Introduction

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 and cause vibriosis that leads to big yield losses in aquaculture (Baker-Austin et al. 2018; Brumfield et al. 2021; Ghosh et al. 2021). V. parahaemolyticus and V. alginolyticus are the most common infectious bacteria in aquaculture (Abd El Tawab et al. 2018), while 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).

In both clinical medicine and aquaculture, antibiotics like quinolone, tetracycline, and erythromycin have been used to control vibriosis (Handayani et al. 2022). With extensive usage of antibiotics, the issue of antibiotic resistance has emerged with complicated cases (Hussain et al. 2020; Li et al.

Materials and methods

General experimental procedures

Optical rotations were measured using an Autopol I polarimeter (Rudolph Research Analytical Inc, Boston, MA, USA) in methanol (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

Endophytic fungi species were isolated from the soil samples collected from Arctic Svalbard Archipelago (73–80 °N, 18–31 °E). 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. Fungal isolates were identified by sequencing the internal transcribed regions (ITS). 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 using BLASTN. The fungus was identified to be a Penicillium sp. (GenBank no. OP536848) 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, the seed culture (~ 15 mL) was inoculated into an Erlenmeyer flask which contained 100 g of sterilized dry rice and 110 mL of distilled water. All the flasks were stacked at room temperature for 14 days. 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, dichloromethane (CH2Cl2), EtOAc, and MeOH. The EtOAc fraction was partitioned by an ODS column eluted with a gradient of MeOH–H2O (30–100% MeOH) and divided into five fractions, A to E. Based on TLC analysis, fractions B and E were chosen for further purification. Fraction B was purified by an ODS column (acetonitrile–H2O, 35:65) and a semi-preparative HPLC with 60% MeOH/H2O isocratic elution. Compounds 1 (8.2 mg), 2 (10.1 mg), 4 (18.6 mg), 6 (17.9 mg), and 7 (6.3 mg) were obtained from fraction B. Fraction E was purified by an ODS column (acetonitrile–H2O, 30:70) and a semi-preparative HPLC with 55% MeOH/H2O. Compounds 3 (6.1 mg) and 5 (14.5 mg) were obtained from fraction E.

Structure determination of the compounds

The seven compounds (1–7) were dissolved in MeOH and tested with high-resolution electrospray ionization mass spectroscopy (HRESIMS) to obtain the molecular formulas. To determine the 2D structures, compounds were dissolved in deuterated chloroform (CDCl3) (compound 1), deuterated methanol (MeOD) (compounds 2, 3, 4, and 5), or deuterated dimethyl sulfoxide (DMSO-d6) (compounds 6 and 7) for NMR spectrometry. Compounds 2, 4, 5 and 6 were dissolved in MeOH 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. The single colony of each strain was inoculated into a 250-mL Erlenmeyer flask containing 100 mL of the NaCl-LB liquid medium and cultured at 37 °C overnight with 200 rpm shaking. 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 dimethyl sulfoxide (DMSO) to make 5-mM stocks. The stocks were diluted to desired concentrations with the NaCl-LB liquid medium for testing. Into each well of a 96-well plate, 100 μL of the 106 CFU/mL bacterial dilution and 100 μL of the compound solution with the desired concentration was added. Concentrations of the compounds tested were 500, 250, 125, 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. 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 units 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 activity at micromolar level. Molecular docking of the compounds suggested that the anti-Vibrio activity 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 an example in that the secondary metabolites of polar microbes are a good source of natural medicine.