RU2380374C1 - Antimicrobial peptide - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: biologically active peptide WAMP-1 having the following amino acid sequence: Ala¹-Gln²-Arg³-Cys⁴-Gly⁵-Asp⁶-Gln⁷-Ala⁸-Arg⁹-Gly¹⁰-Ala¹¹-Lys¹²-Cys¹³-Pro¹⁴-Asn¹⁵-Cys¹⁶-Leu¹⁷-Cys¹⁸-Cys¹⁹-Gly²⁰-Lys²¹-Tyr²²-Gly²³-Phe²⁴-Cys²⁵-Gly²⁶-Ser²⁷-Gly²⁸-Asp²⁹-Ala³⁰-Tyr³¹-Cys³²-Gly³³-Ala³⁴-Gly³⁵-Ser³⁶-Cys³⁷-Gln³⁸-Ser³⁹-Gln⁴⁰-Cys⁴¹-Arg⁴²-Gly⁴³-X, where X-Cys⁴⁴ or Cys⁴⁴-Arg⁴⁵, expresses antimicrobial action.

EFFECT: peptide can be used for protection of plants and foodstuff against pathogenic fungi and bacteria.

3 dwg, 2 tbl, 4 ex

Description

The invention relates to biochemistry, to biologically active peptides with antimicrobial activity, which can be used in biotechnology to create pathogen-resistant forms of crops and in the food industry for preserving food.

Crop losses associated with pathogens (fungi, bacteria, viruses and viroids), pests and nematodes reach 45% [Oeke BC, Dehne HW, Schonbeck P., Weber A. Crop Production and Crop Protection: Estimated Losses in Major Food and Cash Crops. - 1994. - Elsevier, Amsterdam]. In addition to reducing agricultural production, pathogens degrade its quality. So Fusarium sp. Mycotoxins present in infected grains are toxic to humans and animals. The damage caused by pathogens and pests annually reaches 100 billion dollars. Against the backdrop of the rapid growth of the Earth’s population by 1.5% per year, the need for food protein is also increasing, which currently amounts to 230 million tons per year, which makes the problem of increasing crop yields extremely urgent. Traditional selection methods are not always effective due to the lack of appropriate resistance genes, in addition, they are laborious and time consuming. The use of chemical plant protection products poses a significant threat to environmental safety and can reduce crop losses by only 5-10%.

An alternative strategy to increase the resistance of crops to pathogens and insect pests, as well as to environmental stress factors of an abiotic nature (drought, soil salinity, etc.) is genetic engineering, which allows you to embed foreign genes that cause resistance in the genomes of cultivated plants. The spectrum of transgenes used to transform plants is growing. Through genetic transformation, crops resistant to herbicides, pests, and abiotic stress have already been obtained.

A genetically modified papaya variety that is resistant to the ring spot virus is known by incorporating the envelope protein of the virus into the genome [Ferreira SA, Pitz KY, Manshardt R., Zee F., Fitch M., Gonsalves D. Virus coat protein transgenic papaya provides practical control of papaya ringspot virus in Hawaii. // Plant Dis. - 2002 .-- Vol.86. - P.101-106]. A genetically modified potato variety is known that is resistant to leaf curling virus. In this case, resistance to the virus is provided by inhibiting the activity of the replicase gene [Delvas M., Ceriani M.F., Collavita M., Butzonich I., Hopp H.E. Analysis of transgenic potato plants expressing potato leaf-roll virus coat protein gene. // Plant Physiol. - 1993. - Vol.102 - P.174-180].

Currently, genetically modified crops resistant to fungi and bacteria are not known.

According to modern concepts, plants contain a whole arsenal of tools that allows them to fight against pathogens and insect pests. A significant role among them is played by compounds possessing antimicrobial properties, which include secondary plant metabolites - phytoalexins and phytoantisipins, as well as a number of proteins and peptides. An increase in plant resistance can be achieved both by enhancing the expression of native genes involved in defense reactions, and by incorporating genes encoding proteins and peptides with antimicrobial properties from other plant species into the genome. In this sense, antimicrobial peptides with a wide spectrum of antimicrobial activity are of particular interest, because their genes can be directly inserted into the genome of plants sensitive to pathogens using genetic transformation methods [Carlini C.R., Grossi-de-Sa M.F. Plant toxic proteins with insecticidal properties. A review on their potentialities as bioinsecticides. // Toxicon. - 2002 .-- Vol. 40. - P.1515-1539].

Known closest to the claimed in structure and antifungal properties is the peptide EAFP1 isolated from the bark Eucommia ulmoides [Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv. // FEBS Lett. - 2002 .-- Vol.521. - P.87-90]. The substance belongs to the family of cysteine-rich chitin-binding peptides, inhibits the growth of certain pathogenic fungi and bacteria in concentrations of 3.5-30 μM.

The invention solves the problem of expanding the range of peptides of plant origin with antimicrobial activity.

The problem is solved due to the structure of the new WAMP-1 peptide having the following amino acid sequence:

Ala ¹ -Gln ² -Arg ³ -Cys ⁴ -Gly ⁵ -Asp ⁶ -Gln ⁷ -Ala ⁸ -Arg ⁹ -Gly ¹⁰ -Ala ^ll -Lys ^l2 -Cys ¹³ -Pro ¹⁴ -Asn ^l5 -Cys ^l6 -Leu ^l7 -Cys ^l8 -Cys ^l9 -Gly ²⁰ -Lys ²¹ -Tyr ²² -Gly ²³ -Phe ²⁴ -Cys ²⁵ -Gly ²⁶ -Ser ²⁷ -Gly ²⁸ -Asp ²⁹ -Ala ³⁰ -Tyr ³¹ -Cys ³² -Gly ³³ -Ala ³⁴ -Gly ³⁵ -Ser ³⁶ -Cys ³⁷ -Gln ³⁸ -Ser ³⁹ -Gln ⁴⁰ -Cys ⁴¹ -Arg ⁴² -Gly ⁴³ -X,

where X is Cys ⁴⁴ (WAMP-1a) or Cys ⁴⁴ -Arg ⁴⁵ (WAMP-1b).

The inventive peptide exhibits pronounced antifungal and antimicrobial activity against the following plant pathogens, molds, and pathogenic bacteria: Fusarium solani VKM F-142, Fusarium verticillioides VKM F-670, Fusarium oxysporum TCXA-4, Botrytis cinerea VKMporo Furopo VKMpouropoMporopoMpoMropo VKMporopoMpoMropo VKMpouropro crassa VKM F-184, Pseudomonas syringae pv. tomato, Clavibacter michiganensis subsp. michiganensis and Erwinia carotovora. To completely inhibit the growth of these fungi and bacteria in vitro, micromolar concentrations of the peptide are necessary.

The technical result of the invention is the high antimicrobial activity of the claimed peptide.

The WAMP-1 peptide consists of 44 or 45 amino acid residues and can be obtained by chemical synthesis, biotechnologically, or isolated from the seeds of Triticum kiharae wheat, which belongs to the Roaceae family of cereals, the monocotyledonous Monocotyledones class, the angiosperms or flowering plants of Magnoliophyta (Angiospermae).

The invention is illustrated by the drawings:

Figure 1 - Separation of the total extract of 10 g of wheat seeds by affinity chromatography on a HiTrap ™ Heparin HP 5 ml column (Amersham Biosciences, USA) with a NaCl step gradient, elution rate - 1 ml / min. Detection of proteins and peptides in the eluate by optical density at 280 nm. The gray rectangle indicates the fraction containing the WAMP-1 peptide.

Figure 2 - Separation of a 100 mM fraction containing the WAMP-1 peptide (see Figure 1) by gel filtration on a Superdex Peptide HR 10/30 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) of size 1 × 30 cm. Separation in 5% acetonitrile in 0.05% trifluoroacetic acid, elution rate - 1 ml / min. Detection of proteins and peptides by optical density at 214 nm. The gray rectangle indicates the fraction containing the WAMP-1 peptide.

Figure 3 - Separation of the fraction containing the WAMP-1 peptide (see figure 2) by reverse-phase high-performance liquid chromatography on a Vydac C ₁₈ column with a size of 4.6 × 250 mm, pore size 300 Å, particle diameter 5 μm, ( The Nest Group, Inc., USA) in a linear gradient of acetonitrile in the presence of 0.1% trifluoroacetic acid at an elution rate of 1 ml / min and a temperature of 40 ° C. Registration of the optical density of the eluate at 214 nm. The arrow indicates the fraction containing the WAMP-1 peptide.

The invention is illustrated by the following examples.

Example 1

Isolation of WAMP-1 Peptide

Wheat seeds are ground to flour in a coffee grinder and extracted with 5 volumes of a mixture of 1% trifluoroacetic acid, 1 M HCl, 5% HCOOH and 1% NaCl at room temperature and stirring for 1 h. The suspension is centrifuged at 22000 g for 15 min at room temperature, the precipitate was discarded, and the supernatant was desalted by reverse phase high performance liquid chromatography on an Aquapore RP-300 C ₈ column (10 × 30 mm, pore size 300 Å, particle diameter 7.5 μm ; Applied Biosystems, USA). The following solutions are used: A - 0.1% trifluoroacetic acid in water, B - 80% acetonitrile in 0.1% trifluoroacetic acid. The column is washed with 6 volumes of solution B and equilibrated with 6 volumes of solution A at an elution rate of 1.5 ml / min. Detection is carried out by optical absorption of the eluate at 214 nm. After applying the acid extract to the column, it is washed with solution A until the absorption value of the eluate becomes equal to the initial value. Proteins and peptides are stripped from a column of 70% solution B, evaporated in a vacuum concentrator to remove acetonitrile and freeze-dried.

The resulting dry residue from 10 g of feedstock was dissolved in 5 ml of solution B (10 mM Tris-HCl, pH 7.2) and separated by affinity chromatography on a 5 ml HiTrap ™ Heparin HP column (Amersham Biosciences, USA), previously equilibrated 5 volumes of buffer B. After applying the sample, the sorbent is first washed with buffer B from unbound substances, after which the proteins and peptides are eluted first with 100 mM NaCl in buffer B (fraction I) and then with 500 mM NaCl in buffer B (fraction II) with speed of 1 ml / min. Proteins and peptides are detected by optical absorption of the eluate at 280 nm (Fig. 1). The obtained fractions are desalted and dried as described above, and their biological activity is tested.

Fraction I was dissolved in 1 ml of solution G (5% acetonitrile in 0.05% trifluoroacetic acid) and separated by gel filtration on a Superdex Peptide HR 10/30 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) with a size of 1 × 30 cm, previously balanced with solution G. 200 μl of a solution of fraction I is applied to the column. Separation is carried out at an elution rate of 150 μl / min and room temperature. Proteins and peptides are detected by optical absorption at 214 nm (Fig. 2). The obtained fractions are dried, their biological activity is tested.

Fractions with an elution time of 50-55 min (Fig. 2) with antifungal activity are combined and separated by reverse phase high performance liquid chromatography on a Vydac C ₁₈ column (4.6 × 250 mm, pore size 300 Å, particle diameter 5 μm; The Nest Group, Inc., USA). Fractionation is carried out in a linear gradient of acetonitrile: 10-50% solution B for 60 minutes at an elution rate of 1 ml / min and a temperature of 40 ° C. Detection is carried out by optical absorption at 214 nm (Fig. 3), and the obtained fractions are tested. The WAMP-1 peptide elutes with a retention time of 17.5 minutes. The result is a preparation of the desired peptide with an impurity content of less than 3%. The two molecular forms of the WAMP-1 peptide are separated by rechromatography under the same conditions.

Example 2

Establishment of the amino acid sequence of the peptide WAMP-1

To determine the number of cysteine residues in the amino acid sequence of the peptide, disulfide bonds are restored and the formed thiol groups are alkylated. Reduction and alkylation are carried out according to a modified method [Thomsen J., Bayne S. Microscale alkylation with 4-vinylpyridine. // J. Protein Chem. - 1988 .-- Vol. 7. - P.295-296]. The peptide (1 nmol) was dissolved in 35 μl of a buffer containing 6 M guanidine hydrochloride and 2 mm EDTA in 0.5 M Tris-HCl buffer, pH 8.5), 2 μl of an aqueous solution containing 1 μmol 1.4- was added dithiothreitol, purged with nitrogen, stirred and incubated at 40 ° C for 4 hours. Then, 2 μl of a 50% solution of 4-vinylpyridine in 2-propanol are added to the mixture, stirred and incubated in the dark for 20 minutes at room temperature. 120 μl of 0.1% trifluoroacetic acid was added to the mixture, and the reduced and alkylated peptide was separated by reverse phase high-performance chromatography on a Vydac C ₁₈ column (4.6 × 250 mm, pore size 300 Å, particle diameter 5 μm; The Nest Group, Inc., USA), pre-equilibrated with 0.1% trifluoroacetic acid. The reaction products and reagents are separated by washing the column with the same solvent until the initial optical absorption of the eluate is achieved, the peptide is eluted under the same conditions as described in Example 1 for the purification of the WAMP-1 peptide.

The amino acid sequence of the purified reduced and alkylated WAMP-1 peptide is determined by the Edman stepwise degradation method using a Precise 492 automated sequencer (Applied Biosystems, USA). As a result, the complete amino acid sequence of WAMP-1, consisting of 44 or 45 amino acid residues, is established:

Ala ^l -Gln ² -Arg ³ -Cys ⁴ -Gly ⁵ -Asp ⁶ -Gln ⁷ -Ala ⁸ -Arg ⁹ -Gly ^l0 -Ala ^ll -Lys ^l2 -Cys ¹³ -Pro ^l4 -Asn ^l5 -Cys ^l6 -Leu ^l7 -Cys ^l8 -Cys ^l9 -Gly ²⁰ -Lys ²¹ -Tyr ²² -Gly ²³ -Phe ²⁴ -Cys ²⁵ -Gly ²⁶ -Ser ²⁷ -Gly ²⁸ -Asp ²⁹ -Ala ³⁰ -Tyr ³¹ -Cys ³² -Gly ³³ -Ala ³⁴ -Gly ³⁵ -Ser ³⁶ -Cys ³⁷ -Gln ³⁸ -Ser ³⁹ -Gln ⁴⁰ -Cys ⁴¹ -Arg ⁴² -Gly ⁴³ -X,

where X is Cys ⁴⁴ (WAMP-1a) or Cys ⁴⁴ -Arg ⁴⁵ (WAMP-1b).

Example 3

Determination of the relative molecular weight and the number of free and disulfide-linked cysteine residues in the WAMP-1 peptide

The obtained amino acid sequence, as well as the identity of the purified peptide, is confirmed by mass spectrometric analysis. Mass spectra were obtained on a MALDI time-of-flight mass spectrometer Ultraflex II TOF / TOF (Bruker Daltonik, Germany), with identification of positive ions in a reflex mode. Dihydrobenzoic acid (10 mg / ml in 50% acetonitrile containing 0.1% trifluoroacetic acid) is used as a matrix. To calibrate the device using a standard mixture of peptides and proteins with a molecular weight range of 700-66000 Da (Sigma, USA).

The measured monoisotopic molecular weight of the natural peptide WAMP-1 is 4431.95 Da and within the accuracy of the MALDI mass spectrometer (0.01%) does not differ from the calculated (4431.66 Da). The molecular weight of the alkylated unreduced peptide WAMP-1 is 4431.88 Da, i.e. also does not differ from the measured molecular weight of the natural peptide. Thus, the WAMP-1 peptide does not contain free sulfhydryl groups, and all 10 cysteine residues are involved in the formation of 5 disulfide bonds, the peptide does not contain any other chemical modifications.

Example 4

Biological properties of the peptide WAMP-1

The following fungal cultures are used to determine the antifungal activity of the fractions obtained by separating acid-soluble proteins and peptides of wheat, as well as the purified WAMP-1 peptide: Fusarium solani VKM F-142, Fusarium verticillioides VKM F-670, Fusarium oxysporum TCXA-4, Botrytis cinerea VKM F-85, Neurospora crassa VKM F-184. The following bacterial cultures are used to determine the antibacterial activity of the isolated WAMP-1 peptide: Pseudomonas syringaepv. tomato, Clavibacter michiganensis subsp. michiganensis and Erwinia carotovora.

To study the activity, the isolated fractions and the purified WAMP-1 peptide were frozen in liquid nitrogen, freeze-dried, re-dissolved in water and again dried. Solids are redissolved in 50 μl of water. The concentration is determined by the absorption spectrum in the UV region.

Antifungal activity is determined by the inhibition of spore germination in a liquid nutrient medium (potato dextrose solution) in 96-well plates. Determination of the peptide concentrations necessary for 50% inhibition of fungal spore germination (IC ₅₀ ) is carried out by the double dilution method. A series of twofold dilutions of the test substances in water is prepared. The test sample and the spore suspension of the fungus are placed in the wells. The total sample volume is 100 μl. As a control, a well is used in which sterile distilled water is added instead of the solution of the test sample. The plates were incubated for 24 hours and the inhibition of spore germination was evaluated by the optical density of the suspension at 620 nm. The results for the WAMP-1 peptide are shown in table 1.

Table 1 Antifungal properties of the peptide WAMP-1 * Mushroom IC ₅₀ μm Fusarium solani 5 Fusarium verticillioides thirty Fusarium oxysporum fifteen Botrytis cinerea twenty Neurospora crassa 10 * Data obtained for WAMP-1a peptide

Antibacterial activity is determined by radial diffusion. To test antibacterial activity, 50 μl of solutions of the studied peptides of different concentrations are applied to cotton swabs, which are then placed in 0.5 cm diameter wells on Petri dishes with LB agar medium. Plates are pre-seeded with bacterial cultures grown for 16-18 hours in LB liquid medium. For testing, a bacterial mottling agent strain of tomato Pseudomonas syringae pv is used. tomato, a bacterial cancer pathogen strain of Clavibacter michiganensis subsp.michiganensis and a wet rot pathogen strain Erwinia carotovora. The plates are incubated at room temperature. The test results are evaluated after 24-48 hours by the size of the zone of inhibition of bacterial growth. Claforan antibiotic is used as a control. The results for the WAMP-1 peptide are shown in table.2.

table 2 Antibacterial properties of the peptide WAMP-1 * Peptide concentration (μg / 50 μl) ** The zone of inhibition of bacterial growth, cm (taking into account the size of the hole) *** R. syringae E. carotovora C. michiganensis 10 1.3 (1.4) 1.5 (3.4) 1.7 5 1.2 (1.2) 1.3 (2.7) 1,5 2.5 0.9 (1.0) 1.1 (2.2) 1.3 * Data presented for WAMP-1a ** the volume of all samples is 50 μl *** hole diameter - 0.5 cm

The size of the zones of inhibition of bacterial growth caused by the antibiotic claforan is given in parentheses.

Claims (1)

A peptide having antimicrobial activity having the following amino acid sequence:
Ala ¹ -Gln ² -Arg ³ -Cys ⁴ -Gly ⁵ -Asp ⁶ -Gln ⁷ -Ala ⁸ -Arg ⁹ -Gly ¹⁰ -Ala ¹¹ -Lys ¹² -Cys ¹³ -Pro ¹⁴ -Asn ¹⁵ -Cys ¹⁶ -Leu ¹⁷ -Cys ¹⁸ -Cys ¹⁹ -Gly ²⁰ -Lys ²¹ -Tyr ²² -Gly ²³ -Phe ²⁴ -Cys ²⁵ -Gly ²⁶ -Ser ²⁷ -Gly ²⁸ -Asp ²⁹ -Ala ³⁰ -Tyr ³¹ -Cys ³² -Gly ³³ -Ala ³⁴ -Gly ³⁵ -Ser ³⁶ -Cys ³⁷ -Gln ³⁸ -Ser ³⁹ -Gln ⁴⁰ -Cys ⁴¹ -Arg ⁴² -Gly ⁴³ -X,
where X is Cys ⁴⁴ or Cys ⁴⁴ -Arg ⁴⁵ .

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