Anti-vibriosis bioactive molecules from Arctic Penicillium sp. Z2230

doi:10.21203/rs.3.rs-2329237/v1

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Anti-vibriosis bioactive molecules from Arctic Penicillium sp. Z2230

https://doi.org/10.21203/rs.3.rs-2329237/v1

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published 02 Feb, 2023

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Vibrio species (Vibrio sp.) is a class of gram-negative aquatic bacteria that causes vibriosis in aquaculture and leads to big economic losses. Antibiotics have been used to control vibriosis, but concerns on antibiotic resistance keep raising. Natural products are important sources of drug lead compounds that provide potential antibiotic alternatives. In this study, seven compounds were isolated from the Arctic endophytic fungus Penicillium sp. Z2230 and determined for the structures. Among these seven compounds, compounds 3, 4, and 5 showed anti-Vibrio activity, and the antibacterial mechanisms were explored by molecular docking. The results suggested that the anti-Vibrio activities could come from the inhibition of bacterial peptide deformylase (PDF). The data indicate that these Penicillium-derived compounds are potential lead molecules for developing novel anti-Vibrio agents, and PDF is a promising antibacterial target. This study expands the bioactive diversity of polar endophytic fungi by showing a good example in which the secondary metabolites of a polar microbe are a good source of natural medicine.

Arctic endophytic fungus

secondary metabolites

natural products

anti-vibriosis

aquaculture

antibiotic resistance

Vibrio species (Vibrio sp.) are a class of gram-negative aquatic bacteria characterized by motile rods and facultative anaerobic metabolism. They reside in estuarine and coastal environments as well as freshwaters globally (Baker-Austin et al. 2018; Brumfield et al. 2021). Vibrio sp. can cause vibriosis in aquaculture including the shrimp farming, and result in big yield losses (Ghosh et al. 2021). Recent reports have revealed that V. parahaemolyticus and V. alginolyticus are the most common infectious bacteria in aquaculture (Abd El Tawab et al. 2018). V. cholerae, V. vulnificus and V. parahaemolyticus are also serious foodborne pathogens that cause gastroenteritis, wound infections, and septicaemia in humans (Ina-Salwany et al. 2019).

Currently in both clinical medicine and aquaculture, antibiotics like quinolone, tetracycline, and erythromycin have been used to control vibriosis (Handayani et al. 2022). Although there are many different antibiotics, the multidrug resistance problem has emerged and caused complicated cases (FB et al. 2020; Li et al. 2021; Yano et al. 2014). It has been reported that some V. alginolyticus strains in bivalves displayed resistant to vancomycin, erythromycin, and ampicillin, V. campbellii became resistant to oxytetracycline, ampicillin, and carbenicillin, and V. harveyi became resistant to more than three antibiotic types (Kang et al. 2016). About 64% of the Vibrio strains isolated from marine fishes in Southern China showed resistance to more than three antibiotic types (Nakayama et al. 2006). Alternative anti-Vibrio drugs are urgently needed to solve the antibiotic resistance problem.

Natural products are important sources of new drug lead compounds. Microorganisms in extreme environments (e.g., polar regions) have become a promising source of active natural products because of the complex structural types and rich chemical diversity of their metabolites (Brunati et al. 2009; Robinson 2001). In particular, polar fungi produce antimicrobial agents to kill other microbes to compete for the scarcely available nutrients in the cold and hostile environment (Gonçalves et al. 2013). For examples, Ketidocillinones B from Antarctica sponge-derived fungus Penicillium showed a broad-spectrum antibacterial activity, and the ethanol extract of fungus Purpureocillium lilacinum displayed high antimicrobial activities against a variety of bacteria and fungi (Gonçalves et al. 2015; Shah et al. 2020). Thus, exploring the compounds from polar fungi may provide alternative choices of antibiotics.

In this study, we isolated seven compounds from the Arctic endophytic fungus Penicillium sp. Z2230 (GenBank no. OP536848) and determined their structures. They are 3-benzylidene-3,4-dihydro-4-methyl-H-l,4-benzodiazepine-2,5-dione (1), penicopeptide A (2), viridicatol (3), cyclopenol (4), cyclopenoin (5), fructigenines A (6), and 3-O-methylviridicatin (7). All the compounds were tested for antibacterial activities against four Vibrio species, namely V. parahaemolyticus, V. cholerae, V. vulnificus, and V. alginolyticus. The results showed that compounds 3, 4, and 5 had distinct antibacterial activity against V. parahaemolyticus, V. cholerae, and V. vulnificus. Furthermore, molecular docking studies of compounds 3, 4, and 5 suggested the anti-Vibrio activities could come from the inhibition of bacterial peptide deformylase (PDF). These Penicillium-derived compounds are potential lead molecules for the development of novel anti-Vibrio drugs.

General experimental procedures

Optical rotations were measured using an Autopol I polarimeter (Rudolph Research Analytical Inc, Boston, MA, USA) in MeOH. UV spectra were recorded on a Shimadzu UV-1800 spectrophotometer (Shimadzu Corporation Co Ltd, Tokyo, Japan) in MeOH. 1D NMR spectra were recorded on a Bruker A VIII-600 NMR spectrometer using tetramethylsilane as an internal standard. Column chromatography was performed on silica gel (200–300 mesh, Qingdao Marine Chemical Inc, Qingdao, China) and ODS (50 µm, YMC Co Ltd, Kyoto, Japan) on a Flash Chromatograph System (SepaBen machine, Santai Technologies Inc, Changzhou, China). Preparative high-performance liquid chromatography (Pre-HPLC) was performed on a Shimadzu LC-20 system (Shimadzu Co Ltd, Tokyo, Japan) equipped with a Shim-pack RP-C18 column (20×250 mm, 10 µm, Shimadzu Co Ltd, Tokyo, Japan) with a flow rate of 10 mL/min at 25°C.

Identification Of The Fungus

Fungal isolates were identified by sequencing the internal transcribed regions (ITS). The fungal isolates were grown in a 15-mL potato dextrose broth for 3 days at 28°C, and the mycelia were filtered and used for DNA extraction. Primers ITS1 (5’-TCCGTAGGTGAACCTGCGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC-3’) were used for sequencing the ITS of the fungal genome (Shanghai Personal Biotechnology Co Ltd, Shanghai, China). The sequencing results were aligned with fungal ITS sequences on NCBI (GenBank no. OP536848) using BLASTN. The fungus was identified to be a Penicillium sp. according to the sequence similarity (Schoch et al. 2012).

Fermentation, Extraction, And Isolation Of Fungal Compounds

The fungus was incubated on a potato dextrose agar (PDA) medium at 28°C for 3 days, and the fungal mycelia were inoculated into a 250-mL Erlenmeyer flask containing 100 mL of the potato dextrose broth and fermented at 28°C with 220 rpm. After 2 days of fermentation, every 15 mL of the above seed cultures were inoculated into an Erlenmeyer flask. In every Erlenmeyer flask there were 100 g of sterilized dry rice and 110 mL of distilled water. All the flasks were put at room temperature for 14 days. After the incubation, the fermented rice medium was extracted for three times using ethyl acetate (EtOAc), and the solvents were concentrated by a rotary evaporator to get a crude extract (43.2 g). The crude extract was loaded onto a silica gel column and eluted sequentially with a series of polar solvents (petroleum ether, CH₂Cl₂, EtOAc, and MeOH). The EtOAc fractions were separated on an ODS column with a gradient of MeOH-H₂O (30–100% MeOH) and eluted into five fractions. Fractions A, B and C are basically the same, among which B is the most abundant. Fractions D showed no obvious UV absorption peak. Thus, fractions B and E were further purified. Fraction B was further purified by an ODS column (MeCN-H₂O, 35:65) and a semi-preparative HPLC with 60% MeOH/H₂O to isolate compounds 1 (8.2 mg), 2 (10.1 mg), 4 (18.6 mg), 6 (17.9 mg), and 7 (6.3 mg). Fractions E was further purified by an ODS column (MeCN-H₂O, 30:70) and a semi-preparative HPLC with 55% MeOH/H₂O to isolate compounds 3 (6.1 mg) and 5 (14.5 mg).

Determination Of The Compounds

The seven compounds (1–7) isolated from the Arctic fungus Penicillium sp. Z2230 were dissolved in CH₃OH and tested on HRSIMS to obtain the molecular formulas. To determine the 2D structures, compounds were dissolved in CDCl₃ (compound 1), CH₃OD (compounds 2, 3, 4, and 5), or DMSO-d₆ (compounds 6 and 7) for NMR spectrometry. All the compounds were dissolved in CH₃OH and tested on an Autopol I polarimeter for the optical rotations.

Anti- Vibrio activity

V. parahaemolyticus, V. cholerae, V. vulnificus and V. alginolyticus were cultured on 3% NaCl-LB medium (5 g/L yeast extract, 10 g/L peptone, 30 g/L NaCl, pH 7.4) plates at 30°C for 12 h, and inoculated into 250 mL Erlenmeyer flasks containing 100 mL of the NaCl-LB liquid medium, continued to culture at 37°C overnight with 200 rpm. Then the bacterial cultures were diluted to 10⁶ CFU/mL with the NaCl-LB liquid medium for testing. Streptomycin and the seven compounds were dissolved in DMSO to make 5-mM solutions, and the 5 mM solutions were further diluted to desired concentrations with the NaCl-LB liquid medium for testing. For the anti-Vibrio activity test, each well of a 96-well plate was added with 100 µL of the 10⁶ CFU/mL bacterial dilution and 100 µL of the compound solution. Different concentrations of the compounds were tested, including 500 µM, 250 µM, 125 µM, and 62.5 µM. The test was conducted at 30°C overnight with three independent repeats.

Molecular Docking

Three-dimensional (3D) crystal structures or homology models of the Vibrio-specific targets reported in recent years on RCSB Protein Data Bank (PDB) or UniProt Database were used for molecular docking (Bonardi et al. 2021; Ding et al. 2018; Ragunathan et al. 2018; Raval et al. 2021; Sasikala et al. 2016). The AutoDock Tools 1.5.6 software was used to remove the water molecules, add the nonpolar hydrogen, and calculate the Gasteiger charges for the protein structures. Two-dimensional (2D) structures of the compounds were depicted by the ChemDraw software and transformed into PDB format through the Chem3D software as the docking ligands. In AutoDock Tools 1.5.6, the ligand was set as flexible and the receptor (the protein target) was set as rigid. The search parameters were Genetic Algorithm and the docking parameters were default. A total of 50 conformations were generated for each docking, and the conformation with the best affinity was selected as the final conformation to be visualized in the PyMOL2 software.

Structure determination of the compounds

Seven compounds were isolated from the mycelia extracts of the Arctic endophytic fungus Penicillium sp. Z2230 cultured in glucose-typed rice medium. The structures were determined to be three benzodiazepines (1, 4, 5), two viridicatin derivatives (3, 7), one cyclic peptide (2), and one diketopiperazine (6) (Fig. 1). For structure determination, the spectroscopic data (¹H NMR,¹³C-NMR, and MS) of the seven compounds were comprehensively compared with the previous reports (Fremlin et al. 2009; Ma et al. 2017; Sun et al. 2016; Wang et al. 2020; Xin 2006).

The compound 1 appeared as yellow powders is 3-benzylidene-3,4-dihydro-4-methyl-H-l,4-benzodiazepine-2,5-dione with molecular formula C₁₇H₁₄N₂O₂. This was determined by HRESIMS with the ion peak at m/z 301.0959 [M + Na]⁺ (calcd. for C₁₇H₁₄N₂O₂Na, 301.0953). The detailed data are as follows. ¹H-NMR (400 MHz, CDCl₃): δ_H 8.57, s, 1H (1-NH); 8.03, dd, 1H (H-12, J = 7.9, 1.5 Hz); 7.50, td, 1H (H-8, J = 7.7, 1.6 Hz); 7.43–7.34, m, 5H (H-6, H-7, H-13/15, H-16); 7.27, t, 1H (H-14, J = 7.6 Hz); 7.04, d, 1H (H-9, J = 8.0 Hz); 6.96, s, 1H (H-10); 3.20, s, 3H (4-NMe). ¹³C-NMR (150 MHz, CDCl₃): δ_C 175.6 (C-2), 166.8 (C-5), 135.6 (C-9a), 133.4 (C-8), 132.8 (C-11), 132.1 (C-9), 131.5 (C-14), 131.5 (C-3), 129.9 (C-10), 129.5 (C-13/15), 129.1 (C-12/16), 125.6 (C-5a), 125.2 (C-7), 120.5 (C-6), 35.9 (4-NMe) (Sun et al. 2016)(Figures S1, S2, S3 and S4).

The compound 2 appeared as yellowish powders is Penicopeptide A ([α]20 D-26.0 (c 1.0, MeOH)) with molecular formula C₃₄H₃₁N₄O₄. This was determined by HRESIMS with the ion peak at m/z 583.2329 [M + Na]⁺ (calcd. for C₃₄H₃₁N₄O₄Na, 583.2321). The detailed data are as follows. ¹H-NMR (400MHz, CH₃OD): δ_H 7.98, d, 1H (H-13, J = 7.9 Hz); 7.84, d, 1H (J = 8.3 Hz); 7.61, t, 1H (J = 7.6 Hz); 7.52, t, 1H (J = 7.6 Hz); 7.36, t, 1H (J = 7.6 Hz); 7.15–7.32, overlapped, 8H (H-6 ~ H-8, H-22 ~ H-26); 7.09, d, 1H (H-33, J = 8.1 Hz); 7.02, d, 2H (H-5, H-9, J = 7.2 Hz); 4.44, t, 1H (H-19, J = 7.6 Hz); 4.35, dd, 1H (H-2, J = 10.6, 6.9 Hz); 3.42, dd, 1H (H-20a, J = 14.5, 7.9 Hz); 3.27, dd, 1H (H-20b, J = 14.5, 7.3 Hz); 3.09, s, 3H (H-27); 2.92, s, 3H (H-10); 2.81, dd, 1H (H-3a, J = 13.6, 6.9 Hz); 2.68, dd, 1H (H-3b, J = 13.6, 10.6 Hz). ¹³C-NMR (150 MHz, CH₃OD): δ_C 170.9 (C-1), 169.8 (C-18), 169.3 (C-11), 166.9 (C-28), 136.8 (C-4), 136.6 (C-21),135.8 (C-17), 135.6 (C-34), 132.8 (C-15), 132.4 (C-32), 131.0 (C-13), 130.5 (C-30), 128.7 (C-5/7, C-22/26), 128.7 (C-6/8), 128.5 (C-23/25), 128.3 (C-7/24), 126.9 (C-14), 126.5 (C-31), 124.6 (C-12), 124.5 (C-29), 120.7 (C-16), 120.3 (C-33), 68.4 (C-2), 56.5 (C-19), 38.4 (10-NMe), 33.8 (C-3), 31.6 (C-20), 28.2 (27-NMe) (Sun et al. 2016) (Figures S5, S6, S7 and S8).

The compound 3 appeared as brown powders is Viridicatol with molecular formula C₁₅H₁₁NO₃. This was determined by HRESIMS with the ion peak at m/z 276.0641 [M + Na]⁺ (calcd. for C₁₅H₁₁NO₃Na, 276.0637). The detailed data are as follows. ¹H-NMR (400 MHz, CH₃OD): δ_H 7.39–7.30, m, 3H (H-5, H-5’, H-8); 7.26, d, 1H (H-6, J = 8.1 Hz); 7.14, dq, 1H (H-7, J = 8.3, 4.5, 4.1Hz); 6.89, dd, 1H (H-4’, J = 7.5, 2.2Hz); 6.86–6.80, m, 2H (H-2’, H-6’). ¹³C-NMR (150MHz, CH₃OD): δ_C 159.1 (C-2), 157.1 (C-3’), 141.7 (C-8a), 134.8 (C-1’), 133.0 (C-4), 129.2 (C-5’), 126.6 (C-5), 125.9 (C-7), 125.0 (C-3), 122.4 (C-6), 121.6 (C-4a), 120.8 (C-6’), 116.5 (C-2’), 115.1 (C-8), 114.6 (C-4’) (Ma et al. 2017) (Figures S9, S10, S11 and S12).

The compound 4 appeared as yellow powders is Cyclopenol ([α]20 D-97.5 (c 0.8, MeOH)) with molecular formula C₁₇H₁₄N₂O₄. This was determined by HRESIMS with the ion peak at m/z 311.1037 [M + H]⁺ (calcd. for C₁₇H₁₅N₂O₄, 311.1032). The detailed data are as follows. ¹H-NMR (400MHz, CH₃OD) δ_H 7.59, t, 1H (H-8, J = 7.9 Hz); 7.19, m, 3H (H-6, H-7, H-9); 7.03, t, 1H (H-15, J = 7.9 Hz); 6.73, d, 1H (H-14, J = 7.7 Hz); 6.17, s, 1H (H-12); 6.13, d, 1H (H-16, J = 7.7 Hz); 4.10, s, 1H (H-10); 3.22, s, 3H (4-NMe). ¹³C-NMR (150 MHz, CH₃OD): δ_C 167.3 (C-2), 167.1 (C-3), 157.1 (C-13), 135.1 (C-9a), 132.6 (C-8), 130.8 (C-6), 128.9 (C-15), 126.6 (C-5a), 124.7 (C-7), 121.0 (C-9), 117.1 (C-10), 115.7 (C14), 112.5 (C-12), 70.3 (C-3), 64.6 (C-10), 30.2 (4-NMe) (Fremlin et al. 2009) (Figures S13, S14, S15 and S16).

The compound 5 appeared as yellow powders is Cyclopenoin ([α]20 D-348.1 (c 1, MeOH)) with molecular formula C₁₇H₁₄N₂O₃. This was determined by HRESIMS with the ion peak at m/z 317.0905 [M + Na]⁺ (calcd. for C₁₇H₁₄N₂O₃Na, 317.0902). The detailed data are as follows. ¹H-NMR (400 MHz, CH₃OD): δ_H 7.59, t, 1H (H-8, J = 7.7 Hz); 7.32, m, 1H(; 7.27–7.12, m, 4H (H-6, H-7, H-13/15); 7.08, dd, 1H (H-9, J = 7.9, 1.6 Hz); 6.71, d, 2H (H-12/16, J = 7.9 Hz, 2H); 4.20, s, 1H (H-10); 3.22, s, 3H (4-NMe). ¹³C-NMR (150MHz, CH₃OD): δ_C 167.2 (C-2), 167.0 (C-5), 135.2 (C-9a), 132.7 (C-8), 130.8 (C-11), 130.8 (C-9), 128.7 (C-14), 127.8 (C-13/15), 126.6 (C-5a), 125.9 (C-12/16), 124.7 (C-7), 121.0 (C-6), 70.3 (C-3), 64.6 (C-10), 30.3 (4-NMe) (Wang et al. 2020)(Figures S17, S18, S19 and S20).

The compound 6 appeared as yellowish powders is Fructigenine A ([α]20 D-230.0 (c 0.6, MeOH)) with molecular formula C₂₅H₂₇N₃O₂. This was determined by HRESIMS with the ion peak at m/z 402.2169 [M + H]⁺ (calcd. for C₂₅H₂₈N₃O₂, 402.2182). The detailed data are as follows. ¹H-NMR (400 MHz, DMSO- d₆): δ_H 8.01, s, 1H (H-7); 7.34–7.16, m, 7H (H-8, H-10, H-14 ~ H-18); 7.12, t, 1H (H-9, J = 7.5Hz); 6.13, s, 1H (H-2); 6.03, s, 1H (H-5a); 5.74, dd, 1H (H-20, J = 17.2, 10.8Hz); 5.13, d, 1H (Hcis-21, J = 4.3Hz); 5.09, d, 1H (Htrans-21, J = 10.9Hz); 4.25, dd, 1H (H-3, J = 9.6, 3.6Hz); 3.77, dd, 1H (H-11a, J = 11.6, 5.5Hz); 3.53–3.39, m, 1H (H-12); 2.88, dd, 1H (H-12, J = 14.3, 9.5Hz); 2.66, s, 3H (H-23); 2.53, dd, 1H (H-11, J = 12.4, 5.6Hz); 2.17, t, 1H (H-11, J = 12.0Hz); 1.10, s, 3H (H-19b); 0.96, s, 3H (H-19a). ¹³C-NMR (150 MHz, DMSO-d₆): δ_C 170.2 (C-22), 168.2 (C-4), 164.8 (C-1), 143.4 (C-7a), 143.1 (C-20), 135.4 (C-13), 132.0 (C-10a), 129.4 (C-15/17), 129.2 (C-14/18), 129.1 (C-8), 127.6 (C-16), 124.6 (C-7), 124.6 (C-10), 119.1 (C-9), 114.6 (C-21), 79.4 (C-5a), 61.0 (C-10), 59.1 (C-11a), 56.1 (C-3), 40.4 (C-19), 37.1 (C-11), 36.3 (C-12), 23.7 (C-19a), 23.2 (C-19b), 22.4 (C-23) (Xin 2006) (Figures S21, S22, S23 and S24).

The compound 7 appeared as brown powders is 3-O-methylviridicatin with molecular formula C₁₆H₁₃NO₂. This was determined by HRESIMS with the ion peak at m/z 274.0852 [M + Na]⁺ (calcd. for C₁₆H₁₃NO₂Na, 274.0844). The detailed data are as follows. ¹H-NMR (400 MHz, DMSO- d₆): δ_H 12.12, s, 1H (1-NH); 7.57–7.37, m, 5H (H-2’ ~ H-6’); 7.35–7.31, m, 2H (H-7, H-8); 7.09, t, 1H (H-5, J = 7.5 Hz); 6.99, d, 1H (H-6, J = 8.1 Hz); 3.70, s, 3H (3-OMe). ¹³C-NMR (150 MHz, DMSO-d₆): δ_C 159.0 (C-2), 145.6 (C-3), 137.9 (C-8), 136.3 (C-4), 134.0 (C-1’), 129.6 (C-2’/6’), 129.1 (C-6), 128.9 (C-3’/5’), 128.5 (C-4’), 126.2 (C-4a), 122.5 (C-5), 120.3 (C-8a), 115.6 (C-7), 59.9 (3-OMe) (Ma et al. 2017) (Figures S25, S26, S27 and S28).

Anti- Vibrio activities

The anti-Vibrio activities of the seven compounds were tested with V. parahaemplyticus, V. cholerae, V. vulnificus, and V. alginolyticus using different concentrations of the compounds. In bacterial culturing, the cell density in the culture was related to OD₆₀₀ within a certain range, thus OD₆₀₀ was used to estimate the anti-Vibrio activity. After overnight culturing, we analyzed OD₆₀₀ of the bacterial cultures. The OD₆₀₀ of compounds 1, 2, 6 and 7 with all the concentrations tested had no difference comparing to the control bacterial culture (no addition of any compound), which suggested that these four compounds had no anti-Vibrio activity. On the other hand, the OD₆₀₀ showed that compounds 3, 4 and 5 had anti-Vibrio activity. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the compound that caused the OD₆₀₀ to be lower than the one-half of that of the control bacterial culture. In this study, the OD₆₀₀ of the control bacterial cultures of V. parahaemplyticus, V. cholerae, V. vulnificus and V. alginolyticus were 2.0, 1.7, 1.1 and 1.8, respectively. The results showed that compound 3 had antibacterial activity on all the four Vibrio species, while compounds 4, and 5 had antibacterial activity on V. cholerae, V. vulnificus, and V. alginolyticus (Fig. 2). The MIC of the compounds on the Vibrio species are listed in Table 1.

Table 1

The MIC of anti-*Vibrio* activities of compounds 1–7
Compound	V. parahaemplyticus	V. cholerae	V. vulnificus	V. alginolyticus
1	—	—	—	—
2	—	—	—	—
3	63.2 µg/mL (250 µM)	63.2µg/mL (250 µM)	63.2µg/mL (250 µM)	126.4µg/mL (500 µM)
4	—	77.5µg/mL (250 µM)	77.5µg/mL (250 µM)	155.0µg/mL (500 µM)
5	—	73.5µg/mL (250 µM)	73.5µg/mL (250 µM)	147.0µg/mL (500 µM)
6	—	—	—	—
7	—	—	—	—
Streptomycin (Positive)	36.3 µg/mL (125 µM)	72.6 µg/mL (250 µM)	72.6 µg/mL (250 µM)	145.2 µg/mL (500 µM)

Study On The Antibacterial Mechanism

Molecular docking was used to study the antibacterial mechanism of the three anti-Vibrio compounds. V. parahaemolyticus, V. cholerae, and V. vulnificus were chosen as the models. The docking of multiple Vibrio-specific proteins with the three active compounds was done with the Autodock software. The target proteins were the peptide deformylases (PDF) from the three Vibrio species, the proline dehydrogenase (PDH) and the outer protein J (VopJ) from V. parahaemolyticus, the UDP-N-acetylenolpyruvoylglucosamine reductase (MurB) and the carbonic anhydrase (VchA) from V. cholerae, and the UDP-GlcNAC C4 epimerase (WbpP) from V. vulnificus. The docking results showed that PDF had the lowest binding energy and more hydrogen bonds when docked with the three compounds (Tables S1, S2 and S3). This suggested PDF could be a potential target related to the antibacterial mechanism of the three compounds.

The compound 3 binds to V. parahaemplyticus PDF (PDB ID: 5mte) by interacting with five amino acid residues (Fig. 3). Among these, Gly84 forms a hydrogen bond with the carbonyl oxygen at C-2 position, while Gln47, Leu86, His127 and His131 form four hydrogen bonds with the phenolic hydroxyl at C-3’ position (Fig. 3B). The five H-bonds allows compound 3 and the target to form a stable complex with the binding energy at -8.23 kcal/mol. However, compounds 4 and 5 bind to the protein with less H-bonds and higher binding energies at -5.22 ~ -5.70 kcal/mol (Fig. 3A, C and D).

The compound 3 forms five hydrogen bonds with the binding energy at -7.97 kcal/mol when binds to V. cholerae PDF (PDB ID: 3qu1) (Fig. 4A). Among these, Asp42 and Asn43 form two H-bonds with the phenolic hydroxyl at C-3’ position, Asp96 forms a hydrogen bond with the carbonyl oxygen at C-2 position. Besides, the secondary amine at N-1 position forms two hydrogen bonds with Asp96 and Tyr98 (Fig. 4B). Compounds 4 and 5 exhibits the same binding energy at -7.84 kcal/mol when docked to V. cholerae PDF. Carbonyl oxygen at C-2 and hydrogen at N-1 of compound 4 form two hydrogen bonds with Gly90, and phenolic hydroxyl at C-13 forms a hydrogen bond with carbonyl oxygen of Glu134 (Fig. 4C). The compound 5 forms three hydrogen bonds with three amino acids (Ile45, Gly46 and Leu92) through C-2 carbonyl and C-3,10 epoxy groups, respectively (Fig. 4D).

The three compounds also bind tightly to V. vulnificus PDF (Uniprot: Q597F5 (Q597F5_VIBVL)). The binding energy of compounds 3, 4 and 5 are − 7.97, -7.57 and − 7.36 kcal/mol, respectively (Fig. 5A). For compound 3, Asn43 forms two hydrogen bonds with secondary amine at N-1 and alcohol hydroxyl at C-3, while Cys91 and Val94 form two hydrogen bonds with the phenolic hydroxyl at C-3’ (Fig. 5B). In the complex of compound 4 and the PDF, Gly90 forms a H-bond with amino hydrogen at N-1, and His133, Glu134 and His137 form three H-bonds with phenolic hydroxyl at C-13 (Fig. 5C). Amino hydrogen at N-1 and carbonyl at C-2 of compound 5 form two H-bonds with Gly90 (Fig. 5D).

The low binding energy of these dockings showed strong binding of the compounds to the targets (PDF). Moreover, the results of these dockings are consistent with the anti-Vibrio activities as shown in Table 1.

Microbes in extreme environments are more likely to produce secondary metabolites with complex structures and exceptional biological activities. In this study, seven compounds were isolated and determined from the Arctic-derived Penicillium sp. Z2230, of which compounds 3, 4 and 5 showed particularly anti-Vibrio activities. This suggests that the Penicillium sp. from Arctic may become a solid source of active natural products.

The three active anti-Vibrio compounds offered a glimpse on their structure-activity relationships. For the viridicatin derivatives, compound 3 and 7, the anti-Vibrio activity of compound 3 was significantly higher than that of compound 7, which demonstrated that the hydroxyl at C-3 and the phenyl at C-3’ were important for the activity. Benzodiazepines are commonly known as sedative-hypnotics. This is the first time that benzodiazepines show anti-Vibrio activity. As shown in Table 1, the growth of Vibrio strains can be inhibited by compounds 4 and 5, but not compound 1. The ternary epoxy at C-3 on the diazepine ring of compounds 4 and 5 maybe the key group for their activities.

Recent publications have suggested that peptide deformylase (PDF) is a possible candidate of bacteriostatic target (Grzela et al. 2018). Deformylation is an essential process in microbial cells. The growth and reproduction of pathogenic microorganism will be inhibited once the sequence of PDF has mutations or deletions. The necessity of deformylation makes PDF an attractive target in developing new antibacterial agents (Meinnel et al. 1993, 1994). In this study, the firm binding of the active compounds to PDF may have changed the protein conformation and affected the activity. After PDF inactivation, the formyl group of the nascent peptide chain in Vibrio cells could not be removed smoothly. This indicates the compounds can inhibit bacterial growth by preventing bacterial protein synthesis (Durand et al. 1999). Moreover, the result showed that electron-withdrawing epoxy unit are more likely to form hydrogen bonds than π-rich double bond when binding to the ligand pockets. The discovery that PDF is present in several human parasites also suggests it to be a potential target for antiparasitic agents (Meinnel 2000).

In conclusion, seven compounds were isolated from the Arctic endophytic fungus Penicillium sp. Z2230. Three compounds, viridicatol (3), cyclopenol (4), and cyclopenoin (5), showed potent anti-Vibrio activities at micromolar level. Molecular docking of the compounds suggested the anti-Vibrio activities could come from the inhibition of bacterial peptide deformylase (PDF). These Penicillium-derived compounds are potential lead molecules for the development of novel anti-Vibrio agents. The data also indicate that PDF is a promising target for new antibacterial agents. This study expands the biologically active diversity of polar endophytic fungi, and shows a good example in that the secondary metabolites of polar microbes are a source of natural medicine.

Acknowledgements

The authors are thankful to the Jiangsu Key Laboratory of Marine Biological Resources and Environment and Key Laboratory of Bioreactor Engineering for providing lab facilities and equipment to carry out the research work.

Authors' contributions

JCG and JY carried out the isolation of the fungus and extraction of the secondary metabolites, performed the anti-vibrio assay, structure characterization. BG and HFL performed the molecular docking and collaborated in the analysis of the obtained results. FLA provided the fungus and conceived this work. PW and DZ wrote the manuscript text. SG contributed to the data interpretation and the revision of the draft. All authors read and approved the final manuscript.

Funding

This work was supported by the grants from the Natural Science Foundation of the Jiangsu Higher Education Institutions of China (No. 22KJB170010); the Scientific Research Foundation of Jiangsu Ocean University (No. KQ20047); the Program of Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening (No. HY202001); the Foundation from Jiangsu Province (No. JSSCBS20211307); the Program of MNR Key Laboratory of Coastal Salt Marsh Ecosystems and Resources (No. KLCSMERMNR2021107); the “Blue Project” of Jiangsu Higher Education Institutions of China, and the Priority Academic Program Development of Jiangsu Higher Education Institutions of China.

Availability of data and materials

All data generated or analyzed during this study are included in this research article.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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Anti-vibriosis bioactive molecules from Arctic Penicillium sp. Z2230

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Abstract

Figures

Introduction

Materials And Methods

General experimental procedures

Identification Of The Fungus

Fermentation, Extraction, And Isolation Of Fungal Compounds

Determination Of The Compounds

Molecular Docking

Results

Structure determination of the compounds

Study On The Antibacterial Mechanism

Discussion

Declarations

References

Supplementary Files

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