Purification and identification of two antifungal cyclic dipeptides from Bacillus cereus subsp. thuringiensis associated with a rhabditid entomopathogenic nematode especially against Fusarium oxysporum

Abstract

The cell-free culture filtrate of Bacillus cereus subsp. thuringiensis associated with an entomopathogenic nematode (EPN), Rhabditis (Oscheius) sp., exhibited strong antimicrobial activity. The ethyl acetate extract of the bacterial culture filtrate was purified by silica gel column chromatography to obtain two cyclic dipeptides (CDPs). The structure and absolute stereochemistry of this compound were determined based on extensive spectroscopic analyses (FABMS, ¹H NMR, ¹³C NMR, ¹H--¹H COSY, ¹H--¹³C HMBC) and Marfey’s method. The compounds were identified as cyclo(D-Pro-L-Met) and cyclo(D-Pro-D-Tyr). CDPs showed significantly higher activity than the standard fungicide bavistin against agriculturally important fungi, viz., Fusarium oxysporum, Rhizoctonia solani and Penicillium expansum. The highest activity of 2 µg/ml by cyclo(D-Pro-D-Tyr) was recorded against F. oxysporum, a plant pathogen responsible for causing fusarium wilt followed by R. solani, a pathogen that causes root rot and P. expansum. To our knowledge, this is the first report on the isolation of these compounds from Rhabditis EPN bacterial strain Bacillus cereus subsp. thuringiensis.

• Antimicrobial
• Bacillus cereus subsp. thuringiensis
• cyclic dipeptides
• Fusarium oxysporum

Introduction

Plant pathogenic fungi have evolved many ways to infect their hosts and can have devastating effects on commercial crop production. The fungus Fusarium oxysporum is found in both agricultural and non-cultivated soils throughout the world. The species consists of non-pathogenic and pathogenic isolates, both known as efficient colonizers of the root rhizosphere. The pathogenic isolates, grouped into formae specialis depending on their host range1, cause wilt or rot disease in important agricultural and ornamental plant species, such as tomato, banana, cotton and tulip bulbs, thereby causing serious problems in commercial crop production2,3. Recently, F. oxysporum has also been reported as an emerging human pathogen, causing opportunistic mycoses4. Many strategies to control this fungal pathogen have been investigated5,6. Currently, the most effective method in preventing crops from fusarium wilt is to mix the seed with chemical fungicides. The application of chemical fungicide induces other problems, such as harm to other living organisms and the reduction of useful soil microorganisms7,8. Therefore, public concern is focused on alternative methods of this fungal control, which can play a key role in natural product to reduce the concentration of chemical pesticides9. A promising strategy for the replacement of chemical pesticides has been the implementation of microbial natural product-based compounds.

The entomopathogenic nematode (EPN) Heterorhabditis and Steinernema together with their symbiotic bacteria Photorhabdus and Xenorhabdus, respectively, are obligate and lethal parasites of insects10. Infective juveniles (IJs), the only free-living stage, enter hosts through natural openings (mouth, anus and spiracles), or in some cases, through the cuticle. After entering the host’s hemocoel, nematodes release their bacterial symbionts11. The bacteria multiply rapidly and produce various metabolites that can overcome the insect immune system, kill the insect and inhibit the growth of various fungal and bacterial competitors12–14. By doing so, the bacterial symbionts are believed to prevent putrefaction of the insect cadaver and establish conditions that favor the development of both the nematode and bacterial symbionts. The antimicrobial nature of metabolites produced by Xenorhabdus spp. and Photorhabdus spp. is known, and several compounds with antibiotic activity have been isolated and identified. These include indoles15, stilbenes16, xenorhabdins17, xenocoumacin18, nematophin19, benzylineacetone20, xenortides and xenematide21, and cyclolipopeptide (PAX)22. These metabolites not only have diverse chemical structures, but also have a wide range of bioactivities of medicinal and agricultural interests such as antibiotic, antimycotic, insecticidal, nematicidal, antiulcer, antineoplastic and antiviral.

During our studies on EPN, we isolated a new EPN Rhabditis sp. from sweet potato weevil grubs collected from Central Tuber Crops Research Institute (CTCRI) farm, Thiruvananthapuram. To our knowledge, this is the first time Rhabditis sp. is being studied as an EPN. A specific bacterium isolated from third-stage IJs of the nematode or from the haemolymph of nematode infested Galleria mellonella larvae was found to be pathogenic to a number of insect pests (unpublished results). The antimicrobial substance producing bacterium was isolated from an entomopathogenic, Rhabditis (Oscheius) sp. resembling Rhabditis isolate Tumian 2007 at D2 and D3 expansion segments of 28S rDNA23. Molecular analyses revealed that the bacterium resembles Bacillus sp. (Accession No. CP001407). The bacterium has been deposited in IMTECH (Institute of Microbial Technology, Chandigarh, India) and the accession number is MTCC 5234. The cell-free culture filtrate of the bacteria was found to inhibit several pathogenic bacteria, fungi and a plant-parasitic nematode (Meloidogyne incognita)24, suggesting that it could be a rich source of biologically active compounds. In this paper, we report the isolation, structure elucidation and antimicrobial activity of two diketopiperazines, especially against plant pathogenic fungi from the cell-free culture filtrate of the bacterium.

Materials and methods

Chemicals and media

All the chemicals used for extraction and column chromatography were of analytical grade and high performance liquid chromatography (HPLC) grade methanol was from Merck Limited, Mumbai, India. Silica gel (230–400 mesh) used for column chromatography and precoated silica gel 60 GF₂₅₄ plates used for thin layer chromatography (TLC) were from Merck Limited, Darmstadt, Germany. Microbiological media were from Hi-Media Laboratories Limited, Mumbai, India. All other reagents were of analytical grade and the other chemicals used in this study were of the highest purity. The standard antibiotics ciprofloxacin, cefotaxime and amphotericin B were purchased from Sigma Aldrich (St. Louis, MO). The software used for the chemical structure drawing was Chemsketch Ultra, Advanced Chemistry Development Inc, Toranto, Canada.

Test microorganisms

Agriculturally important fungi: F. oxysporum MTCC 284, Rhizoctonia solani MTCC 4634, and Penicillium expansum MTCC 2006 and medically important fungi: Aspergillus flavus MTCC 183, Candida albicans MTCC 277; gram-positive bacteria: Bacillus subtilis MTCC 2756, Staphylococcus aureus MTCC 902; gram-negative bacteria: Escherichia coli MTCC 2622, Pseudomonas aeruginosa MTCC 2642. All the test microorganisms were purchased from Microbial Type Culture collection Centre, IMTECH, Chandigarh, India.

Isolation of Bacillus cereus subsp. thuringiensis

The bacterium was isolated from third-stage IJs of the nematode sample collected from sweet potato weevil grubs or from the haemolymph of nematode infested G. mellonella larvae. The strain was identified as Bacillus sp. (Accession No. CP001407) based on 16S rDNA and BLAST analysis. The strain was currently deposited in IMTECH (Institute of Microbial Technology, Chandigarh, India) and the accession number is MTCC 5234.

Fermentation and extraction

The bacterial fermentation was carried out using modified Tryptic soya broth (TSB) (tryptone 17 g/l, soytone 3.0 g/l, glucose 2.5 g/l, NaCl 5.0 g/l, yeast extract 10 g/l, water 1000 ml). A single colony of Bacillus sp. N strain from the agar plate was inoculated into the flask containing 100 ml sterile media. The flasks were incubated in a gyrorotatory shaker (150 rpm) at 30 °C in dark for 24 h. When the optical density of the culture at 600 nm was approximately 1.7, the bacterial cultures were transferred aseptically into 400 ml sterile medium and incubated in the gyrorotatory shaker at 30 °C in dark for 96 h. The culture media were then centrifuged (10 000 g, 20 min, 4 °C) followed by filtration through a 0.45 µm filter, to obtain cell-free culture filtrate. Thirty litres of cell-free culture filtrate were neutralized with concentrated hydrochloric acid and extracted with an equal volume of ethyl acetate thrice. The ethyl acetate layers were combined, dried over anhydrous sodium sulphate and concentrated at 30 °C using a rotary flash evaporator.

Purification of bioactive compounds

The oily yellow residue (9.3 g) obtained after drying was then loaded on a silica gel column (25 × 600 mm) previously equilibrated with hexane and eluted successively with 200 ml of 100% hexane, 200 ml of linear gradient hexane: dichloromethane (v/v, 75:25 to 25:75), 200 ml of 100% dichloromethane, 200 ml of linear gradient dichloromethane:ethyl acetate (v/v, 95:5 to 5:95), 200 ml of 100% ethyl acetate and finally with 200 ml of 100% methanol. Two fractions (100 ml each) were collected from each combination. Two fractions yielded white crystal compounds which were further purified by crystallization using hexane and benzene and the antibacterial activity of these fractions were determined by well diffusion assay against B. subtilis, which was selected as initial test microorganism.

TLC and HPLC

An aliquot of crystal compounds was loaded on the silica gel TLC plates (20–20 cm). The plates were developed using benzene: acetone (70:30) solvent for first and benzene:acetone (60:40) solvent for second compound. The spots were located by exposing the plate to iodine fumes. The purified crystal compounds were tested for their purity using HPLC, using LC-10AT liquid chromatograph (LC, Shimadzu, Singapore) equipped with a C-18 column (5 μm, 4.6 × 250 mm) and 100% methanol as a mobile phase with a flow rate of 1 ml/min. Ultraviolet (UV) detection was carried out with a diode array detector (Shimadzu).

Characterization of bioactive antifungal compound

UV spectrophotometer

UV-visible spectrum of the pure compounds was recorded on a Systronics Double Beam Spectrophotometer 2201, Ahmedabad, India at room temperature (scanning range 190–800 nm). About 1 mg of the isolated compound dissolved in 20 ml of chloroform was used to record the spectrum.

Nuclear magnetic resonance

The structure of the compounds was determined using nuclear magnetic resonance (NMR) spectroscopy (Bruker DRX 500 NMR instrument, Bruker, Rheinstetten, Germany) equipped with a 2.5-mm microprobe. NMR spectrometer using CDCl₃ was deployed to measure ¹H and ¹³C and 2D NMR. All spectra were recorded at 23 °C. One-dimensional ¹H NMR experiments as well as two-dimensional ¹H–¹H correlation spectroscopy, ¹H–¹³C heteronuclear multiple bond correlation and ¹H–¹³C heteronuclear multiple quantum coherence (HMQC) experiments were performed according to Bruker standard pulse sequences. Proton chemical shifts were determined from one-dimensional ¹H NMR and from HMQC experiments, and ¹³C chemical shifts were determined from HMQC and ¹H–¹³C heteronuclear multiple bond correlation experiments. Chemical shifts are reported relative to the solvent peaks (CDCl₃: ¹H d 7.24 and 13C d 77.23).

High resolution mass spectrophotometer

High resolution mass spectrophotometer (HRMS) was performed on a Thermo Scientific Exactive Orbitrap LC-Mass Spectrometer (Massachusetts) with an electrospray ionization mode.

Optical rotations

Optical rotation of the compounds was measured using a Rudolph Research Autopol III polarimeter (New Jersey) at 25 °C in acetone.

Differential scanning calorimetry

The melting point of the pure compounds was measured with a differential scanning calorimeter in a Mettler Toledo DSC 822e instrument (Mettler-Toledo, Schcoerfenbach, Switzerland). Temperature ranges from 30 °C to 300 °C were employed.

Absolute configuration determination of compounds

A solution of two compounds (1.5 mg) in 6 M HCl (1 ml) was heated to 120 °C for 24 h. The solution was then evaporated to dryness and the residue redissolved in H₂O (100 μl) and was then placed in a 1 ml reaction vial and treated with a 2% solution of FDAA (200 μl) in acetone followed by 1.0 M NaHCO₃ (40 μl). The reaction mixture was heated at 47 °C for 1 h, cooled to room temperature, and then acidified with 2.0 M HCl (20 μl). In a similar fashion, standard d- and l-amino acids were derivatized separately. The derivatives of the hydrolysates and standard amino acids were subjected to HPLC analysis (Shimadzu LC-20AD, C18 column; 5 μm, 4.6 × 250 mm; 1.0 ml/min) at 30 °C using the following gradient program: solvent A, water +0.2% TFA; solvent B, MeCN; linear gradient 0 min 25% B, 40 min 60% B, 45 min 100% B; UV detection at 340 nm25.

Antimicrobial activity

Antifungal activity

Minimum inhibitory concentration (MIC) was determined using potato dextrose agar media against the standard fungicide bavistin by the poisoned food technique26 against A. flavus, F. oxysporum, R. solani and P. expansum. A stock solution of 2000 µg/ml of the test compound was prepared, which was further diluted with methanol to give the required concentrations 1000 to 1 µg/ml. One tube was used as solvent control. For C. albicans, the broth dilution method27 was adopted using potato dextrose broth against the standard fungicide amphotericin B. All experiments were in triplicate for each treatment against each fungus.

Antibacterial activity

The test compounds and antibiotics were screened for antimicrobial activity using the macro dilution method recommended by the National Committee for Clinical Laboratory Standards, USA28. To determine the MICs, the compounds were dissolved in methanol to give a stock concentration of 2000 μg/ml while the antibiotics were dissolved in sterile distilled water to give stock concentrations of 1000 μg/ml. All stock concentrations of compounds and antibiotics were filter-sterilized using 0.2 μm syringe filter. Two-fold serial dilutions of the antibiotics and compounds were made with nutrient broth to give concentrations ranging from 1 to 1000 μg/ml. Colony suspensions equivalent to a 0.5 McFarland standard were prepared and inoculated onto antibiotic-containing medium to yield a final inoculum of 10⁴ CFU/ml. The tubes were incubated at 35 °C for 24 h. The MIC was defined as the lowest antibiotic concentration showing no growth. Triplicate sets of tubes were maintained for each concentration of test sample.

Agar disc-diffusion method

In vitro antibacterial and antifungal activity of the compounds was measured using agar disc-diffusion assay against the test bacteria and fungi29. The sterile discs were impregnated with MIC concentration of test compounds. The ciprofloxacin (5 µg/disc) was used as positive reference standards for bacteria. Amphotericin B (Sigma) was used as reference standard for C. albicans. Bavistin was used as reference standard against other fungi. The antimicrobial activity was evaluated by measuring the zone of growth inhibition surrounding the discs. All the assays were carried out in triplicate.

Statistical analysis

All statistical analyses were performed with SPSS (Version 17.0; SPSS, Inc., Chicago, IL). Data for disc-diffusion assay was presented as means ± standard deviation. Statistical significance was defined as p < 0.0530.

Results

Isolation and purification of bioactive compounds

The ethyl acetate extract of the cell-free culture filtrate of the bacteria showed antifungal activity against F. oxysporum. Silica gel column chromatography of this extract yielded two crystal compounds eluted at 20% ethyl acetate in DCM (DKP 1) and 55% ethyl acetate in DCM (DKP 2). These crystal compounds were further purified by crystallization using hexane and benzene. Initial bioactivity of these compounds was confirmed by testing against F. oxysporum.

Identification of bioactive compound

The pure compounds were subjected to various spectroscopic analyses, i.e. UV, NMR and HRMS. The structure of these two compounds corresponded to two different cyclic dipeptides (CDPs) (DKP 1--2). The cyclic dipeptide identified are Cyclo (D-Pro-L-Met) and Cyclo (D-Pro-D-Tyr) (Figure 1). The ¹H and 13C NMR spectra in all the DKPs showed the signal at δH 4.10 (t) with J value around 9 Hz with a corresponding δC of 59.0, along with multiplets around δH 2.3, 2.0 and 3.5 ppm, clearly indicated the presence of a proline residue. By comparison of NMRs of standard amino acid residue’s chemical shifts and multiplicities it was clear that DKP 2 contains tyrosine residues. Presence of an amide proton in all the DKPs along with mass spectral information and by comparison of standard DKPs it is decisive that they are all CDPs containing proline residue. Additional spectral information such as HMQC and ¹H–¹H COSY was also used. HMQC information was used to transfer assigned numbers from carbons in 13C NMR to the protons in ¹H NMR. The key COSY cross-peaks between C9-C8, C1-C8 and δH 2.13 (s) in DKP 1 indicated the presence of methionine residue; COSY cross-peaks between C1–C8 and COSY cross-peaks between C1–C8, and δH 6.8 (d, 2H) and 7.0 (d, 2H) DKP 2 indicated the presence of Tyrosine residue. The retention times of the HPLC analysis of the 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA, Marfey’s reagent) derivatives of L-Pro, D-Pro, L-Tyr, D-Tyr, L-Met, D-Met are presented in the Table 1 and Supplementary figures 1–3. FDAA derivatives of the acid hydrolysates of Pro and Met in the hydrolysate of DKP 1 were 18.281 and 22.211 min, respectively (Figure 2A). The FDAA derivatives of Pro and Tyr in the hydrolysate of DKP 2 were 18.326 and 20.727 min, respectively (Figure 2B).

Figure 1. Structure of DKPs: (A) cyclo(D-Pro-L-Met) and (B) cyclo-(D-Pro-D-Tyr).

Figure 2. HPLC profile of FDAA derivatives of the acid hydrolysates of DKPs: (A) cyclo(D-Pro-L-Met) and (B) cyclo-(D-Pro-D-Tyr).

Table 1. HPLC reversed-phase retention times of the standard L and D-amino acid and CDPs derivatives.

Amino acid derivatives	Retention time (min)
L-Pro	17.451
D-Pro	18.394
L-Met	22.225
D-Met	24.404
L-Tyr	20.514
D-Tyr	33.084

DKP 1: Cyclo (D-Pro-L-Met), (3R,8aS)-3-[2(methylsulfanyl)ethyl]hexahydropyrrolo[1,2-a] pyrazine-1,4-dione: 13 mg; white amorphous powder; R_f 0.45 (35% Benzene in acetone); HPLC retention time: 2.711 min (Figure 3A); melting point: 155.03 °C (Figure 4A);  = −85.3° (c = 0.10, EtOH); UV max: 210 nm (MeOH); NMR data (Table 2); HRMS [M + H]⁺ C₁₀H₁₇O₂N₂S calcd. for m/z 229.10053, found 229.10033 (Supplementary figure 4).

Figure 3. HPLC chromatogram of diketopiperazines on a reversed-phase C18 column (LC-20AD). Samples of 15 µl were injected to a column (250 mm × 4.6 mm × 5 mm), eluted with 100% methanol (A) cyclo(D-Pro-L-Met), retention time is 2.711 min. The calculated purity is 98% based on the peak area (B) cyclo-(D-Pro-D-Tyr), retention time is 2.820 min. The calculated purity is 92% based on the peak area.

Figure 4. DSC curves of diketopiperazines. Temperatures corresponding to the onset of transition and midpoint of the transition region and enthalpy (ΔH) were recorded by means of the built-in software: (A) cyclo(D-Pro-L-Met) and (B) cyclo-(D-Pro-D-Tyr).

Table 2. NMR data for DKPs in CDCl₃ (δ in ppm, J in Hz).

	DKP 1		DKP 2
Carbon No.	^1H	^13C	^1H	^13C
1	4.24 (t, J = 5.6)	54.5	4.24 (dd, J = 3.0, 10.0)	56.3
OH			6.88 (s)
NH	7.27 (s)		5.92 (s)
2		165.4		165.2
3	4.17 (t, J = 7.5)	59.0	4.1 (t, J = 7.5)	59.1
4		170.4		169.7
5	2.39	27.9	2.03, 2.34	28.3
6	2.06	21.9	1.93	22.5
7	3.59	45.7	3.58, 3.65	45.4
8	2.03	28.0	2.77 (dd, J = 10.0, 14.5), 3.49 (dd, J = 4.0, 14.5)	35.9
9	2.76 (td, J = 2.5, 7.5)	29.1		126.9
10	2.16	15.2	7.06 (d, J = 8.0, H10,10′)	130.3
11			6.79 (d, J = 8.5, H11,11′)	116.1
12				155.6
Ph				165.2

DKP 2: Cyclo (D-Pro-D-Tyr), (3R,8aR)-3-(4-hydroxybenzyl)hexahydropyrrolo[1,2-a]pyrazine-1,4-dione: 16 mg; white amorphous powder; R_f 0.32 (60% Benzene in acetone); HPLC retention time: 2.820 min (Figure 3B); melting point: 148.93 °C (Figure 4B);   = +209.0° (c = 0.11, EtOH); UV max: 278 nm (MeOH); NMR data (Table 2); HRMS [M + H]⁺ C₁₄H₁₇O₃N₂ calcd. for m/z 261.12337, found 261.12333 (Supplementary figure 5).

Antimicrobial activity

The data of antimicrobial activity of DKPs are shown in Tables 3–5. DKPs recorded considerably good activity against test organisms. DKPs showed significantly higher activity against agriculturally important fungi viz. F. oxysporum, R. solani and P. expansum. The highest activity of 2 µg/ml by DKP 2 was recorded against the plant pathogen F. oxysporum, which causes vascular wilt in plants. DKPs were also found to be very effective against R. solani and significant MIC was recorded by DKP 1 (4 µg/ml).The two DKPs were found to be having superior antifungal activity than the standard fungicide bavistin against F. oxysporum, R. solani and P. expansum. DKP 2 recorded lowest MIC against E. coli (16 µg/ml).

Table 3. MIC of diketopiperazines against test fungi.

		MIC (µg/ml)
Test organisms	DKP 1	DKP 2	Bavistin	Amphotericin B
A. flavus	32	64	100	–
C. albicans	64	32	–	64
F. oxysporum	4	2	32	–
R. solani	4	8	32	–
P. expansum	16	8	64	–

The values represents mean of three replications.

“–” denotes not tested.

Table 4. MIC and MBC of diketopiperazines against test bacteria.

	MIC (µg/ml)
	DKP 1		DKP 2		Cefotaxime		Ciprofloxacin
Test organisms	MIC	MBC	MIC	MBC	MIC	MBC	MIC	MBC
B. subtilis	32	64	32	64	100	100	5	5
S. aureus	16	16	64	64	250	250	5	5
E. coli	125	250	16	32	100	250	5	5
P. aeruginosa	32	64	16	16	500	1000	10	10

The values represent mean of three replications.

Table 5. Antimicrobial activity of diketopiperazines.

	Zone of inhibition (diameter in mm)
Test organisms	DKP 1	DKP 2	Ciprofloxacin	Bavastin	Amphotericin B
A. flavus	18 ± 1	20 ± 0	*	24 ± 1.52	*
C. albicans	11 ± 1	17 ± 0.57	*	*	24 ± 1.73
F. oxysporum	34 ± 1.52	32 ± 0	*	16 ± 0	*
R. solani	31 ± 1	32 ± 0.57	*	19 ± 1.52	*
P. expansum	20 ± 0.57	30 ± 0.57	*	24 ± 1.15	*
B. subtilis	20 ± 1	21 ± 0	31 ± 0.57	*	*
S. aureus	18 ± 0	23 ± 0.57	31 ± 0	*	*
E. coli	15 ± 1	25 ± 0.57	28 ± 1.52	*	*
P. aeruginosa	19 ± 1.52	23 ± 0	25 ± 0.57	*	*

(*) not tested.

Discussion

CDPs [also known as 2,5-dioxopiperazines; 2,5-diketopiperazines; cyclo(dipeptides); or dipeptide anhydrides] are relatively simple compounds and, therefore, are among the most common peptide derivatives found in nature. CDPs are more than simple curiosities31 and are well known for their economically beneficial biological activities32. CDPs are relatively simple compounds, and therefore are among the most common peptide derivatives found in nature32. They have been isolated from microorganisms, sponges and from a variety of tissues and body fluids33–35.

However, with a growing awareness of the diversity and biological roles played by many of the diketopiperazines found in nature, interest in these compounds has grown36. Due to their relative simplicity37 and stability32, diketopiperazines provide excellent models for theoretical studies as well as the development of pharmaceutical compounds. The linear form of the dipeptide is often less stable in vivo than its cyclic counterpart, therefore making the latter variety far more promising in terms of drug candidacy36. Both natural and synthetic diketopiperazines have a wide variety of biological activities, including antitumor38, antiviral39, antifungal40 and antibacterial41. Especially, 2,5-diketopiperazines recently have attracted attention due to their biological properties42. The wide spectrum of their biological properties points to various therapeutic possibilities.

The cyclo(Pro-Tyr) was first isolated from Alternaria alternate35,43. Cyclo(Pro-Tyr) was also isolated from Streptomyces sp. TN25644. Cain et al.44 reported that cyclo(Pro-Tyr) exhibits no activity against strains of Micrococcus luteus, Mycobacterium smegmatis, Sacharomyces cerevisiae, Candida neoformans, Candida albicans and Aspergillus niger. Smaoui et al.45 reported the antimicrobial activity of Cyclo(Pro-Tyr) against Micrococcus luteus LB 14110, S. enterica ATCC43972 and Fusarium sp. Cyclo(Pro-Tyr) demonstrated inhibitory effects against bioluminescence by V. harveyi46.

Cyclo-(L-Pro-L-Met) was first reported from Antarctic sponge-associated bacterium, P. aeruginosa47 and marine-derived actinomycete, Nocardiopsis sp. 03N6748. Nothing is known about the antimicrobial activity of this compound in literatures. To our best knowledge this is the first report of the antimicrobial activity of cyclo(D-Pro-L-Met).

The diketopiperazines are heterocyclic compounds which exist in DD, DL, LD and LL forms. The stereochemistry of the compounds play an important role in biological activity. Li et al.49 reported the production of two stereoisomeric CDPs, cyclo(L-Tyr-L-Pro) and cyclo(L-Tyr-D-Pro) from Lactobacillus reuteri, where cyclo(L-Tyr-L-Pro) is more effective against S. aureus which causes menstruation-associated toxic shock syndrome (TSS) in human.

We report herewith the antimicrobial effects of the CDPs DKPs 1–2 assayed against both medicinally and agriculturally important bacterium and fungi viz. B. subtilis, S. aureus, E. coli, P. aeruginosa, A. flavus, C. albicans, F. oxysporum, R. solani and P. expansum. The results showed that our DKPs exhibit potent inhibitory values in the range of µg/ml. The plant pathogen F. oxysporum, causing fusarium wilting, chlorosis, necrosis, premature leaf drop, browning of the vascular system, stunting and damping-off etc, and R. solani, associated with plant diseases such as collar rot, root rot, damping-off, wire stem etc, P. expansum, which causes post-harvest decay of stored apples and oranges, were strongly inhibited by three DKPs and the activity is superior than the standard antifungal agent bavistin.

Origin of DKPs has been questioned, once several DKPs have been found in fermentation broths and cultures of yeast, as well as in lichens and fungi31. It is known that DKPs can be generated via non-enzymatic cyclization of linear dipeptides at extreme temperatures50. In our study, such a possibility of formation of DKPs during the fermentation process was ruled out by heat sterilization and incubation of the LB medium without Bacillus sp. N strain since we did not observe any detection of DKPs in the HPLC profiles of the corresponding ethyl acetate extracts.

Conclusion

In conclusion, cyclo(D-Pro-L-Met), cyclo (D-Pro-D-Tyr) and other diketopiperazines are the smallest cyclic peptides, commonly biosynthesized from amino acids by different organisms, including mammals, and are considered to be secondary functional metabolites or side products of terminal peptide cleavage and mainly act as cell signaling molecules. The antifungal activities of the compounds especially against plant pathogenic fungi F. oxysporum, R. solani and P. expansum have never been known. Here, we identified these compounds as a new antifungal agent against plant pathogenic fungi. Antifungal activity of compounds is an encouraging bioprobe to develop new antimicrobial therapeutics from such type of small molecules in near future.

Supplementary materials

Supplementary materials includes mass spectrum of three compounds, HPLC profile of FDAA derivatives of l and d standard amino acids and HRMS of DKPs.

Declaration of interest

The authors are grateful to Indian Council Medical Research (ICMR), Government of India for funding.

Acknowledgements

We thank the Director, CTCRI, for providing facilities for the work.