Structure and properties of antimicrobial peptides produced by antagonist microorganisms isolated from Siberian natural objects

: Introduction. Public healthcare urgently needs new pharmaceuticals – alternative to traditional antibiotics – that pathogens develop no resistance to. Of special interest in this regard are antimicrobial, ribosomally synthesized bacterial peptides or bacteriocins. In this work, we aimed to study the structure and properties of antimicrobial peptides produced by antagonist microorganisms isolated from the natural objects of the Siberian region. Study objects and methods . The study objects were bacteria isolated from the natural sources of Kuzbass. After culturing bacteria, total protein was precipitated from the culture fluid and separated into fractions by gel permeation HPLC. Their amino acid sequences were determined by MALDI-TOF mass spectrometry. The antibacterial (against Bacillus pumilus and Escherichia coli ) and fungicidal (against Aspergillus flavus and Aspergillus niger ) properties of the peptides were studied by the disk diffusion method. Results and discussion. Seven peptides with different amino acid sequences were isolated from the culture fluid of bacteria, five of which had no analogues in the PepBank and Uniprot data banks. The peptide with an amino acid sequence of VMCLARKCSQGLIVKAPLM (2061.66 Da) was homologous to the cysteine membrane protein Giardia lamblia P15, and the peptide with an amino acid sequence of AVPSMKLCIQWSPVRASPCVMLGI (2587.21 Da) showed a homology with the Planctomycetes bacterium I41 peptides. We found antibacterial (against gram-positive and gram-negative bacteria) and fungicidal (against Aspergillus ) properties in the peptide fractions. Conclusion . Antimicrobial peptides produced by bacteria isolated from the natural objects of the Siberian region can be used to create pharmaceuticals as an alternative to traditional antibiotics to treat infectious diseases.


INTRODUCTION
Pathogenic microorganisms resistant to traditional antibiotics are a serious problem of modern healthcare. There is evidence that over 70% of all pathogenic bacteria are resistant to at least one of the most commonly used antibiotics. Therefore, there is an urgent need for new drugs and therapeutic approaches to overcome their resistance [1][2][3][4][5].
Antimicrobial peptides produced by various organisms from bacteria to mammals are an ideal alternative to antibiotics due to their antimicrobial, antiinflammatory, angiogenic, and immunomodulatory properties, as well as low bacterial resistance [6]. However, their use is limited by toxicity and stability in vivo [7].
Antimicrobial peptides act against various types of pathogens, including Gram-positive and Gram-negative bacteria, viruses, and microscopic fungi, through the destruction of the cytoplasmic membrane, intracellular penetration, and immunomodulation [8,9]. Structurally, antimicrobial peptides are classified into linear cationic amphipathic peptides and macrocyclic peptides [10]. As a rule, antimicrobial peptides are short peptides consisting of 10-50 amino acids [11,12]. They have common features despite differing in length, amino acid sequences, and conformation [13]. Typical antimicrobial peptides are composed of positively charged residues such as arginine, lysine, and histidine [14]. Cationic peptides with a positive charge ranging from +2 to +11 can interact with the membranes of microbial cells. Besides, a significant part of antimicrobial peptides is hydrophobic, contributing to the formation of amphipathic secondary or quaternary structures [15].
Antimicrobial peptides have several advantages over traditional antibiotics [16]. First of all, they have a broad spectrum of antimicrobial activity, against even multidrug-resistant pathogens [8,16]. Secondly, antimicrobial peptides are highly active against gramnegative bacteria, which are more serious targets than gram-positive bacteria [17]. Another advantage is a rather low likelihood of drug resistance.
Potential sources of bacteria producing bacteriocins are dairy products, cow rumen, feed, as well as natural objects such as soils, plant waste, rhizosphere of plants, bottom sediments of water bodies, etc. [18,25,26].
In this study, we aimed to examine the structure and properties of antimicrobial peptides produced by antagonist microorganisms isolated from the natural objects in Siberia.

STUDY OBJECTS AND METHODS
Our study objects were bacteria isolated from the natural sources of Kuzbass (Table 1).
Microorganism cultures. To obtain enrichment cultures of microorganisms, we crushed the samples of soil, bottom sediments, and plant waste under sterile conditions and rubbed their small amounts on Petri dishes with nutrient agar. The Petri dishes were incubated for three days at 26°C. Two nutrient media were used: lactobacilli were cultured on MRS agar; Bacillus and Geobacillus bacteria were cultured on a medium (pH 7.4 ± 0.2) containing 10.0 g/L casein hydrolysate, 2.5 g/L yeast extract, 5.0 g/L glucose, 2.5 g/L potassium hydrogen phosphate, and 12.0 g/L bacteriological agar.
Pure cultures of microorganisms were obtained from enrichment cultures by streaking. Microorganisms were cultivated on the media described above for 24 h: Lactobacillus, Leuconostoc and Pediococcus bacteria at 37°C, and Bacillus and Geobacillus at 30°C.
At the end of cultivation, cell debris was removed from all suspension cultures. The cultures were centrifuged at 3900 rpm in plastic flasks. The resulting supernatant was dried in a Labcocnco Triad freeze dryer (Labcocnco, USA) at a freezer temperature of -80°С, supernatant temperature of -20°С, and 0.05 mbar vacuum.
Protein fractions. To separate protein into individual fractions, the dried biomass was dissolved in 1 mL of 0.25 M phosphate buffer and the total protein was precipitated by adding 2 mL of concentrated ammonium sulfate solution. The resulting protein suspension was separated by centrifugation at 8000 rpm. The protein precipitate was dissolved in 1 mL of 0.025 M Tris buffer solution (pH 4.5). The precipitate was applied to an Enrich 650 10 mm × 300 mm column (Biorad, USA) for a gel permeation high performance liquid chromatography (HPLC) at 280 nm using a direct injection system. Fractionation was performed using an NGC fraction collector (Biorad, USA). Additionally, each protein fraction was purified on hydrophobic Amberlite XAD X-6 resins by chromatography. A glass column was filled with 10 g of Amberlite XAD-2 resin equilibrated with 10 mL of 20 mM trifluoroacetic acid solution. A protein solution in an acetate buffer was applied to the column and eluted in a methanol gradient from 0 to 15%, with a gradient rise of 5% for every 10 fractions. Fractions containing proteins were determined by taking 50 μL of each fraction and mixing it with a solution of Bradford's reagent in a 1:1 ratio. The resulting solution was measured on a Biorad SmartSpec Plus Spectrophotometer (USA). Fractions with an optical absorption of 0.06 or more were selected for further drying and identifying the amino acid sequence by the MALDI-TOF method using a MALDI TOF/TOF BRUKER Autoflex Speed mass spectrometer (Bruker Corporation, USA) Trypsinolysis. Peptides were precipitated by adding an equal volume of methanol/chloroform mixture to an aliquot of a 200 μL fraction. The resulting precipitate was separated by centrifugation at 4000 rpm. The precipitate was dissolved in 100 μL of 6 M urea solution, to which 5 μL of dithiothreitol (DTT) solution was added to keep for 60 min at room temperature. Then, we added 20 μL of iodoacetamide solution and kept the mixture for 60 min at room temperature. After that, we added 20 μL of a DTT solution and kept the mixture again for 60 min at room temperature. After adding 775 μL of MiliQ H 2 O and 50 μL of trypsin solution, the mixture was stirred by pipetting and kept in a thermostat at 37°C for 12 h. The enzyme was inactivated by adding 10 μL of trifluoroacetic acid. The peptides were purified by chromatography on C18 cartridges. The reaction mixture was applied to a cartridge and eluted with a solution of 0.1% trifluoroacetic acid in a 1:1 H 2 O/acetonitrile mixture. Analysis and Top-Dawn sequencing were performed on 1 μL of a purified peptide solution.
The antibacterial properties of the peptides against Bacillus pumilus and Escherichia coli were measured by the disk diffusion method. For this, we used suspensions of night cultures grown on a standard liquid nutrient LB medium with a titer of 0.5. The number of microorganisms (titer) in the suspension was determined by optical density at 595 nm. 200 μL of the pathogen culture was dropped onto a 90 mm Petri dish, rubbed with a sterile spatula by the spread plate method, and left to dry for 20 min under a laminar with the lid ajar. Then, 0.5 cm sterile filter disks soaked in the peptide solutions under study and dried at room temperature for 10 min were placed on the Petri dishes in the radial direction. The Petri dishes were left for 30 min at room temperature and then incubated in a thermostat at 37°C for 12 h. Then, we identified a bacterial inhibition zone around the disc and measured its diameter with a vernier caliper. Ampicillin at a concentration of 5 mg/mL was used as a positive control, and a disc soaked in a liquid medium was used as a negative control.
The fungicidal activity of the peptides against the microscopic fungi Aspergillus flavus and Aspergillus niger was measured by the disk diffusion method. The fungi were cultivated for 7 days, with an inoculation density of 6×10 7 conidia per 1 mL of medium. The results were analyzed with time intervals (3,9,12,24,48, 72 h, etc.) and by the fungus growth phase (stationary, accelerated growth, logarithmic), i.e., during the periods of exponential cell growth, decreased growth, and death or autolysis. At the end of the incubation, the inhibition zone around the disc was measured with a vernier caliper (mm), which indicated the degree of biocidal activity or its absence. A negative control was the samples with filters impregnated with the medium, and a positive control was the pharmaceutical preparation Irunin® (Veropharm, Russia) with itraconazole as an active ingredient.
Statistical data were analyzed in Microsoft Office Excel 2007. All the experiments were carried out in triplicate. Statistical analysis was performed using a one-sample Student's t-test. The differences were considered statistically significant at P < 0.05.

RESULTS AND DISCUSSION
Several protein fractions were isolated from the culture fluid of all the studied samples (Table 2).
According to The results of the MALDI TOF mass spectrometry of protein fractions are presented in Figs. 1-7. We found some identical mass spectra of protein fractions synthesized by different bacteria.  Having analyzed the mass spectra, we determined the molecular masses and amino acid sequences of seven peptides (Table 3). Table 3 also shows the presence of analogues for the studied peptides in the PepBank and Uniprot databases. We established a homology of fractions Pp-11_1, Pp-11_2, Pp-11_3, Pp-11_4, Pp-11_5, Pp-11_6, Pp-11_7, Pp-11_8, Lp-7_1, Pd-16_1, Pd-16_2, Pd-16_3, and Pd-16_4 with the cysteine membrane protein Giardia lamblia P15 (Fig. 8), as well as a homology of peptides Pa-9_1 and Pa-9_2 with the Planctomycetes bacterium I41 peptides (Fig. 9). The rest of the peptides had no analogues in the PepBank and Uniprot databases. According to Table 4       Unlike biocidal properties, which do not depend on the pathogen growth phase and naturally decrease over time, fungicidal properties need to be determined at each stage of the fungus life cycle since fungal pathogens have a complex growth cycle. We found that the peptide fractions under study did not stop fungal growth, but only inhibited it, which was indicated by a change in the mycelium color. The results were analyzed with time intervals (3,9,12,24,48, 72 h, etc.) and by the fungus growth phase (stationary, accelerated growth, logarithmic), i.e., during the periods of exponential cell growth, decreased growth, and death or autolysis. The samples with filters impregnated with a nutrient medium were used as a control.
Having analyzed the peptides' fungicidal activity ( Table 5, Fig. 11), we identified those peptides which could inhibit Aspergillus growth, rather than stop it completely. They were Bs-1_1, Bc-20_1 (identical Lf-13_1, Lf-13_2, and Lf-13_3) and Bs-19_2, with a lysis zone diameter of 0.1-0.   Thus, the fact that peptides produced by microorganisms inhabiting the natural ecosystems of Kuzbass exhibit antagonistic activity against opportunistic strains opens up prospects for their use in the production of pharmaceutical substances with antimicrobial action, alternative to traditional antibiotics.

CONCLUSION
We identified amino acid sequences and molecular masses of peptide fractions produced by bacteria (Lactobacillus, Leuconostoc, Pediococcus, Bacillus, and Geobacillus) isolated from the natural objects of the Siberian region (soil, rhizosphere of plants, bottom The peptides obtained from the culture fluid of bacteria isolated from natural sources of the Siberian Federal District were analyzed for antibacterial properties against Bacillus pumilus and Escherichia coli. We identified one peptide that exhibited no antagonistic activity against either gram-negative or gram-positive bacteria. One peptide fraction showed high antibacterial properties against both B. pumilus and E. coli. One peptide was active against E. coli, but not against B. pumilus (gram-positive bacteria). Finally, four out of seven peptides under study exhibited moderate and pronounced antagonism against B. pumilus, but no antibacterial activity against E. coli.
Our study of the peptides' antifungal activity revealed three peptides that could inhibit the growth of the microscopic fungi Aspergillus niger and Aspergillus flavus, without stopping it completely (0.1-0.2 mm lysis zone). Four peptide fractions showed high fungicidal activity against Aspergillus (0.3-0.5 mm lysis zone).
According to our results, antimicrobial peptides produced by bacteria isolated from the natural objects of the Siberian region can be used as promising agents in the production of pharmaceutical substances and drugs (after safety trials) to treat infectious diseases, such as gastrointestinal, respiratory, blood and skin, as well as fungal infections.

CONTRIBUTION
The authors are equally responsible for the research results and the manuscript.