METHODS FOR INHIBITING FUSARIUM MYCOTOXIN PRODUCTION

Abstract

The present invention relates to peptides for inhibiting the production of mycotoxins by fungi of the genus Fusarium and also to compositions comprising them and methods implementing them.

Claims

1-14. (canceled)

15. A method for inhibiting the production of mycotoxins by fungi of the genus Fusarium on a plant, involving placing the plant in contact with a composition comprising a peptide, the peptide comprising a sequence selected from: TABLE-US-00019 (SEQ ID NO: 18) C/S-G/S-N/G-F/R/I-L/W/I-K/T-R/L/Q/T-T-C/S-I/T-C/ S-V/I/Y-K/M/R/T-K/N/T; (SEQ ID NO: 19) C/S-G/S-N/G-F/I-L/I-K/T-R/Q/T-T-C/S-I/T-C/S-V/Y-K/ R/T-K/T; (SEQ ID NO: 20) C/S-G/S-N/G-F/I-L/I-K/T-R/T-T-C/S-I/T-C/S-V/Y-K/ T-K/T; and (SEQ ID NO: 21) C/S-G/S-N/G-F/I-L/I-K/R-R/K-T-C/S-ET-C/S-V/Y-K/ R-K/R.

16. The method according to claim 15, wherein the peptide comprises not more than 20 or 30 amino acids.

17. The method according to claim 15, wherein the peptide comprises a sequence selected from the following sequences with the sequence optionally comprising 1, 2, 3, 4 or 5 substitutions, additions, deletions or a mixture thereof: TABLE-US-00020 (SEQ ID NO: 2) C G N F L K R T C I C V K K; (SEQ ID NO: 3) C S G I I K Q T C T C Y R K; (SEQ ID NO: 6) C G N F L T R T C I C V K K; (SEQ ID NO: 7) C G N F L K T T C I C V K K; (SEQ ID NO: 8) C G N F L K R T C I C V T K; (SEQ ID NO: 9) C G N F L K R T C I C V K T; (SEQ ID NO: 10) C G N F L T T T C I C V T T; (SEQ ID NO: 11) S G N F L K R T S I S V K K; (SEQ ID NO: 12) S S G I I K Q T S T S Y R K; (SEQ ID NO: 13) S G N F L T R T S I S V K K; (SEQ ID NO: 14) S G N F L K T T S I S V K K; (SEQ ID NO: 15) S G N F L K R T S I S V T K; (SEQ ID NO: 16) S G N F L K R T S I S V K T;  and (SEQ ID NO: 17) S G N F L T T T S I S V T T.

18. The method according to claim 15, wherein the peptide is PEGylated.

19. The method according to claim 15, wherein the peptide does not comprise any disulfide bridges.

20. The method according to claim 15, wherein the peptide comprises a sequence selected from SEQ ID NOs: 22 and 23, the sequence optionally comprising 1, 2, 3, 4 or substitutions, additions, deletions or mixtures thereof.

21. The method according to claim 15, wherein the peptide consists of SEQ ID NO: 22 or 23.

22. The method according to claim 15, wherein the mycotoxin is a category B trichothecene, deoxynivalenol (DON) and/or acetylated deoxynivalenol (A-DON).

23. The method according to claim 15, wherein the fungus of the genus Fusarium is selected from Fusarium culmorum, Fusarium graminearum, Fusarium tricinctum, Fusarium avenaceum, Fusarium poae, Fusarium sporotrichioides, Fusarium verticilioides, Fusarium proliferatum, Fusarium langsethiae, Fusarium oxysporum, Fusarium roseum, Fusarium arthrosporioides and Fusarium avenaceum.

24. The method according to claim 15, wherein the plant is selected from cereals, wheat, barley, corn, oat, triticale, rice, fruits, and vegetables.

25. The method according to claim 24, wherein the plant is selected from tomato, melon, cucumber, zucchini, Jerusalem artichoke, bell pepper, potato, asparagus, sweet potato, celery, garlic, onion, cabbage, ginger, banana, manioc and vanilla and date palm.

26. A phytosanitary composition intended for treating a plant comprising a peptide or for inhibiting the production of mycotoxins by fungi of the genus Fusarium on the plant, the peptide comprising a sequence selected from: TABLE-US-00021 (SEQ ID NO: 18) C/S-G/S-N/G-F/R/I-L/W/I-K/T-R/L/Q/T-T-C/S-I/T-C/ S-V/I/Y-K/M/R/T-K/N/T; (SEQ ID NO: 19) C/S-G/S-N/G-F/I-L/I-K/T-R/Q/T-T-C/S-I/T-C/S-V/  Y-K/R/T-K/T; (SEQ ID NO: 20) C/S-G/S-N/G-F/I-L/I-K/T-R/T-T-C/S-I/T-C/S-V/Y-K/  T-K/T; and (SEQ ID NO: 21) C/S-G/S-N/G-F/I-L/I-K/R-R/K-T-C/S-ET-C/S-V/Y-K/ R-K/R, the peptide comprising not more than 30 amino acids and the peptide not being an unmodified peptide having the sequence CGNFLKRTCICVKK (SEQ ID NO: 2) or CSGIIKQTCTCYRK (SEQ ID NO: 3).

27. The composition according to claim 26, wherein the peptide comprises a sequence selected from: C G N F L K R T C I C V K K (SEQ ID NO: 2), the peptide bearing a PEGylation, a C-terminal amidation, an N-terminal acetylation, a modified peptide bond, a D-form amino acid, a modified cysteine or a combination of these modifications; C S G I I K Q T C T C Y R K (SEQ ID NO: 3), the peptide bearing a PEGylation, a C-terminal amidation, an N-terminal acetylation, a modified peptide bond, a D-form amino acid, a modified cysteine or a combination of these modifications; TABLE-US-00022 (SEQ ID NO: 6) C G N F L T R T C I C V K K; (SEQ ID NO: 7) C G N F L K T T C I C V K K; (SEQ ID NO: 8) C G N F L K R T C I C V T K; (SEQ ID NO: 9) C G N F L K R T C I C V K T; (SEQ ID NO: 10) C G N F L T T T C I C V T T; (SEQ ID NO: 11) S G N F L K R T S I S V K K; (SEQ ID NO: 12) S S G I I K Q T S T S Y R K ; (SEQ ID NO: 13) S G N F L T R T S I S V K K; (SEQ ID NO: 14) S G N F L K T T S I S V K K; (SEQ ID NO: 15) S G N F L K R T S I S V T K; (SEQ ID NO: 16) S G N F L K R T S I S V K T;  and (SEQ ID NO: 17) S G N F L T T T S I S V T T.

28. The composition according to claim 26, wherein the peptide comprises not more than 20 or 30 amino acids.

29. The composition according to claim 26, wherein the peptide is PEGylated.

30. The composition according to claim 26, wherein the peptide does not comprise any disulfide bridges.

31. The composition according to claim 26, wherein the mycotoxin is a category B trichothecene, deoxynivalenol (DON) and/or acetylated deoxynivalenol (A-DON).

32. The composition according to claim 26, wherein the fungus of the genus Fusarium is selected from Fusarium culmorum, Fusarium graminearum, Fusarium tricinctum, Fusarium avenaceum, Fusarium poae, Fusarium sporotrichioides, Fusarium verticilioides, Fusarium proliferatum, Fusarium langsethiae, Fusarium oxysporum, Fusarium roseum, Fusarium arthrosporioides and Fusarium avenaceum.

33. The composition according to claim 26, wherein the plant is selected from cereals, wheat, barley, corn, oat, triticale, rice, fruits, and vegetables or selected from tomato, melon, cucumber, zucchini, Jerusalem artichoke, bell pepper, potato, asparagus, sweet potato, celery, garlic, onion, cabbage, ginger, banana, manioc and vanilla and date palm.

Description

FIGURES

[0082] FIG. 1: Antifungal and antimycotoxin activity of TickCore3, native and oxidized forms. After 10 days of incubation in media supplemented or not supplemented with TickCore3 (native form) or TickCore3Ox (oxidized forms), the mycelia of F. graminearum were separated out and weighed. The mycelium weight values are given in grams (g) (FIG. 1a). The TCTB mycotoxins, DON (FIG. 1b) and 15-ADON (FIG. 1c), were quantified and expressed in μg of mycotoxins per ml of medium, then divided by the weight of mycelium and expressed in μg of TCTB mycotoxins per g of dry mycelium biomass (μg/g).

[0083] FIG. 2: Antifungal and antimycotoxin activity of linear and cyclic TickCore3 peptides. After 10 days of incubation in media supplemented or not supplemented with TickCore3 CH3-1 Ox, TickCore3 CH3-2 Ox, TickCore3 CH3-3 Ox, or TickCore3 CH3-123, the F. graminearum mycelia were separated out and weighed. The mycelium weight values are given in grams (g) (FIG. 2a). The TCTB mycotoxins, DON (FIG. 2b) and 15-ADON (FIG. 2c), were quantified in μg of mycotoxins per ml of medium, then divided by the weight of mycelium and expressed in μg of TCTB mycotoxins per g of dry mycelium biomass (μg/g).

[0084] FIG. 3: Antifungal and “antimycotoxin” 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 with T, an uncharged amino acid. After 10 days of incubation in media supplemented or not supplemented with TickCore3, TickCore3-K6T, TickCore3-R7T, TickCore3-K13T, and TickCore3-K14T, the F. graminearum mycelia were separated out and weighed. The mycelium weight values are given in grams (g) (FIG. 3a). The TCTB mycotoxins, DON (FIG. 3b) and 15-ADON (FIG. 3c), were quantified in μg of mycotoxins per ml of medium, then divided by the weight of mycelium and expressed in μg of TCTB mycotoxins per g of dry mycelium biomass (μg/g). Three different concentrations of peptide (12.5 μM, 25 μM and 50 μM) were tested.

[0085] FIG. 4: Antifungal and antimycotoxin activity of TickCore3 peptide and of a PEGylated version TickCore3-PEG. After 10 days of incubation in media supplemented or not supplemented with TickCore3 or TickCore3-PEG, the F. graminearum mycelia were separated out by centrifugation and weighed. The mycelium weight values are given in grams (g) (FIG. 4a). The TCTB mycotoxins, DON (FIG. 4b) and 15-ADON (FIG. 4c), were quantified in μg of mycotoxins per ml of medium, then divided by the weight of mycelium and expressed in μg of TCTB mycotoxins per g of dry mycelium biomass (μg/g).

[0086] FIG. 5: Antimycotoxin activity of the tick defensin DefMT3. After 10 days of incubation in media supplemented or not supplemented with DefMT3, the TCTB mycotoxins, DON (FIG. 5A) and 15-ADON (FIG. 5B), were quantified in μg of mycotoxin per ml of medium, divided by the mycelium weight, and expressed in μg of TCTB mycotoxin per g of dry mycelium biomass (μg/g). Two concentrations of DefMT3 were tested: 25 and 50 μM.

EXAMPLES

[0087] The inventors have shown that the conserved linear gamma core moiety, with reduced cysteine (Cys) residues, of the tick defensin DefMT3, referred to hereinbelow as “TickCore3”, inhibits the production of category B trichothecenes (TCTB) by F. graminearum. Alkylation of all the Cys residues ofTickCore3 with methyl groups (TickCore3-CH3) had little effect on the inhibitory effect affecting TCTB production. Conversely, oxidation of all the Cys residues of TickCore3 strongly decreased the inhibitory effect on TCTB production. A major loss of inhibitory function was observed when the specific Cys4-Cys6 and Cys4-Cys5 disulfide bridges were formed. The inventors also showed that the cationic charge of the peptide was an important factor in its biological activity. Replacement of the positively charged amino acids with a neutral residue occasionally led to a very significant reduction in antifungal activity and in inhibition of TCTB production.

[0088] Results

[0089] TickCore3 is a Highly Effective Inhibitor of TCTB Production by F. graminearum

[0090] FIG. 1 shows the results obtained after 10 days of culturing the strain F. graminearum CBS 185.32 in MS medium supplemented with TickCore3 and its oxidized variant (TickCore3 Ox) at three different concentrations (12.5, 25 and 50 μM). Untreated culture media (control) were not supplemented with any of the TickCore3 peptides. Although TickCore3 Ox had no effect on the dry fungal biomass, TickCore3 significantly inhibited fungal growth at all the concentrations tested compared to the control condition (FIG. 1a). The inhibitory effect on fungal growth of TickCore3 was dose-dependent since increasing the concentration of the peptide had a proportional effect on the amount of dry biomass. As regards mycotoxin production, 15-ADON was the main TCTB produced in the liquid media by the strain studied. Under the control conditions, 15-ADON production (mean 21867 SD±3259 μg/g dry biomass) was 26 times higher than that of DON (835±68 μg/g). Supplementation with TickCore3 led to non-detectable levels of DON (FIG. 1b) and 15-ADON (FIG. 1c) for all the concentrations tested. However, residual levels of 15-ADON were observed with the 12.5 μM concentration (FIG. 1c). In contrast, a 12.5 μM concentration of TickCore3 Ox did not significantly reduce DON (FIG. 1b) or 15-ADON (FIG. 1c) production.

[0091] Cyclization of TickCore3 Reduces the Antifungal Activity

[0092] The inventors hypothesized that cycle formation in TickCore3 may have effects on inhibiting TCTB production. In order to test this hypothesis, TickCore3 derived peptides 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).

[0093] 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, in comparison with the control (FIG. 2a). The mycotoxin production by F. graminearum was then measured in 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 μM (FIG. 2b). Increasing the concentrations of TickCore3-CH3-2 Ox and TickCore3-CH3-3 Ox resulted in a significant increase in DON (FIG. 2b) and 15-ADON (FIG. 2c) production, except for the fungi exposed to TickCore3-CH3-2 Ox for which 15-ADON production did not change significantly. Moreover, treatment with TickCore3-CH3-1 Ox at 50 μM resulted in a significant reduction in DON and 15-ADON levels (FIG. 2bc). An additional peptide, TickCore3-CH3-CH3-123, with all the Cys residues alkylated with methyl groups, was synthesized. F. graminearum exposed to TickCore3-CH3-123 produced significantly less DON and 15-ADON in comparison with the control conditions.

[0094] The Cationic Charge is a Key Element of the Antifungal and “Antimycotoxin” Activity of TickCore3.

[0095] The inventors hypothesized that the cationic charge of TickCore 3 was an important element of its biological activity. In order to test this hypothesis, substituted TickCore3 peptides were synthesized by replacing the “basic” amino acids (arginine and lysine, R and K) with an uncharged residue, threonine (T). The following peptides were thus generated: TickCore3-K6T (replacement of lysine at position 6 with a threonine residue), TickCore3-R7T (replacement of arginine at position 7 with a threonine residue), Tickcore3-K13T (replacement of lysine at position 13 with a threonine residue), and TickCore3-K14T (replacement of lysine at position 14 with a threonine residue). For all the substituted peptides, the antifungal activity was reduced compared to that of the native peptide (FIG. 3a). The substituted forms (TickCore3-K13T, TickCore3-K14T and TickCore3-R7T) showed strong antifungal activity only at 50 μM, whereas TickCore3-K6T had no antifungal activity below the concentration at 50 μM. The mycotoxin production 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 μM (FIGS. 3b and 3c). For all the substituted peptides, whether with respect to DON (FIG. 3b) or 15-ADON (FIG. 3c) production, the ability to inhibit mycotoxin production is lower than that of the native form TickCore3. This reduction in the ability to inhibit TCTB production is particularly pronounced for TickCore3-K6T and to a lesser extent for TickCore3-K13T, suggesting that the lysine amino acids at positions 13 and 6 play an important role in the “antimycotoxin” activity.

[0096] PEGylation of TickCore3 Increases the Antifungal Activity

[0097] The peptide TickCore3-PEG was synthesized in the same way as TickCore3, except that two PEG units were sequentially attached to the N-terminal end of the peptide. Compared with TickCore3, TickCore3-PEG at 50 μM showed a greater antifungal effect with a 3.3-fold decrease in fungal growth (FIG. 4a). Furthermore, TickCore3-PEG is also considered to have improved inhibitory activity on mycotoxin production (FIG. 4bc).

[0098] The Defensin DefMT3 is Capable of Inhibiting Mycotoxin Production.

[0099] The peptide DefMT3 was synthesized in the same manner as TickCore3. This peptide shows inhibitory activity on production of the mycotoxins DON and 15-ADON (FIGS. 5a and 5b).

[0100] Materials and Methods

[0101] Synthesis of the Gamma Core of TickCore3

[0102] TickCore3 (CGNFLKRTCRTCICVKKK, SEQ ID NO: 2) is the conserved gamma core moiety of DefMT3 (GenBank accession number: JAA71488; Tonk et aL., 2015). The peptide synthesis was commissioned from Pepmic (Suzhou, China), which used 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). Briefly, the peptide synthesis was performed using 2-chlorotrityl chloride resin as a solid support, and 9-fluorenyl-methyloxy-carbonyl (Fmoc) labile base as a protecting group. The amino acids were protected as follows: Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Asn(Trt)-OH, Fmoc-Phe-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH and Fmoc-Val-OH. All the peptide sequences were synthesized according to SPPS principles. The peptides were purified by high performance liquid chromatography (HPLC) according to the standard peptide chemical coupling protocols.

[0103] The peptides purified by reverse-phase HPLC were then optionally oxidized using a 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 the Cys residues was confirmed by the Ellman reaction and disulfide bond formation was characterized by electrospray mass spectroscopy (ESI-MS) using an LCMS-2020 mass spectrometer (Shimadzu, Kyoto, Kyoto Prefecture, Japan). The sequence composition 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-CH3-1 (C(CH3)GNFLKRTCRICVKK), TickCore3-CH3-2 (CGNFLKRTC(CH3)ICVKK) and TickCore3-CH3-3 (CGNFLKRTCIC(CH3)VKK) were synthesized as described previously, except that Fmoc-Cys(Me)-OH was added in replacement for Fmoc-Cys(Trt)-OH. Alkylation of the sulfur (S) of the thiol groups of all the Cys residues prevents the cyclization of these residues (i.e. no disulfide bridges can be formed). Conversely, alkylation of the S of the thiol group of a single Cys residue, followed by its oxidation, directs the cyclization of the reduced Cys residues (i.e. those which have not been alkylated).

[0104] The peptide TickCore3-PEG was synthesized as for TickCore3, but two PEG units were sequentially attached to the N-terminal end of the peptide using protected Fmoc-NH-PEG2-CH2CH2COOH groups.

[0105] The peptide DefMT3 having the sequence GGYYCPFRQDKCHRHCRSFGRKAGYCGNFLKRTCICVKK (SEQ ID NO: 22) was synthesized according to a protocol identical to that of TickCore3 by the company Pepmic (Suzhou, China).

[0106] Test of Inhibition of Mycotoxin Production

[0107] Fusarium Strain and Culture Conditions

[0108] The strain F. graminearum CBS 185.32 which produces DON and 15-ADON (Centraal bureau voor Schimmelkulturen, The Netherlands) was used throughout this study. The fungal culture was maintained at 4° C. on potato dextrose agar (PDA) (Difco, Le Ponts de Claix, France) in slanted tubes under mineral oil. For the inoculum realization, the strain was cultured at 25° C. in the dark on PDA slants for 7 days and the spore suspension was prepared by adding 6 mL of sterile distilled water to the PDA slant with gentle agitation.

[0109] Liquid culture experiments were performed using 24-well static plates. Each well containing 2 ml of a synthetic medium (SM medium) which promotes rapid induction of TCTB production, prepared as previously published (Boutigny et aL., 2009, Mycol. Res., 113, 746), supplemented or not supplemented with the peptide, was inoculated with 2×10.sup.4 spores/mL. The fungal liquid cultures were incubated at 25° C. in the dark for 10 days. After incubation, the mycelia were recovered by centrifugation and the fungal biomass was measured by weighing the mycelia after 48 hours of freeze-drying (Flexi-Dry®, OErlikon Leybold, Germany). The culture media were stored at −20° C. until the time of TCTB analysis. All the peptides were tested at three concentrations: 12.5, 25 and 50 μM. Five repetitions were performed for each condition. DefMT3 was tested at 25 and 50 μM. Suitable controls using peptide-free control media and non-inoculated control media were included.

[0110] TCTB Extraction and Analysis

[0111] A 1.5-mL sample of culture medium was extracted with 3 mL of ethyl acetate. A volume of 2.5 mL of the organic phase was evaporated to dryness at 45° C. under a stream of nitrogen. The dried samples were dissolved in 200 μL of methanol/water (1/1, v/v) and filtered through a 0.2 am filter before analysis. The 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 ChemStation chromatography management software (Agilent, France). Separation was performed on a Kinetex XB-C18 100 Å column (4.6×150 mm, 2.6 μm) (Phenomenex, France) maintained at 45° C. The mobile phase consisted of water acidified with orthophosphoric acid at pH 2.6 (solvent A) and acetonitrile (solvent B). The flow rate was maintained at 1 mL.Math.min.sup.−1. The injection volume was 5 μL. The TCTBs were separated out using an elution gradient: 7 to 30% B in 10 min, 30-90% B in 5 min, 90% B for 5 min, 90 to 7% B in 2 min, 7% B for 5 min. The UV-Vis spectra were recorded from 190 to 400 nm and the peak areas were measured at 230 nm. Quantification was performed by external calibration with standard solutions (Romer Labs, Austria). The toxin yields were expressed in μg.Math.g.sup.−1 of dry biomass.

[0112] Statistical Analyses

[0113] All the values presented are means±standard deviation including four biological replications. Since the data did not follow a normal distribution (Shapiro-Wilk normality test), Kruskal-Wallis one-way analysis was used, with comparisons of means performed using the Connover-Inman test. Statistical analysis was performed with the XLSTAT 2017 software (Addinsoft, Rennes, France). The statistical significance level p=0.05 was used throughout the study.