Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/50—Isolated enzymes; Isolated proteins
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
  • A01N63/10—Animals; Substances produced thereby or obtained therefrom
  • A01N63/14—Insects
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4723—Cationic antimicrobial peptides, e.g. defensins

Definitions

• the present invention relates to the phytosanitary field.
• it relates to means for combating fungi of the Fusarium genus, in particular the biocontrol or phytosanitary control of contamination by category B trichothecene type mycotoxins produced during Fusarium wilt.
• Fusarium graminearum is a major causative agent of Fusarium wilt of wheat and Fusarium wilt of corn. These two fungal diseases have a significant economic impact in many grain-producing regions around the world.
• F. graminearum can produce mycotoxins, mainly category B trichothecenes or TCTBs (the major representatives of which are deoxynivalenol / DON and its acetylated forms 15 and 3 acetyldeoxynivalenol / 15 and 3-ADON), toxic to humans. and animals. Indeed, these toxins have a proven acute toxicity at the origin of serious food poisoning sometimes fatal and a very strongly suspected chronic toxicity (hematotoxicity and immunotoxicity).
• thermostable molecules As thermostable molecules, TCTBs, accumulated in harvested grains, are not completely eliminated during the manufacture of food and feed from cereals.
• the agronomic practices recommended to limit the contamination of crops do not guarantee their compliance with the regulatory thresholds set by the EU (N ° 1126/2007). It is therefore urgent to put in place new pre-harvest control strategies to limit the accumulation of TCTBs in grains, which are sustainable and respectful of the environment.
• Defensins are low molecular weight cyclic peptides with a conserved motif called gamma-core, important for their antimicrobial activity.
• tick defensins called DefMT3 and DefMT6 have been described as having antibacterial and antifungal activity, in particular against Fusarium culmorum and Fusarium graminearum (Tonk et al, 2015, Developmental and Comparative Immunology, 53, 358 - 365).
• the present invention describes a method for reducing the production of mycotoxins by fungi of the genus Fusarium, in particular category B trichothecenes (TCTB).
• TCTB trichothecenes
• the inventors have identified the capacity of certain peptides to inhibit the production of these mycotoxins. They observed that the DefMT3 peptide but also a peptide carrying the conserved gamma motif of DefMT3 or variants thereof severely inhibited the production of the DON (Deoxynivalenol) and 15-ADON (15-Acetyldéoxynivalenol) toxins by Fusarium graminearum. They have shown that the linear peptide forms exhibit more potent inhibitory activity on the production of mycotoxins than the cyclic forms. In addition, the linear forms exhibit fungal growth inhibiting activity which is almost not present for the cyclic forms. It has also been shown that the cationic charge of peptides was an important element for their antifungal activity and inhibitory of TCTBs biosynthesis. Finally, the inventors have shown that the PEGylated forms exhibited improved antifungal activity.
• the present invention relates to the use of a peptide for inhibiting the production of mycotoxins by fungi of the Fusarium genus on a plant, the peptide comprising a sequence chosen from among
• It also relates to a method for inhibiting the production of mycotoxins by fungi of the Fusarium genus on a plant comprising bringing the plant into contact with a composition comprising a peptide, the peptide comprising a sequence chosen from among
• the peptide comprises at most 30 amino acids.
• the peptide comprises a sequence chosen from the sequences SEQ ID Nos: 22 and 23, consists of a sequence chosen from the sequences SEQ. ID Nos: 22 and 23 with the sequence possibly comprising 1, 2, 3, 4 or 5 substitutions, additions, deletions or mixture thereof, or consists of a sequence chosen from the sequences SEQ ID Nos: 22 and 23.
• the peptide consists of the sequence SEQ ID NO: 22.
• the peptide implemented in this use or this method can comprise, consist essentially of or consist of a sequence chosen from among
• phytosanitary composition intended for the treatment of a plant comprising a peptide, in particular for inhibiting the production of mycotoxins by fungi of the Fusarium genus on the plant, the peptide comprising a sequence chosen from among
• the peptide of the phytosanitary composition can comprise, consist essentially of or consist of a sequence chosen from
• the peptide comprises at most 20 amino acids.
• the peptide is PEGylated.
• the peptide does not include a disulfide bridge.
• the mycotoxin is Class B trichothecene, preferably deoxynivalenol (DON) and / or acetylated deoxynivalenol (A-DON).
• the fungus of the genus Fusarium is chosen from Fusarium culmorum, Fusarium graminearum, Fusarium tricinctum, Fusarium avenaceum, Fusarium poae, Fusarium sporotrichioides, Fusarium verticillioides, Fusarium proliferatum , Fusarium langsethiae, Fusarium oxysporum, Fusarium roseum, Fusarium arthrosporioides, and Fusarium avenaceum, preferably Fusarium culmorum or Fusarium graminearum_.
• the plant is selected from cereals, such as wheat (soft and hard), barley, corn, oats, triticale and rice, or fruits and vegetables such as tomato, melon, cucumber, zucchini, Jerusalem artichoke, chili, potato, asparagus, sweet potato, celery, garlic, onion , cabbage, ginger, banana, cassava, vanilla, and fruit trees such as date palm, preferably cereals chosen from wheat (soft and hard), barley, corn, oats, triticale and rice.
• cereals such as wheat (soft and hard), barley, corn, oats, triticale and rice.
• peptide oligopeptide
• polypeptide refers to a chain of amino acids linked by peptide bonds, regardless of the number of amino acid residues constituting this chain.
• amino acid refers to the 20 naturally occurring standard amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E, D, S and T), with rare natural amino acid residues (for example hydroxyproline, hydroxylysine, allohydroxylysine, 6-N-methylysine, N-ethylglycine, N-methylglycine, N-ethylasparagine, allo-isoleucine, N-methylisoleucine, N-methylvaline, aminobutyric acid) and unnatural amino acids (for example norleucine, norvaline and cyclohexyl-alanine ).
• this term refers to the 20 naturally occurring standard amino acid residues (G, P, A, V, L, I, M, C, F, Y, W, H, K, R, Q, N, E , D, S and T).
• substitution refers to the replacement of one amino acid residue with one selected from among 20 naturally occurring standard amino acid residues, rare naturally occurring amino acid residues and amino acid residues. unnatural.
• substitution refers to the replacement of an amino acid residue by another chosen from among the 20 naturally occurring standard amino acid residues (G, P, A, V, L, I, M, C , F, Y, W, H, K, R, Q, N, E, D, S and T).
• the substitution (s) can be conservative or non-conservative substitutions.
• conservative substitution refers to a substitution of one amino acid residue for another which exhibits similar chemical or physical properties (size, charge or polarity). Examples of conservative substitutions are shown in the following tables.
• the peptide may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at the level of the N- and / or C-terminal, preferably 1, 2, 3, 4 or 5 additional amino acids, and / or 1, 2 or 3 additional amino acids.
• the number of substitutions, additions, deletions or a mixture thereof depends on the length of the sequence.
• the percentage of substitutions, deletions, additions or a mixture thereof may not exceed 30%, preferably not more than 25%.
• substitution refers to the exchange of a single amino acid for another in a sequence.
• the term “deletion” refers to the removal of a single amino acid in a peptide sequence
• the term “insertion” or “addition” is equivalent and refers to the addition of a single amino acid in a peptide sequence.
• sequence identity refers to the number (%) of matches (identical amino acid residues) at positions resulting from an alignment of two polypeptide sequences. Sequence identity is determined by comparing the sequences when aligned so as to maximize overlap and identity while minimizing sequence interruptions. In particular, the sequence identity can be determined using any of a number of global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (eg Needleman & Wunsch, J. Mol.
• Biol 48: 443, 1970 which optimally align the sequences along the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm, for example the Smith and Waterman algorithm (Smith and Waterman, Adv. Appl. Math. 2: 482, 1981) or the AltschuI algorithm. (Altschul et al (1997) Nucleic Acids Res. 25: 3389-3402; Altschul et al (2005) FEBS J. 272: 5101-5109). Alignment for the purpose of determining the percentage identity of amino acid sequence can be carried out by any method known to those skilled in the art, for example by using software available on Internet sites such as http: // blast.ncbi.nlm.
• the inventors have identified peptides exhibiting an activity which inhibits the production of mycotoxins, in particular of category B trichothecenes, by a fungus of the genus Fusarium.
• the inhibitory activity of the production of mycotoxins, in particular of category B trichothecenes can be measured by any technique known to those skilled in the art. For example, it can be measured as described in detail in the examples in the section “Mycotoxin production inhibition test” by measuring the amounts of DON and 15-ADON with the Fusarium graminearum CBS 185.32 strain.
• a peptide exhibits inhibitory activity on the production of mycotoxins when this production is reduced by 20, 30, 40, 50, 60, 70, 80, 90 or 100% relative to the production in the absence of the peptide.
• the peptide exhibiting an activity for inhibiting the production of mycotoxin by a fungus of the genus Fusarium comprises, consists essentially of or consists of
• C / S indicates a cysteine (C) or a serine (S), the cysteine possibly being modified so as to prevent the formation of a disulfide bond;
• Xi is a small amino acid, for example chosen from G, S, A, V, T, D, N and P, in particular G, S or A, more particularly G or S;
• X2 is a small amino acid, for example chosen from G, S, A, V, T, D, N and P, in particular G, S, A or N, more particularly still G or N;
• X 3 is chosen from I, L, F, Y, W, M, R or K, in particular I, L, F or Y, more particularly I or F;
• X4 is chosen from I, L, F, Y, W, or M, in particular I, L, W or F, more particularly I or L;
• Xs is chosen from K, R, H, T, S, or Q., in particular K, R, S or T, more particularly still K or T, K, R or T, or K or R;
• Ce is chosen from R, K, L, I, Q. or T, in particular R, K, Q. or T, more particularly still Q, R or T, K, R or T, or K or R;
• X7 is chosen from I, L, M, T or S, in particular I, L or T, more particularly I or T;
• Xs is chosen from V, I, L, Y, F, W or M, in particular V, I, L or Y, more particularly still V or Y;
• Xg is chosen from R, K, H, T, S, N, M or 3 ⁇ 4 in particular R, K, T or M, more particularly R, K or T, preferably K or R; and Xio is chosen from S, T, Q, K, R, N or H, in particular K, R, N or T, more particularly still K or T, K, R or T, or K or R.
• the peptide comprising at most 30 amino acids.
• cysteines when the peptide comprises at least 2 cysteines, these cysteines are in reduced form (SH), that is to say that they do not together form disulfur bridges.
• the peptide comprises serines when the sequence indicates C / S.
• the peptide comprises one or more cysteines modified so as to prevent disulfide bonding.
• the cysteines can be alkylated on the thiol function, in particular methylated (S-CH3), or by carboxyamidomethylation (addition of a CH2-CONH2 group).
• S-CH3 methylated
• carboxyamidomethylation addition of a CH2-CONH2 group.
• all of the cysteines of the peptide or all of the cysteines except one are modified or replaced by cysteines.
• the peptide can comprise a sequence identity with the peptide of SEQ ID NO: 2 or 3 of at least 50, 60, 70, 80, 90 or 95%.
• the peptide can comprise 1, 2, 3, 4, or 5 substitutions relative to the peptide of SEQ. ID NO: 2 or 3, in particular at the level of the positions of the residues chosen from Xi, X2, X3, X4, Xs, Cd, X7, Xs, X9, and X10.
• the peptide comprises, consists essentially of or consists of a sequence
• the peptide comprises, consists essentially of or consists of a sequence chosen from the group consisting of
• TickCore3 CGNFLKRTCICVKK SEQ ID NO: 2
• TickCore6 CSGIIKQTCTCYRK SEQ ID NO: 3
• TC3K6T CGNFLTRTCICVKK SEQ ID NO: 6
• TC3R7T CGNFLKTTCICVKK SEQ ID NO: 7
• TC3K13T CGNFLKRTCICVTK
• TickCore3 C G N F L K R T C I C V K K SEQ ID NO: 2
• TickCore6 C S G I I K Q T C T C Y R K SEQ ID NO: 3
• TC3K6T C G N F L T R T C I C V K K SEQ ID NO: 6
• TC3R7T C G N F L K T T C I C V K K SEQ ID NO: 7
• TC3K13T C G N F L K R T C I C V T K SEQ ID NO: 8 and TC3K14T: C G N F L K R T C I C V K T SEQ ID NO: 9; or in the group consisting of
• TickCore3 C G N F L K R T C I C V K K SEQ ID NO: 2
• TickCore6 C S G I I K Q T C T C Y R K SEQ ID NO: 3 TC3R7T: C G N F L K T T C I C V K K SEQ ID NO: 7 TC3K13T: C G N F L K R T SEQ C T C T C Y R K: SEQ ID NO: 3 TC3R7T: C G N F L K T T C I C V K K SEQ ID NO: 7 TC3K13T: C G N F L K R T SEQ T C I C V K SEQ ID NO: 7 TC3K13T: C G N F L K R T SEQ T C I C V T K: C C T 8 and SEQ C I C V T: C K 8 and SEQ C N C T14: C K 8
• the peptide may also comprise, consist essentially of or consist of a sequence selected from the group consisting of
• the peptide is not or does not comprise the sequence of a peptide chosen from among
• the peptide molecule can further comprise other peptide sequences, on the N-terminal or C-terminal side.
• the peptide can comprise, at the N-terminal or C-terminal, a tag (or label) useful for the purification or the immobilization of the peptide.
• tags are well known to those skilled in the art and include for example the tags histidine (His6), FLAG, HA (epitope derived from the hemagglutinin of the influenza virus), MYC (epitope derived from the human proto-oncoprotein MYC) or GST (glutathione-S-transferase).
• the peptide can comprise a cleavage site by a protease or a chemical agent making it possible to delete this tag.
• the peptide is less than 30 amino acids in length, preferably less than 25, 20, 19, 18, 17, 16 or 15 amino acids.
• the peptide is not cyclic. Thus, it does not contain a covalent interaction between the side chains of two amino acids or between the ends
• the peptide bond (s) of the peptide can be modified to make them resistant to proteolysis.
• all peptide bonds can be replaced.
• the peptide molecule can comprise either a carboxylic (-COO-) or an amidated (-CONH2) C-terminus.
• the C-terminus can also be esterified.
• the peptide can also be optionally modified at its N-terminus, for example with an acetyl radical.
• the peptide can be PEGylated (polyethylene glycol), partly at its N-terminal end.
• the peptide can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more PEG groups.
• the L and D isomers of amino acids are contemplated.
• the D isomers are not sensitive to proteases and the present invention also comprises molecules comprising D amino acids, in particular molecules comprising only or essentially D amino acids.
• the L amino acids are preferred.
• the peptide can comprise only D amino acids and be in a retro-inverted configuration (same sequence but inverted).
• the present invention relates to a novel peptide exhibiting an activity which inhibits the production of mycotoxin by a fungus of the genus Fusarium.
• a new peptide according to the present invention is a peptide as defined above but is not an unmodified peptide of sequence
• a new peptide can be a peptide of sequence SEQ ID NO: 2 or 3 which carries a PEGylation, a C-terminal amidation, an N-terminal acetylation, a modified peptide bond, an amino acid of form D, a modified cysteine or a combination of these modifications.
• the peptide comprises a sequence chosen from the sequences SEQ ID Nos: 22 and 23, consists of a sequence chosen from the sequences SEQ ID Nos: 22 and 23 with the sequence possibly comprising 1, 2, 3, 4 or 5 substitutions, additions, deletions or mixture thereof, or consists of a sequence chosen from the sequences SEQ ID Nos: 22 and 23.
• the peptide can consist of the sequence SEQ. ID NO: 22.
• the present invention relates to a composition
• a composition comprising a novel peptide as described in the present application or a mixture thereof, the composition preferably being a phytosanitary composition, in particular for combating a fungus of the genus Fusarium, and more particularly against the production of mycotoxins.
• the present invention also relates to the use of a peptide as described in the present application or a mixture thereof for inhibiting the production of mycotoxins by fungi of the genus Fusarium on a plant.
• It finally relates to a method for inhibiting the production of mycotoxins by fungi of the Fusarium genus on a plant comprising bringing the plant into contact with a composition comprising a peptide as described in the present application or a mixture thereof. .
• the fungus of the Fusarium genus can in particular be chosen from Fusarium culmorum, Fusarium graminearum, Fusarium tricinctum, Fusarium avenaceum, Fusarium poae, Fusarium sporotrichioides, Fusarium verticillioides, Fusarium proliferatum, Fusarium langsethiae, Fusarium oxysporum, Fusarium roseum, Fusarium, Fusarium avenacarium, preferably from Fusarium culmorum, Fusarium graminearum, Fusarium tricinctum, Fusarium avenaceum, Fusarium poae, Fusarium sporotrichioides, Fusarium verticillioides, Fusarium proliferatum, Fusarium langsethiae and Fusarium oxysporum.
• the plants concerned by this treatment can be cereals, such as wheat (soft and hard), barley, corn, oats, triticale and rice, but also fruits and vegetables such as tomatoes, melons, cucumber, zucchini, Jerusalem artichoke, chili, potato, asparagus, sweet potato, celery, garlic, onion, cabbage, ginger, banana, cassava, vanilla , and fruit trees like the date palm.
• cereals such as wheat (soft and hard), barley, corn, oats, triticale and rice, but also fruits and vegetables such as tomatoes, melons, cucumber, zucchini, Jerusalem artichoke, chili, potato, asparagus, sweet potato, celery, garlic, onion, cabbage, ginger, banana, cassava, vanilla , and fruit trees like the date palm.
• the mycotoxins are in particular category B trichothecenes.
• DON deoxynivalenol
• A- DON acetylated deoxynivalenol
• 3-acetyl-DON 3-acetyl-DON
• 15-acetyl-DON 15-acetyl-DON
• the phytosanitary composition or the peptide is applied to the aerial part of the plant. Preferably, it is applied at the time of flowering or before flowering of the plant. Alternatively, she / he is applied at the time of grain formation.
• the phytosanitary composition or the peptide can be used at a concentration where it has an inhibitory effect on the production of mycotoxins by fungi of the genus Fusarium and has little or no effect on the production of mycotoxins. growth of fungi of the genus Fusarium.
• the concentration will be chosen so as to obtain an optimal effect on the production of mycotoxins.
• the concentration of peptides may for example be less than 100 mM, 75 pM, 50 pM, 25 pM or 15 pM.
• the phytosanitary composition or the peptide can be used at a concentration where it has an effect on the production of mycotoxins by fungi of the genus Fusarium and also has an inhibitory effect on the growth of the fungi of the genus. genus Fusarium.
• the peptide is preferably a new peptide as defined in the present application.
• FIG 1 Antifungal and anti-mycotoxin activity of TickCore3, native and oxidized form. After 10 days of incubation in media supplemented or not with TickCore3 (native form) or TickCore30x (oxidized forms), the mycelia of F. graminearum were separated and weighed. The weight values of the mycelium are presented in grams (g) (Figl a). The TCTB-type mycotoxins, DON (Fig lb) and 15-ADON (Fig le), were quantified and expressed in ⁇ g of mycotoxins per ml of medium, then divided by the weight of the mycelium and expressed in ⁇ g of TCTB mycotoxins by g of dry mycelium biomass (pg / g).
• FIG 2 Antifungal and antimycotoxin activity of linear and cyclic TickCore3 peptides. After 10 days of incubation in media supplemented or not with TickCore3 CH3-1 Ox, TickCore3 CH3-2 Ox, TickCore3 CH3-3 Ox or TickCore3 CH3-123, the mycelia of F. graminearum were separated and weighed. Mycelium weight values are presented in grams (g) (Fig 2a).
• TCTB-type mycotoxins DON (Fig 2b) and 15-ADON (Fig 2c), were quantified by mg of mycotoxins per ml of medium, then divided by the weight of the mycelium and expressed as pg of TCTB mycotoxins per g of dry mycelium biomass (pg / g).
• FIG 3 Antifungal and “anti-mycotoxin” activity of Tickcore3, native form or substituted forms in which the basic amino acids (K in position 6, 13 and 14, and R in position 7) have been replaced by T, a uncharged amino acid.
• TCTB-type mycotoxins DON (Fig 3b) and 15-ADON (Fig 3c), were quantified in ⁇ g of mycotoxins per ml of medium, then divided by the weight of the mycelium and expressed in ⁇ g of TCTB mycotoxins per g of dry mycelium biomass (pg / g). Three different concentrations of peptide (12.5 mM, 25 mM and 50 mM) were tested.
• FIG 4 Antifungal and antimycotoxin activity of TickCore3 peptide and of a tickCore3-PEG pegylated version. After 10 days of incubation in media supplemented or not with TickCore3 or TickCore3-PEG, the mycelia of F. graminearum were separated by centrifugation and weighed. The weight values of the mycelium are presented in grams (g) (Fig 4a).
• TCTB-type mycotoxins DON (Fig 4b) and 15-ADON (Fig 4c), were quantified in ⁇ g of mycotoxins per ml of medium, then divided by the weight of the mycelium and expressed in ⁇ g of TCTB mycotoxins per g of dry mycelium biomass (pg / g).
• FIG 5 Antimycotoxin activity of tick defensin DefMT3. After 10 days of incubation in media supplemented or not with DefMT3, the mycotoxins of type TCTB, DON (Fig 5A) and 15-ADON (Fig 5B), were quantified in pg of mycotoxins per ml of medium, then divided by the weight of the mycelium and expressed in pg of TCTB mycotoxins per g of dry mycelium biomass (pg / g). Two concentrations of DefMT3 were tested, 25 and 50 mM.
• TickCore3 the conserved linear gamma core motif, with reduced cysteine (Cys) residues, of the tick defensin DefMT3, here called “TickCore3”, inhibits the production of category B trichothecenes (TCTB) by F. graminearum. . Alkylation of all Cys residues of TickCore3 with methyl groups (TickCore3-CH3) had little effect on the inhibitory effect affecting TCTB production. Conversely, the oxidation of all Cys residues of TickCore3 greatly reduced the inhibitory effect on TCTB production. A loss of Major inhibitory function was observed when the specific disulfide bonds Cys4-Cys6 and Cys4-Cys5 were formed. The inventors have also shown that the cationic charge of the peptide is an important factor in its biological activity. Substitution of the positively charged amino acids with a neutral residue led to a sometimes very significant reduction in antifungal activity and inhibition of TCTB production.
• TickCore3 is a very effective inhibitor of TCTB production by F. graminearum.
• FIG. 1 shows the results obtained after 10 days of culture of the F. graminearum CBS 185.32 strain in MS medium supplemented with TickCore3 and its oxidized variant (TickCore3 Ox) at three different concentrations (12.5, 25 and 50 mM).
• the untreated (control) culture media were not supplemented with any of the TickCore3 peptides.
• TickCore3 Ox had no effect on dry fungal biomass, TickCore3 significantly inhibited fungal growth at all concentrations tested compared to the control condition (Fig. La).
• TickCore3 may have effects on the inhibition of TCTB production.
• peptides derived from TickCore3 were synthesized by alkylating each Cys residue individually with a methyl group (CH3), followed by an oxidation protocol which generated peptides with disulfide bridges established between Cys5-Cys6 (TickCore3 CH3-1 Ox), Cys4-Cys6 (TickCore3 CH3-2 Ox) and Cys4- Cys5 (TickCore3 CH3-3 Ox).
• TickCore3-CH3-1 Ox, TickCore3-CH3-2 Ox and TickCore3-CH3-3 Ox had no effect on fungal growth at any of the concentrations tested, compared to the control (Fig. 2a).
• the production of mycotoxins by F. graminearum was then measured in the media supplemented with TickCore3-CH3-1 Ox, TickCore3-CH3-2 Ox and TickCore3-CH3-3 Ox at three different concentrations: 12.5, 25 and 50 mM. (figure 2b).
• the increase in the concentrations of TickCore3-CH3-2 Ox and TickCore3-CH3-3 Ox resulted in a significant increase in the production of DON (Fig. 2b) and 15-ADON (Fig.
• TickCore3-CH3-2 Ox for which the production of 15-ADON did not change significantly.
• treatment with 50 mM PickCore3-CH3-1 Ox resulted in a significant reduction in the levels of DON and 15-ADON (Fig. 2bc).
• the cationic charge is a key element of the antifungal and "anti-mycotoxin" activity of.
• TickCore 3 The inventors hypothesized that the cationic charge of TickCore 3 was an important element of its biological activity.
• substituted peptides of TickCore3 were synthesized by substituting the so-called basic amino acids (arginine and lysine, R and K) by an uncharged residue, threonine (T).
• TickCore3-K6T substitution of lysine in position 6 by a threonine residue
• TickCore3-R7T substitution of arginine in position 7 by a threonine residue
• Tickcore3-K13T substitution of lysine at position 13 by a threonine residue
• TickCore3-K14T substitution of lysine at position 14 by a threonine residue
• the substituted forms exhibit strong antifungal activity only at 50mM while TickCore3-K6T has no antifungal activity below the concentration at 50mM.
• the production of mycotoxins by F. graminearum was then measured in media supplemented with TickCore3, TickCore3-K13T, TickCore3-K6T, TickCore3-K14T and TickCore3-R7T at three different concentrations: 12.5, 25 and 50 mM ( Figures 3b and 3c).
• TickCore3 pegylation increases antifungal activity
• TickCore3-PEG peptide was synthesized in the same way as TickCore3, except that two PEG units were sequentially attached to the N-terminus of the peptide.
• TickCore3-PEG at 50 mM showed a greater antifungal effect with a 3.3-fold decrease in fungal growth (Fig 4a).
• TickCore3-PEG would also exhibit improved inhibitory activity on mycotoxin production (Fig 4bc).
• Defensin DefMT3 is able to inhibit the production of mycotoxins.
• the DefMT3 peptide was synthesized in the same way as TickCore3. This peptide exhibits inhibitory activity on the production of DON and 15-ADON mycotoxins (Figs 5a and 5b).
• TickCore3 TickCore3 (CGNFLKRTCRTCICVKKK, SEQ ID NO: 2) is the conserved gamma core motif of DefMT3 (GenBank accession number: JAA71488; Tonk et al., 2015).
• the peptide synthesis was commissioned from Pepmic (Suzhou, China) which uses the solid phase peptide synthesis (SPPS) to obtain peptides with a high degree of purity as described previously in Cabezas-Cruz et al., 2016, (Front Microbiol., 7, 1682).
• SPPS solid phase peptide synthesis
• peptide synthesis was carried out using a 2-chlorotrityl chloride resin as a solid support, and a labile 9-fluorenyl-methyloxy-carbonyl (Fmoc) base as a protecting group.
• Peptides purified by reverse phase HPLC were then optionally oxidized using refolding buffer containing 1 M urea, 100 mM Tris (pH 8.0), 1.5 mM oxidized glutathione, 0.75 mM reduced glutathione and 10 mM methionine.
• Oxidation of Cys residues has been confirmed by Ellman's reaction and the formation of disulfide bonds was characterized by electrospray mass spectroscopy (ESI-MS) using an LCMS-2020 mass spectrometer (Shimadzu, Kyoto, Kyoto Prefecture, Japan).
• the composition of the sequence was also verified by ESI-MS using an LCMS-2020 mass spectrometer (Shimadzu, Kyoto, Kyoto Prefecture, Japan).
• the Peptides TickCore3-CH3-123 (C (CH3) GNFLKRTC (CH3) IC (CH3) VKK), TickCore3-CH3-l (C (CH3) GNFLKRTCRICVKK), TickCore3-CH3-2 (CGNFLKRTC (CH3) ICVKK) and TickCore3 -CH3-3 (CGNFLKRTCIC (CH3) VKK) were synthesized as described previously, with the difference that Fmoc-Cys (Me) -OH was added to replace Fmoc-Cys (Trt) -OH. Alkylation of the sulfur (S) thiol groups of all Cys residues prevents cyclization of these residues (i.e.
• TickCore3-PEG peptide was synthesized as TickCore3 but two PEG units were sequentially attached to the N terminal end of the peptide using protected groups Fmoc-NH-PEG2-CH2CH2COOH.
• the DefMT3 peptide exhibiting the sequence GGYYCPFRQDKCHRHCRSFGRKAGYCGNFLKRTCICVKK (SEQ. I D NO: 22) was synthesized according to a protocol identical to that of TickCore3 by the company Pepmic (Suzhou, China).
• the F. graminearum CBS 185.32 strain which produces DON and 15-ADON was used throughout this study.
• the fungal culture was maintained at 4 ° C. on dextrosed potato agars (PDA) (Difco, Le Ponts de Claix, France) in inclined tubes, under mineral oil.
• PDA dextrosed potato agars
• the strain was grown at 25 ° C in the dark on tilted PDA agars for 7 days and the spore suspension was prepared by adding 6 mL of sterile distilled water to the PDA agar. tilted with a gentle agitation.
• TCTBs were quantified by HPLC-DAD using an Agilent Technologies 1100 Series Liquid Chromatograph equipped with an automatic sampling system, an Agilent Diode Array Detector (DAD) and software. management of ChemStation chromatography (Agilent, France).

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