect the peptides having undergone post-translational glycosylation and, if necessary,  - fractionation of the isolated extract in order to selectively separate the glycopeptides according to the level of their antibacterial activity.  6 / Glycopeptides, according to one of claims 1 to 5, characterized in that they exhibit activity against Gram-negative germs.  7 / Glycopeptides according to one of claims 1 to 6, characterized in that they are of the type of those developed by the dipterans, in particular in brachycera.  8 / Glycopeptides according to claim 7, characterized in that their peptide sequence responsible for their antibacterial activity, which comprises at least one substituted amino acid as defined in claim 1, is contained in a domain comprising at least approximately 30% of proline groups .  9 / Glycopeptides according to claim 8, characterized in that said sequence comprises at least one motif of proline-threonine / serine-Xaal- O-glycosylation Xaa2-proline in which the Xaal and Xaa2 motifs, identical or different, are variable amino acids.  10 / Glycopeptides according to claim 8 or 9, characterized in that said sequence comprises at least one hydrophobic amino acid such as threonine, substituted by a glycosyl group, in particular by an N-acetyl hexosamine group or (N-acetylhexosamine) - hexose.  11 / Glycopeptides according to one of claims 7 to 10, as inducible in Drosophila.  12 / Glycopeptides according to claim 11, characterized in that they comprise or that they consist of an amino acid sequence corresponding to the following sequence (I) MKFT IVFLLLACVFAMAVATPGKPRPYSPRPlSHPRP IRVRREALAI EDHLAQAAIRPP PILPA more particularly to the sequence (II) GKPRPYSPRPTSHPRPIRV  the underlined threonine units corresponding to those substituted by a glycosyl group.  13 / Glycopeptides according to one of claims 7 to 10, as inducible in Phormia terranovae.  14 / Glycopeptides according to claim 13, characterized in that they comprise or that they consist of an amino acid sequence, as determined according to the degradation of Edman, corresponding to the sequence (III) DEKPKLILPTPAPPNLPOLVGGGGGNRKDG FGVSVDAHQKVWTSDNGGHSIGVSPGYSQH LPGPYGNSRPDYRIGAGYSYNE threonine in position 10 and / or that in position 54, being substituted (s) by a glycosyl group or that their N-terminal part comprises or consists of at least part of the following sequences (IV) and (V) D E K P K L V L P (S) X A P P N L P Q L V G G G G G N N K X GXXVS INAAQKV or D E K (P) K L I X P X X A (P) X N L X Q L V G G G G G N N K KXXGVXVXXAO  15 / Glycopeptides according to one of claims 7 to 10, as inducible in hymenoptera, in particular in bees.  16 / The fragments, mutants and functional derivatives of the glycopeptides according to one of claims 1 to 15.  17 / Nucleotide sequences comprising the genetic information corresponding to the amino acids of the glycopeptides according to one of claims 1 to 16, the sequences capable of hybridizing with these under stringent conditions, the complementary sequences and the corresponding RNAs.  18 / Expression and / or cloning vectors comprising at least one fragment of the nucleotide sequences according to claim 17 and hosts transformed by these vectors.  19 / The expression products of the vectors according to claim 18.  20 / A process for obtaining glycopeptides according to one of claims 1 to 16, characterized in that one proceeds to the chain of amino acids by synthesis, at least one of the amino acids used being substituted as defined in claim 1 and being introduced so as to occupy the desired position in the sequence.  21 / Antibacterial agents characterized in that they contain at least one glycopeptide according to one of claims 1 to 16.  22 / Antibacterial compositions, characterized in that they contain an effective amount of glycopeptides according to one of claims 1 to 16, in association with an inert vehicle.  23 / Compositions according to claim 22, characterized in that they are used in human or veterinary therapy, the inert vehicle being suitable for this use.  24 / Compositions according to claim 22, characterized in that they are used in agrochemistry, the inert vehicle being suitable for this use.  25 / Compositions according to claim 22, characterized in that they are used in the food industry.  26 / Plant cells and plants whose genome is modified by the presence of genes capable of coding for the peptide sequences of the glycopeptides according to one of claims 1 to 16, these plant cells and plants being capable of ensuring the glycosylation of the peptide sequences so as to give them antibacterial activity.