KR100915852B1 - Antimicrobial peptide KCM11 against phytopathogenic bacteria - Google Patents

Abstract

The present invention provides a method for sterilizing or microbial growth of a plant pathogenic microorganism comprising administering an antimicrobial peptide KCM11 and an antimicrobial or microbial growth inhibiting effective amount of the antimicrobial peptide KCM11 against a plant pathogenic microorganism consisting of the amino acid sequence of SEQ ID NO: 1. It is about a suppression method.

Phytopathogenic, Antimicrobial Peptides, Microbial Growth Inhibition

Description

Antimicrobial peptide KCM11 against phytopathogenic bacteria}

The present invention provides a method for sterilizing or microbial growth of a plant pathogenic microorganism comprising administering an antimicrobial peptide KCM11 and an antimicrobial or microbial growth inhibiting effective amount of the antimicrobial peptide KCM11 against a plant pathogenic microorganism consisting of the amino acid sequence of SEQ ID NO: 1. It is about a suppression method.

 Infectious diseases are the leading cause of death worldwide (25%), and their value increases by 65% for children. Conventional antibiotics have been used to control pathogens. However, their mass use has caused serious problems such as the emergence of drug resistant strains (MRSA, VRSA) that threaten public health. AMPs (antimicrobial peptides) have been cautioned as potent alternatives, which are generally nontoxic and are often very effective against known drug resistant strains, although the development of drug resistant strains for AMP probably interacts with the membrane, but its This is because rarely occurs due to membrane-based mechanisms of action that do not require any specific receptor protein for action. The continuous use of pesticides and antibiotics also poses a global problem. This not only changes the normal environmental system, but also directly threatens human health.

Korean Patent Publication No. 2002-0049443 discloses a novel antibiotic peptide derived from the ribosomal protein L1 of Helicobacter pylori, but differs from the peptide of the present invention.

Korean Patent Publication No. 2006-0057271 describes a thermostable peptide isolated from potato of the Golden Valley variety, but is different from the peptide of the present invention.

Korean Patent Publication No. 2006-0028676 describes a novel antimicrobial peptide having heparin binding activity, but is different from the peptide of the present invention.

Korean Patent Publication No. 1999-0082383 describes an antimicrobial peptide and its use, but differs from the peptide of the present invention.

In the present invention, in an attempt to use AMP for crop protection, screening synthetic peptide combinatorial libraries, identifying four out of twelve active antimicrobial hexa-peptides that effectively kill phytopathogenic microorganisms, their effectiveness, cytotoxicity and To check plant disease control.

In order to solve the above problems, the present invention provides an antimicrobial peptide KCM11 against phytopathogenic microorganism consisting of the amino acid sequence of SEQ ID NO: 1.

The present invention also provides a method for sterilizing or inhibiting microbial growth of a plant pathogenic microorganism comprising administering to the plant an effective amount of the antimicrobial or microbial growth inhibitory effect of the antimicrobial peptide KCM11.

According to the present invention, the antimicrobial peptide KCM11 of the present invention can effectively kill phytopathogenic microorganisms.

In order to achieve the object of the present invention, the present invention provides an antimicrobial peptide KCM11 against phytopathogenic microorganism consisting of the amino acid sequence of SEQ ID NO: 1. The phytopathogenic microorganism may be selected from the group consisting of gram positive bacteria, gram negative bacteria, fungi and viruses, preferably the phytopathogenic microorganism is Clavibacter michiganensis subsp. Michiganensis , Pseudomonas Syringe F. Tomato DC3000 ( Pseudomonas syringae pv. Tomato DC3000), Pectobacterium cartoborum subspecies Cartovorum ( Pectobacterium carotovorum subsp. c arotovorum ) , Xanthomonas Campestris fV . Bessictoria 833 ( Xanthomonas campestris pv. vesicatoria 833), Xanthomonas Campestris FV. Xanthomonas campestris pv.vesicatoria 833 pila , Pectobacterium carotovorum subsp.Atrosepticum , Acidovorax konjaci , Xanthomona albil Enshi ( Xanthomonas albilineans ) , Xanthomonas oriza fib . Orissa 90 ( Xanthomonas oryzae pv. Oryzae 90 ) , Xanthomonas Orissa fib. Orissa 599 ( Xanthomonas oryzae pv. Oryzae 599 ) , Xanthomonas orissa fib . Xanthomonas oryzae pv.oryzae 710 , Janibacter melonis , Ralstonia solanacearum race 1, Ralstonia solanacearum race 3 3), Candida glabrata , Candida krusei , Candida tropicalis , Saccharomyces cerevisiae 5312 , Berkholderia Glume SL 2870 ( Burkholderia glumae SL 2870), Burkholderia glumae SL 2399 or Burkholderia glumae R1, but is not limited thereto.

Peptides of the invention can be modified by amidation, esterification, acylation, acetylation, PEGylation (PEGylation) or alkylation, in particular antimicrobial peptides are acylation, acetylation, PEGylation (PEGylation), alkylation Modification at the N-terminus and the like and at the C-terminus by amidation or esterification.

The present invention also provides a method for sterilizing or inhibiting microbial growth of a plant pathogenic microorganism comprising administering to the plant an effective amount for inhibiting the sterilization or microbial growth of the antimicrobial peptide KCM11 of the present invention. As used herein, the term “sterilization” or “inhibiting microbial growth” refers to the ability of an antimicrobial peptide to kill or critically damage a target microorganism. The term "inhibiting microbial growth" as used herein refers to inhibiting the growth of a target microorganism without dying. When the antimicrobial peptide of the present invention is administered to a plant in which the microorganism is growing, it is possible to kill or inhibit the growth of the microorganisms present in the plant. The subject microorganism can be selected from the group consisting of Gram-positive bacteria, Gram-negative bacteria, fungi and viruses, preferably the phytopathogenic microorganisms are Clavibacter michiganensis subsp. Michiganensis , Pseudomonas. Syringe F. Tomato DC3000 ( Pseudomonas syringae pv. Tomato DC3000), Pectobacterium carotoborum Subspecies carotoborum ( Pectobacterium carotovorum subsp. c arotovorum ) , Xanthomonas Campestris fV . Bessictoria 833 ( Xanthomonas campestris pv. vesicatoria 833), Xanthomonas Campestris fV . Xanthomonas campestris pv.vesicatoria 833 pila , Pectobacterium carotovorum subsp.Atrosepticum , Acidovorax konjaci , Xanthomona albil Nions ( Xanthomonas albilineans ) , Xanthomonas oriza fib . Orissa 90 ( Xanthomonas oryzae pv. Oryzae 90 ) , Xanthomonas Orissa fib. Orissa 599 ( Xanthomonas oryzae pv. Oryzae 599 ) , Xanthomonas orissa fib . Xanthomonas oryzae pv.oryzae 710 , Janibacter melonis , Ralstonia solanacearum race 1, Ralstonia solanacearum race 3 3), Candida glabrata , Candida krusei , Candida tropicalis , Saccharomyces cerevisiae 5312 , Berkholderia Glume SL 2870 ( Burkholderia glumae SL 2870), Burkholderia glumae SL 2399 or Burkholderia glumae R1, but is not limited thereto.

Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example

Experiment method

Peptide Library and Synthesis of Individual Peptides

The PS-SPCL package was purchased from Peptide Library Support Facility PLSF, Postech, Pohang, Korea. The peptide library was synthesized by solid multiple method and simultaneous multiple peptide synthesis using N- (9-fluorenyl) methoxycarbonyl chemistry (Fields and Noble 1990). Hexa-peptide SCL was synthesized in the so-called PS format as previously described (Houghten, Pinilla et al. 1991; Houghten, Pinilla et al. 1999; Houghten 2000). The mixture (X) position consisted of a mixture of 19 amino acids with relative proportions appropriately adjusted in yields close to the same molar ratio (excluding cysteine). Each amino acid was synthesized with C-terminal amidation. One set of libraries consisted of 114 tubes (6 positions X 19 amino acids) with a total peptide concentration of 30 mM, while individual peptide concentrations were 12 nM (30 mM / 19 ⁵ ).

Individual peptides were identified by Peptron Co. It was purchased from (Daejeon, Korea). Each of these was purified by prep reverse phase high pressure liquid chromatography with Shiseido capcell pak C18 column. The peptide entity was confirmed by mass spectrometry using HP 1100 series LC / MSD. Each peptide except for the water soluble KRS22 was dissolved in 50% dimethyl sulfoxide. The final concentration of the 10 × stock solution was 5 mM, which was stored at −20 ° C. after aliquot.

Microbial Strains and Culture Conditions

The strains used in the present invention are listed in the table below. All fungal strains were grown in YPD medium (10 g yeast extract, 20 g peptone, 20 g dextrose in 1 L distilled water). MNA medium (Modified NA: 1.0 g of Lab-Lemcobeef extract in 1 L distilled water, 2.0 g of yeast extract, 5.0 g of peptone, 5.0 g of NaCl, 0.45 g of KH ₂ PO ₄ , 2.39 g of Na ₂ HPO ₄ 12H ₂ O, pH 6.8 Janibacter melonis ) . Was used for the culture of Ralstonia and Burkholderia spp. Was grown in King's B medium (KB: 20 g proteose peptone # 3, 1.5 g MgSO ₄ .7H ₂ O, 1.5 g KH ₂ PO ₄ , 15 ml glycerol, 15 g agar in 1 L distilled water). All other strains were grown in Luria-Bertani medium (LB: 10 g tryptone in 1 L distilled water, 5 g yeast extract, 10 g NaCl). All cultures were grown at 30 ° C.

Strain names Habitat / Host Source Abbrebation Clavibacter michiganensis subsp. michiganensis Papper KACC20122 CM Pseudomonas syringae pv. tomato DC3000 Tomato [One] DC3000 Pectobacterium carotovorum subsp. carotovorum Potato KACC10226 PCC Xanthomonas campestris pv. vesicatoria 833 Papper [One] XCV833 Xanthomonas campestris pv. vesicatoria 833 pilA Papper pilA mutant of XCV833 XCV833m Pectobacterium carotovorum subsp . atrosepticum Potato [One] PCA Acidovorax konjaci Soil KACC10652 AC Xanthomonas albilineans Sugar cane [One] XAL Xanthomonas oryzae pv. oryzae 90 Rice [One] X090 Xanthomonas oryzae pv. oryzae 599 Rice [One] X599 Xanthomonas oryzae pv. oryzae 710 Rice [One] X710 Janibacter melonis Melon / Human [One] JM Ralstonia solanacearum race 1 Potato [One] RS1 Ralstonia solanacearum race 3 Potato [One] RS3 Candida glabrata Human [2] CGL Candida krusei Human [2] CKR Candida tropicalis Human [2] CTR Scharomyces cerevisiae 5312 Human [2] S5321 Burkholderia glumae SL 2870 Rice grain [One] BG2870 Burkholderia glumae SL 2399 Rice grain [One] BG2399 Burkholderia glumae R1 Rice grain [3] BGR1 Escherichia coli DH5α Human BRL com. DH5α Pseudomonas syringae pv. putida Soil [One] PSP

KACC: Korean Agricultural Culture Collection, Suwon Korea, [1]; from Dr. Heu, SG in National Institute of agricultular science and technology, Suwon, Korea., [2]; from Dr. Kim, WK in Dept. of Microbiology, Medical school, Suwon, Korea., [3]; from Dr. Hwang, IG in Dept of Applied biology and chemistry, Seoul national university, seoul, Korea., BRL com: BRL Life Technologies ,. Gaithersburg, MD

In Vitro Antimicrobial Activity Assay

In vitro antimicrobial activity of peptides was determined using the microtiter plate assay described previously (Lopez-Garcia, Perez-Paya et al. 2002). Bacterial growth was measured at 595 nm (A ₅₉₅ ) in the Bio-Rad microplate reader 550 (Japan). In all treatments, at least 3 replicates were made. Was turnovers the blanks average A ₅₉₅ values from mock inoculated one row from the A ₅₉₅ value of each well, the average and standard deviation (SD) were calculated for each treatment.

In the analysis of peptide libraries, 90 μl of freshly diluted overnight cultures (final concentration ⁵ × 10 ⁵ cells after 16-24 hours incubation) were mixed with 10 μl peptide mixture from PS-SCL. Growth rate was measured in 16 or 48 hours incubation depending on the strain.

The analysis of sequence-defined peptides was essentially identical. Two final concentrations of peptide were analyzed: 100 μΜ and 10 μΜ. In vitro For bacterial and fungal growth, 180 μl of freshly diluted overnight culture ( ⁵ × 10 ⁵ Cell controlled) was mixed with 20 μl of 10 × peptide stock solution and incubated for 16-48 hours with gentle shaking (80 rpm). After the measurement, the OD is shown as a 595 nm relative value.

Inhibition zone analysis

In order to visually observe the activity, growth inhibition ring analysis was performed. 1 mM peptide stock solution was diluted 7 times in sterile water (twice). A 5 μl aliquot of each sample was then spotted onto the plate and dried for 15 minutes. The first spot of each series contained 5 nmoles of peptide in 5 μl solution. The plate was overlayed with 7 ml top layer soft agar (0.7% wt / vol) mixed with 100 μl of indicator strain (10 ⁵ cells). Pictures were taken after 24 hours incubation at 30 ° C.

MIC and MBC Analysis

MIC (minimal inhibition concentration) was determined by Concannon et. al. The method of (2003) was measured with a slight modification. Freshly grown cultures in the log phase were used as inoculum. The MIC assay procedure was essentially the same as the analysis of sequence-defined peptides, but seven different 2-fold dilution series ranging from 3.13 to 200 μM were used. MIC was defined as the lowest concentration of peptide that prevents visible turbidity, measured at 595 nm using a micro-plate reader (Bio-Rad).

Minimum bactericidal concentration (MBC) was determined by spotting 10 μl from each clear well on agar medium. Cultures (5 μl) were taken from each treated well with a multi-pipette, transferred to completely dried plates and incubated for 16-36 hours. MBC was defined as the lowest concentration that does not allow visual growth on the surface of agar.

Leaf inoculation test

Five leaves of fresh cabbage ( Brassica rapa subsp wonbok ) were separated and surface sterilized by spraying a 5% commercial bleach solution. The surface of the leaves was washed with sterile water spray and gently pated with a paper towel. After 15 minutes of air drying, the leaves were punctured in three places using a pencil cap (0.7 cm in diameter) and wounded. Pectobacterium carotovorum subsp. A 10 μl bacterial suspension (1 × 10 ⁷ cfu / ml) of overnight culture of Carotovorum (typical pathogenic pathogen) was applied to the wound leaf surface. For peptide treatment, three concentrations of KCM21 solution (200, 100, 50 μM) were applied. 10 μl of peptide solution was applied simultaneously with the bacterial suspension or before and after inoculation at 1 hour intervals. For each treatment, 5 replicates were inoculated. Treated leaves were maintained at 28 ° C. and 85% relative humidity. Symptom development was recorded for 2 days. Mock inoculation or bacterial free peptide inoculation did not show any symptoms under these conditions.

In vitro Cytotoxicity Test

Three human cell lines were used for in vitro toxicity testing: human endometrial cancer cells (Hec1A, from ATCC Manassas, VA, USA), and normal human gum cells (HGF, from ATCC Manassas, VA, USA), human uterus Primary cultures of endometrial stromal cells (From Dr. Min Dept. of Biological science, Ajou university, Suwon, Korea). Human cell lines were incubated in RPMI 1640 medium (GibcoBRL) containing 5% fetal bovine serum for 24 hours at 37 ° C. in a CO ₂ incubator prior to exposure to 100 μM peptide dissolved in 1% DMSO solution. After 24 hours treatment, 50 μl of MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) (Sigma) was added and incubated for 3 hours. Then 150 μl of DMSO was added and the plate was shaken for 10 minutes to release formazan. Treated cells were examined for viability measured by their ability to reduce MTT. Detection of cellular conversion of MTT to water insoluble colored formazan was performed according to the procedure described previously (Mosmann 1983). For HGF, 200 μM peptides dissolved in 2% DMSO solution were additionally tested. Cells exposed to 1% or 2% DMSO were used as controls. Compared to the mock control, for HGF cells, the 1% DMSO control showed a 6.8% reduction and the 2% DMSO control showed a 13.4% reduction.

Example 1 Effect of Peptide Library on the Growth of XCV 833

Xanthomonas Campestris FV. Besciatoria ( Xanthomonas campestris pv.vesicatoria ) The effect of peptide library on the growth of strain 833 (XCV833) was examined. Using 96-well microplates, 104 combinational library tubes from Pohang University were treated with 3 replicates each and bacterial growth was measured using a spectrophotometer. If a specific amino acid in a specific position is inhibited growth, for example, the presence of a sequence such as K, W, R in the graph O ₁ XXXXX, it can be seen that the growth is inhibited.

Example 2: Effect of Peptide Library on Growth of CM

The effect of peptide libraries on the growth of Clavibacter michiganensis subsp. M ichiganensis (CM) was investigated. Using 96-well microplates, 104 combinational library tubes from Pohang University were treated with 3 replicates each and bacterial growth was measured using a spectrophotometer. Growth is inhibited when a specific amino acid is present at a specific position, for example, when W, R, etc., are present in the graph XXO ₃ XXX, the antimicrobial activity is shown and growth is inhibited (FIG. 2). Based on the graphs of FIGS. 1 and 2, hexamers may be present in various combinations. The most likely 12 hexamers were selected through repeated experiments or experiments with other bacteria, and individual synthesis was performed.

Table 1 below shows twelve hexa-peptides selected based on the PS-SPCL (Positional-Scanning Synthetic Peptide Combinatorial Library). Each peptide was synthesized with C-terminal amidation (Peptron co.). Of these four effective peptides (underlined KCM11, KCM12, KCM21 and KRS22) were selected for further study.

TABLE 1 Twelve hexa-peptides selected based on PS-SPCL.

One ECM1 EDDDDD 2 ECM2 WEDDDG 3 KCM11 TWWRWW 4 KCM12 KWRWIW 5 KCM21 KWWWRW 6 KCM22 KWWWWR 7 KCM23 NFFFWR 8 KPC22 NRFFWH 9 KRS22 WRWFIH 10 KC21 KRFFRF 11 KC22 WKFFRH 12 KIIC-1 KXXXRX

Example  3: 12 things about bacterial growth Hexa Effect of Peptides

The effects of 12 hexa-peptides on bacterial growth were examined. Whether 12 individual hexamers are effective and which ones are most effective are also incubated for 24 to 48 hours with microorganisms and bacteria and individual hexamers. The relative value was obtained by comparing with the value. 3 shows the effect of twelve hexa-peptides on bacterial growth. A value of 1 on the y-axis of the graph is the OD of the culture without hexamer. Except for ECM peptides, all 10 selected hexamers showed growth inhibition against three bacterial strains with different efficacy. Interestingly, Ralstonia solanacearum (RS) was the most resistant strain to hexa-peptide.

Example  4: 4 kinds Hexa Selective inhibition of peptides

We examined whether four hexamers showed different growth inhibition effects, that is, selectivity, depending on the bacteria. 4 shows selective inhibition of four hexa-peptides. The OD value of the growth rate was compared with the control and replaced with a relative value. It can be seen that the four selected hexamers each have different selectivity for the three species of bacteria. KCM21 is effective against E. coli DH5a but less effective in Ralstonia species. KCM11 and KRS22, in contrast, were effective against RS strains. All results were compared with the growth rate indicated by the OD value of the spec. If the growth rate is 20% or less, the antibacterial effect is relatively strong.

Example 5: Effect of four hexa-peptides on fungal growth

The effect of four hexa-peptides on fungal growth was examined. Experiments comparing the growth rate as in Example 3, this example used a fungus instead of bacteria. 5 shows the effect of four hexa-peptides on fungal growth. In Figure 5, A, B, C and D represent KCM11, KCM12, KCM21 and KRS22 peptides, respectively. As can be seen in Figure 5, it can be seen that the effect of four hexa-peptides against fungi (fungus). Specifically, the effect is slightly reduced to the yeast, in the case of candida can be seen to inhibit normal growth. In particular, KCM11 and KRS22 peptides showed a relatively good effect, KCM12 and KCM21 peptides showed good effects on bacteria, but relatively less effective against fungi.

Example 6 Effect of Four Hexa-Peptides on Growth Inhibitory Rings

The effects of four hexa-peptides on growth inhibitory rings were examined. Previous results showed that the growth rate was inhibited on the medium, but this example visually confirmed that there was actually bacteriocidal (killing) against bacteria. Dilute each peptide (KCM11, KCM12, KCM21, and KRS22) up to 7 times with 1/2 dilution, and then spot them in dry media with 5ul each, mix and mix bacteria and media (0.7% soft agar) on it. Pictures were taken after 24 hours. If there is a bactericidal effect that actually kills the bacteria, a clear zone appears, and the stronger the effect, the larger the size of the transparent ring. Dilution several times at low concentrations also revealed a clear ring.

Table 2 below shows growth inhibition ring analysis for 20 bacterial strains. The lowest concentration (uM) that inhibited bacterial growth on the plate was measured after 24 hours incubation. In Table 2, ng is no growth, x is no clear ring at highest concentration, A: KCM11, B: KCM12, C: KCM21, D: KRS22, LB stands for Luria-Bertani medium, KB stands for Kings B medium Indicates. Table 2 is a table summarizing the experiments performed in FIG. It was difficult to describe the concentration because spotting a certain solution on the medium to spread the medium was counted in terms of the total number of moles of the peptide applied. The lower the number, the better the effect.

Table 2. Growth inhibitory assay results for 20 bacterial strains.

Strains Media A B C D One Acidovorax konjaci LB 2.5 1.3 0.6 5 2 Pseudomonas syringae pv. tomato DC3000 LB 5 0.3 0.6 2.5 3 Ralstonia solanacearum race 1 KB x x x x 4 Pectobacterium carotovorum subsp. carotovorum LB 1.3 0.6 0.6 2.5 5 Escherichia coli DH5a LB x 1.3 1.3 5 6 Clavibacter michiganensis subsp. michiganensis LB 0.3 0.6 0.3 2.5 7 Ralstonia solanacearum race 1 LB ng ng ng ng 8 Pseudomonas syringae pv. putida LB x 0.6 1.3 x 9 Burkholderia glumae SL2387 KB x x x x 10 Burkholderia glumae R1 KB x x x x 11 Pectobacterium carotovorum subsp. atrosepticum LB 5 0.6 0.6 2.5 12 Ralstonia solanacearum race 3 KB x x x x 13 Burkholderia glumae SL2395 KB x x x x 14 Burkholderia glumae SL2399 KB x x x x 15 Xanthomonas campestris pv. vesicatoria 833 LB 0.3 0.6 0.3 2.5 16 Xanthomonas campestris pv. vesicatoria 833 pil A LB 0.3 0.3 0.2 2.5 17 Xanthomonas albilineans LB 1.3 2.5 0.6 5 18 Xanthomonas oryzae pv. oryzae 90 LB 1.3 0.2 0.3 1.3 19 Xanthomonas oryzae pv. oryzae 599 LB 1.3 0.2 0.2 0.6 20 Xanthomonas oryzae pv. oryzae 710 LB 1.3 0.2 0.3 1.3

Example 7: Effect of Four Hexa-Peptides on Cytotoxicity

MTT test was performed to determine if the human body is cytotoxic. 7 shows cytotoxicity assessed using the MTT test. The results were replaced with relative values and the control was treated only with medium. Hexamer at 100 mM concentration was dissolved in 1% DMSO and treated. As previously known, DMSO has a function of lysing cells at room temperature. Thus, it was the comparison using 1% DMSO as a 2 ^nd control. If the relative value is lower than the M control, cytotoxicity can be suspected, almost all higher than the 2 ^nd control. When treated with KCM11 peptide in primary cells, it was slightly lower but was not statistically significant. Therefore, since the peptides of the present invention could not find a cytotoxicity potential, it can be seen that the peptides are likely to be safe for humans.

Example 8 Effect of KCM21 Peptide on Disease Development

Indeed, it was examined whether the peptide of the present invention can prevent disease progression in vivo without using a fungicide that is toxic to humans or an antibiotic that has environmental problems. Figure 8 is caused by Pectobacterium carotovorum subsp. C arotovorum Show the effect of KCM21 on symptom development. Panel AC is not well distinguished since it is only one day of inoculation. Panel D is inoculated at the same time with the peptide and bacteria, showing that the dose-dependent symptoms are reduced depending on the concentration. Panel E shows that treating the peptides prior to inoculation also has a control effect. Panel F shows that treatment of the peptide about one hour after inoculation is not very effective.

Table 3 below shows MIC and MBC using 21 bacteria.

Table 3. MIC and MBC results for 21 strains.

KCM11 Test strain MIC (μM) MBC (μM) Clavibacter michiganensis subsp. m ichiganensis 13 25 Pseudomonas syringae pv. tomato DC3000 50 50 Pectobacterium carotovorum subsp. carotovorum 200 < 200 < Xanthomonas campestris pv. vesicatoria 833 25 50 Xanthomonas campestris pv. vesicatoria 833 pila 25 50 Pectobacterium carotovorum subsp. atrosepticum 50 50 Acidovorax konjaci 25 25 Xanthomonas albilineans 25 25 Xanthomonas oryzae pv. oryzae 90 6.25 25 Xanthomonas oryzae pv. oryzae 599 6.25 6.25 Xanthomonas oryzae pv. oryzae 710 6.25 6.25 Janibacter melonis 3.13 6.25 Ralstonia solanacearum race 1 200 < 200 < Ralstonia solanacearum race 3 200 < 200 < Candida glabrata 200 < 200 < Candida krusei 200 200 < Candida tropicalis 200 < 200 < Scharomyces cerevisiae 5312 200 < 200 < Burkholderia glumae SL 2870 200 < 200 < Burkholderia glumae SL 2399 200 < 200 < Burkholderia glumae R1 200 < 200 <

When observed on a microplate, the concentrations that do not grow at all in the liquid medium (confirmed by using a spectrophotometer, MIC) were measured at the same time by removing some of the ungrown wells and plating them on the medium. (MBC). Although there was a definite bactericidal effect on the plant bacterial pathogens in question in most of Korea, some bacteria such as Burkholderia, Ralstonia, and Pectobacterium had no bactericidal effect (at 200uM concentration). This means that as shown in FIGS. 1 to 5, growth inhibition is possible but not complete sterilization.

Although the bactericidal effect on the fungus is insignificant, the bactericidal effect was confirmed when the KRS22 peptide was treated with candida krusei.

references

Concannon, S. P., T. D. Crowe, et al. (2003). "Susceptibility of oral bacteria to an antimicrobial decapeptide." J Med Microbiol 52 (Pt 12): 1083-93.

Fields, GB and RL Noble (1990). "Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids." Int J Pept Protein Res 35 (3): 161-214.

Houghten, RA (2000). "Parallel array and mixture-based synthetic combinatorial chemistry: tools for the next millennium." Annu Rev Pharmacol Toxicol 40 : 273-82.

Houghten, RA, C. Pinilla, et al. (1999). "Mixture-based synthetic combinatorial libraries." J Med Chem 42 (19): 3743-78.

Houghten, RA, C. Pinilla, et al. (1991). "Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery." Nature 354 (6348): 84-6.

Lopez-Garcia, B., E. Perez-Paya, et al. (2002). "Identification of novel hexapeptides bioactive against phytopathogenic fungi through screening of a synthetic peptide combinatorial library." Appl Environ Microbiol 68 (5): 2453-60.

Mosmann, T. (1983). "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays." J Immunol Methods 65 (1-2): 55-63.

1 shows Xanthomonas Campestris fV. Bessictoria ( Xanthomonas campestris pv. Vesicatoria The positional scanning of the synthetic peptide combination library performed for strain 833 (XCV833) is shown. Each panel shows the results obtained for a pool of peptides with known amino acids at each of the six positions of the hexapeptide. The six positions are designated individually for each of the 19 L-amino acids (O1 and O2...). The remaining five positions consist of a mixture (X) of 19 L-amino acids (excluding cysteine). The library consists of 114 peptide pools: The total PS-SPCL consists of 47,045,881 different peptides. Each peptide mixture was analyzed for overnight culture (diluted with 1-5 × 10 ⁶ ) of XCV833 in 96 well microtiter plates. Bacterial cells were incubated at 30 ° C. for 24 hours with shaking (80 rpm). Bacterial growth rates were measured using a microtiter reader (Biorad). * Indicates peptide pools that induce transient bacterial cell lysis or aggregation with turbid color in bacterial cell culture immediately after peptide pool treatment.

FIG. 2 shows positional scanning of the synthetic peptide combination library performed on Clavibacter michiganensis subsp. Michiganensis (CM). Each peptide mixture was analyzed for overnight culture (diluted with 1-5 × 10 ⁶ ) of CM in 96 well microtiter plates. Bacterial cells were incubated at 30 ° C. for 24 hours with shaking (80 rpm). Bacterial growth rate was measured using a microtiter reader (Biorad).

3 shows the effect of twelve hexa-peptides on bacterial growth. Except for ECM peptides, all 10 selected hexamers showed growth inhibition against three bacterial strains with different efficacy. Interestingly, Ralstonia solanacearum (RS) was the most resistant strain to hexa-peptide.

4 shows selective inhibition of four hexa-peptides. Inhibition of bacterial growth ( Ralstonia solanacerum race 1 (RS1), race 3 (RS3) and E. coli DH5α) was measured as OD at 600 nM after 17 h incubation. Values are relative values compared to untreated control. A: KCM11, B: KCM12, C: KCM21, D: KRS22.

5 shows the effect of four hexa-peptides on fungal growth. CK: Candida krusei , CT: Candida tropicalis, SC: Scharomyces cerevisiae 5312.

6 shows the MIC of four hexa-peptides. The first spot of each series contained 5 nmoles of peptide in 5 μl solution. Dilution series (1: 1 dilution) were spotted on the plate and dried for 15 minutes. ⁷ ml top layer soft agar medium containing 10 ⁵ cells of test bacteria was applied to the plate. Pictures were taken after 24 hours incubation at 30 ° C. Panel A: Xanthomonas albilineans, Panel B: Xanthomonas campestris pv. vesicatoria 833 . a: KCM11, b: KCM12, c: KCM21, d: KRS22.

7 shows cytotoxicity assessed using the MTT test. Primary culture cell lines (human endometrial stromal cells) and human endometrial cancer cell lines (Hec1A) were used. a: KCM11, b: KCM12, c: KCM21, d: KRS22, M: 1% DMSO control.

Figure 8 is caused by Pectobacterium carotovorum subsp. C arotovorum Show the effect of KCM21 on symptom development. On each cabbage leaf, the top, middle and bottom spots were simultaneously (A, D) with 200 μM, 100 μM and 0 μM KCM21 or before (B, E) or 1 hour intervals after pathogen inoculation (C , F) injected. AC : 1 day after inoculation. DF: 2 days after inoculation.

<110> AJOU UNIVERSITY INDUSTRY-ACADEMIC COOPERATION FOUNDATION <120> Antimicrobial peptide KCM11 against phytopathogenic bacteria <130> PN07008 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> KCM11 peptide <400> 1 Thr Trp Trp Arg Trp Trp   1 5  

Claims (5)

Clavibacter michiganensis subsp. Michiganensis , Pseudomonas syringe fib , consisting of the amino acid sequence of SEQ ID NO: 1. Tomato DC3000 ( Pseudomonas syringae pv. Tomato DC3000), Xanthomonas campestris fib. Bessictoria 833 ( Xanthomonas campestris pv.vesicatoria 833), Zantonamonas Campestris fV . Besiccataria 833 pila ( Xanthomonas campestris pv.vesicatoria 833 pila ), Pectobacterium carotovorum subsp.Atrosepticum , Acidovorax konjaci , Xanthomonas Abilinins ( Xanthomonas albilineans ), Xanthomonas Orissa F.B. Oriza 90 ( Xanthomonas oryzae pv. Oryzae 90 ), Xanthomonas Orissa F.B. Oriza 599 ( Xanthomonas oryzae pv. Oryzae 599 ), Xanthomonas Orissa F.B. Oriza 710 ( Xanthomonas oryzae pv. Oryzae 710 ) and Johnny bakteo Mello varnish (Janibacter melonis) antimicrobial peptide KCM on phytopathogenic microorganism is selected from the group consisting of (K illing related peptide with C lavibacter m ichiganensis sub. Michiganensis) 11. delete delete The antimicrobial peptide KCM11 of claim 1 wherein the peptide is modified by amidation, esterification, acylation, acetylation, PEGylation, or alkylation. A method for inhibiting the growth of a plant pathogenic microorganism according to claim 1, comprising administering to the plant an effective amount of the microbial growth inhibitory effect of the antimicrobial peptide KCM11 of claim 1.

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