Evaluation of the potential of endophytic Trichoderma sp. isolated from medicinal plant Ampelopsis japonica against MRSA and bioassay-guided separation of the anti-MRSA compound

Abstract

Endophytic fungi have been recognized as a valuable source for the production of biologically active compounds with potential applications in various domains. This study aimed to isolate endophytic fungi from Ampelopsis japonica (Thunb.) Makino and assess their anti-MRSA activity. Meanwhile, chromatographic separation techniques were applied to analyze the constituents of endophytic fungal secondary metabolites. The isolate BLR24, which exhibited strong inhibition activity against MRSA, was identified as Trichoderma virens based on morphological characteristics and ITS sequence analyses. The ethyl acetate extract of BLR24 (EA-BLR24) showed good anti-MRSA activity with the MIC and MBC values of 25 μg/mL and 50 μg/mL, separately. The inhibition of biofilm formation was up to 34.67% under MIC concentration treatment. Meanwhile, EA-BLR24 could significantly reduce the expression of biofilm-related genes (icaA, sarA, and agrA) of MRSA. Based on LC-MS/MS analysis, twenty compounds in EA-BLR24 could be annotated using the GNPS platform, mainly diketopiperazines. The anti-MRSA compound (Fr.1.1) was obtained from EA-BLR24 by bioassay-guided fractionation and determined as gliotoxin. The results indicated that endophytic Trichoderma virens BLR24 isolated from the medical plant A. japonica roots could be a promising source of natural anti-MRSA agents. Endophytic fungal secondary metabolites are abundant in biologically active compounds. Endophytic fungi from medicinal plants could be a source yielding bioactive metabolites of pharmaceutical importance.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-024-01250-z.

Introduction

Infection with drug-resistant microorganisms is one of the greatest risks to public health, and antibiotic resistance infection kills about 0.7 million people each year worldwide [1]. MRSA (methicillin-resistant Staphylococcus aureus) represents a global epidemic pathogenic microorganism. The manifestations of S. aureus infections vary from mild skin and soft tissue infections, such as abscesses, boils, and cellulitis [2], to severe, life-threatening invasive infections, such as pneumonia, bacteremia, endocarditis, and osteoarticular infections [3–5]. The data show that bloodstream infections caused by MRSA are increasing by 3.9% annually in the community in the USA [6]. Somewhat ominously, strains had emerged with resistance to newer antimicrobials, including vancomycin [7]. Thus, it is urgent to develop new anti-MRSA agents to combat these drug-resistant bacteria.

Medicinal plants have a long history of being widely used to cure diseases by diverse ethnic groups around the globe. The World Health Organization (WHO) reported that over 80% of the world’s population uses medicinal plants for healthcare purposes [8], especially in antimicrobial therapy. The demand for medicinal plants is being escalated due to improved living standards and growing healthcare awareness. Nevertheless, many medicinal plants are associated with long periods of growth, high cultivation expenses, low yields, sluggish promotion of artificial cultivation, and extinction due to wildcrafting and overcollection [9]. Thus, it is particularly urgent to develop alternative approaches for exploiting the beneficial properties of medicinal plants. Recent research has found that medicinal plants contain abundant endophytic resources. Endophytic fungi are ubiquitous and are commonly found within host plant tissues of leaves, stems, or roots, where they cause no apparent harm or visible symptoms [10]. Several recent studies have shown that endophytic fungi in plants are capable of producing an array of metabolites, including alkaloids, terpenoids, and polyketides, which have various biological activities and serve as promising new sources of bioactive compounds and drugs [11]. Due to the mutualistic relationship with their host plants, endophytic fungi in medicinal plants have the ability to produce biologically active secondary metabolites that are identical or similar to those of the host, which hold great potential as pharmaceuticals or other valuable property products [12]. Additionally, some of them have the potential to produce novel compounds or lead compounds for new drug research [13]. For example, Puri et al. [14] first reported that the endophytic fungus Entrophospora infrequens from the medical plant Nothapodytes foetida produced camptothecin which was the precursor molecule for synthesizing the clinical anti-cancer drug topotecan. Therefore, endophytic fungi of medicinal plants could be promising sources of natural compounds for therapeutic diseases [15, 16], which may be used as alternative resources for exploiting the beneficial properties of medicinal plants. Endophytic fungi could serve as a promising source for the discovery of novel natural compounds to confront MRSA.

The dried root of Ampelopsis japonica (Thunb.) Makino is a traditional Chinese medicine that is used for treating dermatitis caused by bacteria, fever, pain, and wound healing [17]. It was initially recorded in Sheng Nong’s herbal classic with clearing away heat and removing toxic materials, eliminating carbuncles and dispersing knots properties. It has been reported in the literature that A. japonica has numerous pharmacological properties such as anti-tyrosinase, antioxidant, anti-melanogenesis, anti-inflammatory, antimicrobial, and antitumor activities [18–20]. However, rare research on the biological activities of endophytic fungi in A. japonica has been reported. Therefore, the objectives of this study were to isolate endophytic fungi from the root of A. japonica and assess their biological activities for anti-MRSA effects. Furthermore, the composition of endophytic fungal secondary metabolites was investigated based on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique, and the metabolites were annotated using the Global Natural Product Social Molecular Networking (GNPS) platform. The anti-MRSA compound was directly separated by bioassay-guided fractionation.

Materials and methods

Collection of plant samples and endophytic fungi isolation

Healthy wild Ampelopsis japonica (Thunb.) Makino plants were collected from the mountainous regions in Neixiang, Nanyang, Henan Province of China (33° 15′ 44.3″ N, 111° 55′ 56.0″ E). The plants were carefully dug up, placed in a sterile sampling bag, and immediately transported to the laboratory at 4°C. The plants were identified by Professor Yizhu Chen, South China Botanical Garden, Chinese Academy of Sciences. The endophytic fungi isolation procedures were performed according to the method of Cao et al. [21] with slight modifications. Briefly, the plant roots were first washed in tap water for 30 min to remove soil particles. Then, the roots were cut into small segments (about 2 cm), followed by successive immersion in 75% ethanol solution for 1 min, 5% NaClO (v/v) for 5 min, and 75% ethanol solution for 0.5 min. After being rinsed three times in sterile distilled water, the root samples were cut into small slices (0.5 cm × 0.5 cm) without two terminals, placed in potato dextrose agar medium (PDA), and incubated at 28°C for 7–14 days.

Preliminary screening for strains of anti-methicillin-resistant Staphylococcus aureus

The agar well diffusion method [22] was used to evaluate different fungal potentials against methicillin-resistant Staphylococcus aureus (MRSA, ATCC 43300, mecA positive, Fig. S1) activity. The strain with the most extraordinary capacity to suppress the pathogen was selected for further investigation. Concretely, the fermentation broth of different endophytic fungi was produced by inoculating the hyphal respectively to a 100-mL flask containing 25 mL potato dextrose broth (PDB) medium. The culture was shaken at 28°C and 120 rpm for 7 days, and the resulting broth was subsequently filtered through the 0.22 μm membrane filter. After that, the test bacteria at the exponential phase were adjusted to 10⁶ colony-forming units per milliliter (CFU/mL) through McFarland turbidity, and 100 μL was spread over the surface of sterilized Luria-Bertani (LB) agar plates using the sterile cotton swab. A sterile borer was used to drill 6-mm wells, and 40 μL of cell-free supernatant was added to each well. The negative control was treated with only PDB, while vancomycin (VAN, 100 μg/mL) was used as the positive control. The plates were then incubated at 37°C for 18 h. Finally, the diameter of inhibition zones was measured. All the experiments were carried out in triplicate.

Morphological and molecular identification

Micromorphological analyses were performed on PDA using the microculture technique described previously by Jamal et al. [23]. The structure of mycelia, conidiophores, and conidia was microscopically observed under a light microscope at ×40. Subsequently, the conidia were subjected to observation by lactophenol cotton blue staining.

Genomic DNA was extracted using a Rapid Fungi Genomic DNA Isolation Kit (Sangon, Shanghai, China) following the manufacturer’s instructions. The molecular analysis was carried out through sequencing of the region of internal transcribed spacer (ITS1-5.8S-ITS2 rDNA) with the universal primer pair of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). The polymerase chain reaction (PCR) was conducted under conditions in a 25 μL final volume consisting of 12.5 μL of Premix Taq (TaKaRa Biotechnology Co., Dalian, China), 1 μL of genomic DNA template (20 ng), 1 μL of forward and reverse primers (10 μM), and RNase-free water. The amplified was performed under the following conditions: an initial denaturation cycle at 94°C for 3 min, followed by 30 circles of 94°C at 60 s, 56°C at 60 s, and 72°C at 60 s, and a final extension for 72°C at 10 min. The PCR product was visualized on 1% (w/v) agarose gel by electrophoresis followed by purification and Sanger sequencing by Sangon Biotech Co., Ltd. The sequence obtained for the sample was used as the query sequence to search for similar sequences, align, and analyze using Basic Local Alignment Search Tool (BLAST) of National Center for Biotechnology Information (NCBI) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A phylogenetic tree was constructed using MEGA 7.0 software using the neighbor-joining method with 1000 replicates as bootstrap value. Additionally, the sequence was deposited at GenBank (NCBI), and accession numbers were obtained.

Scale-up fermentation and extraction of the secondary metabolites

The endophytic strain was subcultured on a PDA plate at 28°C for 3 days. To prepare the fungal seed culture, six agar plugs (6 mm) with mycelia were inoculated into an Erlenmeyer flask containing 50 mL PDB and incubated in a shaker incubator at 28°C, 120 rpm for 2 days. Then, the fungal seed culture was transferred to 5 × 2-L Erlenmeyer flasks, each containing 1 L of PDB, and incubated for 7 days under the same conditions [24]. After incubation, the mycelia were removed by filtering through the sterile carbasus, and the fungal broth culture was filtered with a 0.22 μm membrane, then extracted with ethyl acetate at a ratio of 1:1, filtered, and evaporated with a rotary evaporator to yield the crude ethyl acetate extract (EA). The endophytic extracts were dissolved in dimethyl sulfoxide (DMSO) as a stock solution (100 mg/mL).

Phytochemical screening of EA

Screening of phytochemicals of the extract was done based on the standard procedures to assess the class of secondary metabolites, such as alkaloids, phenols, and flavonoids [25], and evaluate by visual detection the colorimetric properties of the reactions or fluorescence appearing under ultraviolet light.

In vitro assessment of anti-MRSA activity of EA

Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of EA against MRSA

Minimum inhibitory concentration (MIC) was defined as the lowest concentration of the antimicrobial that inhibited the growth of a microorganism after overnight incubation, determined by broth microdilution assay. Minimum bactericidal concentration (MBC) was the lowest concentration of antimicrobial that showed no bacterial growth after subculture onto antibiotic-free media. The MIC of EA was determined by using the standard microdilution method according to the procedures [26]. Briefly, the logarithmic phase bacteria were harvested and resuspended to a final cell density of approximately 10⁶ CFU/mL. Subsequently, a twofold serial dilution of EA, ranging from 200 μg/mL to 0.391 μg/mL (final concentration), was prepared in a 96-well plate with 100 μL volume. Subsequently, an equal volume (100 μL) of the bacterial suspension was added to each well, along with appropriate positive and negative controls. After 24-h incubation at 37°C, the MBC was determined by seeding 10 μL liquid from all visible clear wells to LB plates. All wells were incubated with 20 μL of resazurin (0.2 mg/mL) for 3 h to ascertain the MIC further. The MIC finally is defined as the lowest concentration that showed no discernible change in the dark blue coloration.

Effects of EA on the cell morphology of MRSA

Scanning electron microscope (SEM) was used to investigate the effects of EA treatment on the morphology of MRSA cells according to Chan and Chong [27] with some modifications. The logarithmic phase cultured of MRSA was adjusted to OD₆₀₀ = 0.2, then centrifuged at 5000 × g for 10 min, and further treated with 1 mL MIC concentration of EA, meanwhile setting an untreated control. The sample was incubated for 6 h at 37°C, 150 rpm, and harvested bacterial cells by centrifugation at 5000 × g for 10 min, followed by fixation in 2.5% (v/v) glutaraldehyde at 4°C for 12 h. After being washed three times with PBS, the samples were dehydrated for 20 min at 30°C on a silicon substrate. The samples were observed under Gemini SEM 300 (Carl Zeiss, Jena, Germany) at an accelerating voltage of 15.0 kV after gold coating.

Effects of EA on biofilm formation of MRSA

The inhibitory effect of EA on biofilm formation was examined by using a previously described protocol with few modifications [28]. Briefly,1/4 MIC, 1/2 MIC, MIC, and 2 MIC (final concentrations) of EA were distributed into 96 wells containing LB supplemented with 1% glucose in a 200 μL system, and then, 180 μL of logarithmic phase MRSA (OD₆₀₀ = 0.1) was added. After static incubation at 37°C for 24 h, it washed twice gently with phosphate-buffered saline (PBS) to discard the planktonic bacteria before being air-dried for a period of 3 h. Then, the biofilms were stained with 200 μL of 0.1% crystal violet for 30 min. The dye was removed, and the wells were gently washed with PBS until clear to remove excess stain, then light microscopic imaging after air-dried. The stained wells were dissolved using 0.2 mL of 30% acetic acid, and the absorbance was measured at 570 nm using a microplate reader. The percentage of biofilm inhibition was calculated using the following equation:

$$\mathrm{Biofilm}\mathrm{inhibition}\left(\%\right)=\frac{\mathrm{Control}\mathrm{OD}570\text{--}\mathrm{Treated}\mathrm{OD}570}{\mathrm{Control}\mathrm{OD}570}\times 100$$

Effects of EA on the expression of biofilm-related genes of MRSA

After MRSA was grown to exponential phase, collected cells, and treated with EA at MIC concentration for 6 h, LB was used as solvent groups. After culturing, the cells were centrifuged at 5000 × g, 4°C for 10 min to collect all bacteria, washed twice with PBS, and ground under liquid nitrogen. According to the manufacturer’s instructions, total RNA was extracted from MRSA using TransZol reagent (TransGen Biotech Co., Ltd, Beijing, China). The RNA samples were quantified using a microplate reader with μDrop Plate. Total RNA was reverse transcribed into the cDNA template using a HiScript® III All-in-one RT SuperMix Perfect for quantitative real-time polymerase chain reaction (qPCR) (Vazyme Biotech Co., Ltd, Nanjing, China). The reverse transcription reaction was composed of 4 μL total RNA (20 ng), 4 μL 5× All-in-one RT SuperMix, 1 μL enzyme mix, and 11 μL RNase-free water in a 20 μL reaction system. The reactions were conducted with the thermal cycler ETC-811 (Eastwin, Beijing, China) at 50°C for 15 min and 85°C for 5 s.

Quantification of three target genes expression (icaA, sarA, and agrA) of MRSA was performed using the qPCR technique on CFX 96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The PCR bulk reaction mixture (25 μL) was prepared with 12.5 μL 2× TB Green® Premix Ex TaqTM II (Tli RNaseH Plus) (TaKaRa Biotechnology Co., Dalian, China), 1 μL cDNA template, 1 μL each forward and reverse primers (10 μM), and 9.5 μL RNase-free water. The one-step thermal cycling program settings were as follows: the initial denaturation at 95°C for 30 s, and then, it was followed by 40 cycles of 95°C for 5 s and 60°C for 30 s; fluorescence collected meanwhile. The product specificity was detected using a melting curve at the last phase: ranging from 65 to 95°C with an increment of 0.5°C per 5 s and fluorescence signal acquisitions. The RpoB gene, which encoded RNA polymerase β-subunit, was selected as the internal reference. The results of the relative expression were calculated using the 2^−ΔΔCt methodology. The list of primers that were used is presented in Table S1. All the primers were synthesized by Sangon Biotech Co., Ltd.

LC-MS/MS analysis of EA

LC-MS/MS analyses of EA were performed using a Vanquish UHPLC system coupled with an Orbitrap Q Exactive™ HF mass spectrometer (Thermo Fisher, Berlin, Germany). EA was dissolved in 53% HPLC grade methanol and filtered through a 0.22 μm membrane filter and then injected 3 μL onto a Hypersil Gold column (C18, 100 × 2.1 mm, 1.9 μm) at 40°C using a 12-min linear gradient at a flow rate of 0.2 mL/min. The eluents were eluent A (0.1% formic acid in water) and eluent B (methanol). The chromatographic solvent gradient was set as follows: 2% B, 1.5 min; 2-85% B, 1.5–3 min; 85–100% B, 3–10 min; 100–2% B, 10–10.1 min; and 2% B, 10.1–12 min. The Q Exactive™ HF mass spectrometer was operated in positive polarity mode with a spray voltage of 3.5 kV, a capillary temperature of 320°C, a sheath gas flow rate of 35 psi, an aux gas flow rate of 10 L/min, an S-lens RF level of 60, and aux gas heater temperature of 350°C. The instrument was operated in the data-dependent acquisition mode.

The molecular networking of EA was processed using the Global Natural Product Social Molecular Networking (GNPS) platform (https://gnps.ucsd.edu/ProteoSAFe/static/gnps-splash.jsp) according to the protocol [29]. The MS/MS raw data was converted to GNPS supported “.mzXML” format files using the MSConvert package from the ProteoWizard 3.0.22132 (ProteoWizard Software Foundation, Palo Alto, CA, USA). Subsequently, the files were uploaded on the GNPS platform using recommended Core FTP client. Then, molecular network was created using the online workflow. The parameters were set with some modification: the precursor ion mass tolerance and the fragment ion tolerance were set to 0.02 Da, respectively. The matched fragment ion was kept to a minimum of 4 and the cosine score was set above 0.7 to produce the molecular network. For library search, minimum matching peaks were set at 4, with a score threshold of 0.7. The spectral network result was imported into Cytoscape 3.8.1 and visualized for various features. Known secondary metabolites annotated by the GNPS spectral library underwent a careful evaluation through manual assessment.

Bioassay-guided separation of the anti-MRSA compound

Thin layer chromatography (TLC) bioautography was performed to determine the anti-MRSA fraction present in EA by the method described by others, with minor modifications [30]. The sample was dissolved in dichloromethane (DCM) (50 mg/mL), and then, 10 μL was spotted on the silica gel G-precoated plate (Qingdao Haiyang Chemical Factory, Qingdao, China). The plate was eluted in the solvent system (DCM/methanol = 95:5). Drying completely then exposed to iodine and UV_{365 nm} for visualizing, labeling zones, and calculating the retention factor (R_f) value.

The plate was placed in a Petri plate, covered with LB agar medium, and spread the microorganism (10⁶ CFU/mL), then incubated at 37°C for 18 h. After incubation, MTT solution (0.5 mg/mL, GlpBio, Montclair, CA, USA) was sprayed over the plate, coloring reacting at 37°C for 30 min. The clear inhibition zone indicated the antibacterial activity compound on the TLC plate. The activity spot silica gel in equal R_f value was scraped and eluted with ethyl acetate for 1 day, filtered and evaporated, and collected material (Fr.1) for further high performance liquid chromatography (HPLC) analysis.

HPLC analysis, purification, and structural identification

Typically, the sample solutions of EA and Fr.1 were prepared in acetonitrile (ACN) and then filtered. HPLC analysis of Fr.1 was carried out using an Inertsil ODS-3 column (4.6 × 250 mm, 5 μm, GL Sciences) on a Waters e2695 HPLC system equipped with a Waters 2998 photodiode array detector. The UV/vis data were acquired from 190 to 400 nm. The mobile phase A was 0.05% (v/v) trifluoroacetic acid (TFA) in water, while B was ACN. The gradient elution program was set for 0–5 min 10% B, 5–55 min 30–100% B, and 55–60 min 10% B (isocratic) at a flow rate of 0.8 mL/min.

According to the result of gradient HPLC analysis, the separation was performed under a specific eluted program and manually collected the peak fractions (Fr.1.1) at the same retention time. Compound Fr.1.1 was analyzed by LC-ESI-MS (LCMS-2020, Shimadzu, Kyoto, Japan). ¹H and ¹³C NMR spectral datasets in CDCl₃ were recorded on a Bruker 600 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). The spectrometer was operated at a frequency of 600 MHz for ¹H NMR and 151 MHz for ¹³C NMR. Subsequently, the NMR data were processed by using MestReNova software (Mestrelab Research S.L., Santiago de Compostela, Spain).

Statistical analysis

All the biological activity experiments were carried out and repeated in triplicate, and results are presented as mean ± SD from replicates of the experiments. Data were performed by one-way ANOVA followed by Dunnett’s multiple comparison tests, using GraphPad Prism 8.3.0 and plotted. In statistical analysis, p < 0.05 was used to assess significant differences wheresoever applicable.

Results

Isolation of endophytic fungi and preliminary screening of the anti-MRSA strains

A total of nine strains of fungal endophytes with diverse colony morphologies were obtained from the surface-sterilized roots of Ampelopsis japonica (Thunb.) Makino (Fig. S2). The results of fermentation broth supernatant screening for antibacterial activity showed that two strains (BLR17 and BLR24) could antagonize MRSA (Table 1), especially strain BLR24, showing the maximum sensitivity with 17.68 ± 0.28 mm inhibition zone diameter (Fig. S3). Hence, fungal endophyte strain BLR24 was selected for further investigation of its potential in anti-MRSA.

Table 1.

Inhibitory activity of the fermentation supernatant of endophytic fungal

Strains	MRSA
BLR11	−
BLR13	−
BLR16	−
BLR17	++
BLR18	−
BLR21	−
BLR24	+++
BLR27	−
BLR29	−
VAN	+++

−, no antibacterial activity; +, inhibition zone diameter (d) ≤ 10 mm, mildly sensitive; ++, 10 mm < d ≤ 15 mm, moderately sensitive; +++, d > 15 mm, highly sensitive

Identification of endophytic fungi BLR24

On PDA medium, strain BLR24 exhibited rapid radial growth of up to 90 mm in 96 h at 28°C and formed divergent, green conidia in concentric rings throughout the colony. Under the micromorphological characteristics observed with a light microscope, its conidiophores were dendritic; the phialides were subtranslucent, branching toward the tip with a conidial ball at the apex; and the dispersed conidia were round or oval (Fig. 1). The morphological characteristics of BLR24 coincided with the Trichoderma genus.

Fig. 1.

Morphological identification of endophytic fungal strain BLR24. A Conidiophore. B Conidia

The results of BLAST analysis showed that the ITS gene of BLR24 had 99.82% pairwise similarity with Trichoderma virens (ON927121.1). The phylogenetic analysis based on rDNA-ITS sequence showed that the strains BLR24 (ON979472) clustered together with the eight representative taxa of Trichoderma. Within this clade, the strain BLR24 and two representative taxa of Trichoderma virens formed a subclade with a relatively strong bootstrap support of 100% (Fig. 2). According to the morphological characteristics and molecular identification, the stain of BLR24 could be identified as Trichoderma virens.

Fig. 2.

Phylogenetic tree of endophytic fungal strain BLR24 based on the ITS. The phylogenetic tree constructed using the neighbor-joining method, Sphaerostilbella aureonitens strain GJS 74-87 (FJ442633.1), was used as the out-group; the serial number in the bracket was the GenBank accession number of the strain. Values at branch nodes represented support value. Scale length represented the evolutionary distance

Preliminary phytochemical screening of EA-BLR24

As shown in Table 2 and Fig. S4, the conventional qualitative test indicated that endophytic Trichoderma virens BLR24 had the capacity to produce several secondary metabolites such as alkaloids, phenols, and flavonoids.

Table 2.

Phytochemical screening of ethyl acetate extract of isolated strain BLR24 (EA-BLR24)

Phytochemical	Reagent	Analysis results
Alkaloids	Dragendorff’s reagent	+1, yellowish orange precipitate
Phenols	5% ferric chloride solution	+, bluish black color
Flavonoids	1% aluminum chloride ethanolic solution (UV365 nm)	+, yellow fluorescence

¹Positive test

Anti-MRSA activity of EA-BLR24

SEM observation of ultrastructural changes in MRSA upon exposure to EA at MIC concentration for 6 h showed that treatment with EA-BLR24 significantly disrupted the structure of MRSA cells by making them pitted and shriveled compared to the control group (Fig. 3). The result indicated the detrimental impact of EA-BLR24 on MRSA cell morphology.

Fig. 3.

The SEM photography of MRSA treated with EA-BLR24 after 6 h under different magnifications. A Control group. B Treatment group. Yellow arrowheads point to the damaged position

EA-BLR24 showed significant inhibition activities against MRSA with MIC and MBC values that were 25 μg/mL and 50 μg/mL, respectively (Fig. 4). As for positive control (VAN), the MIC and MBC values were 1.563 μg/mL and 1.563 μg/mL, respectively. The negative control (solvent group) had no effect on the experiment.

Fig. 4.

Determining MIC and MBC values of EA-BLR24 against MRSA. A Resazurin dye for measuring the MIC. B Spreading to determine MBC

As shown in Fig. 5A, EA-BLR24 could efficiently inhibit bacteria cell attachment. Concentrations of 6.25 μg/mL (1/4 MIC), 12.50 μg/mL (1/2 MIC), 25.00 μg/mL (MIC), and 50.00 μg/mL (2 MIC) of EA-BLR24 inhibited the biofilm formation of MRSA by 22.08%, 24.18%, 34.67%, and 51.94%, respectively. In addition, the micrograph demonstrated that the higher concentration, the larger the blank area, indicating the smaller the number of biofilms (Fig. 5B). EA-BLR24 inhibited MRSA biofilm formation in a dose-dependent manner.

Fig. 5.

Inhibition of MRSA biofilm formation potential of different concentrations of EA-BLR24. A Biofilm inhibition of EA-BLR24. B Optical observation biofilms after crystal violet dye. The different letters represent a significant difference between them (p < 0.05)

The expression of genes related to MRSA biofilm formation by EA-BLR24 treatment was investigated by the qPCR method. The results indicated that EA-BLR24 could dramatically inhibit the expression of biofilm-related genes icaA, sarA, and agrA compared with the control groups (p < 0.01) (Fig. 6).

Fig. 6.

The effect of EA-BLR24 (25 μg/mL) on biofilm-related gene expression levels of MRSA. Note: compared with the control group, **p < 0.01 and ***p < 0.001

LC-MS/MS analyses and molecular network predict of EA-BLR24

LC-MS/MS analysis of EA-BLR24 was performed (Fig. S5). The tandem mass spectrometry data were processed, and then, the molecular network was established using the GNPS platform. The data files were deposited in MassIVE Dataset (MassIVE ID: MSV000090625). A total of 711 parent ions were detected in EA-BLR24. Clusters were constructed in the generated visual molecular networking (Fig. 7), revealing the presence of diverse types of natural products in EA-BLR24. Most clusters have not matched the compounds in the current GNPS mass spectral library. And only 20 compounds could be annotated based on MS/MS data analysis (Table 3), which mainly belonged to diketopiperazine compounds, such as cyclo(proline-leucine), bisdethiobis(methylthio)gliotoxin, and gliotoxin.

Fig. 7.

GNPS molecular network of EA-BLR24. Nodes represented features detected by MS/MS, and the number over lines represented cosine similarity scores. The values in nodes are m/z [M+H]⁺ of compounds identified. The number in the circle was indexed in Table 3

Table 3.

Putative identification of chemical compounds in EA-BLR24

Compound no.	Molecular formula	Compound name	m/z [M+H]+	MZ error (ppm)
1	C11H14O5	Pyrenocine B	227.082	13.24
2	C11H18N2O3	Cyclo(L-Leu-L-4-hydroxy-Pro)	227.139	0
3	C11H18N2O2	Cyclo(proline-leucine)	211.144	0
4	C10H16N2O2	Cyclo-(L-Val-L-Pro)	197.128	5.03
5	C14H16N2O2	Cyclo(L-Phe-D-Pro)	245.128	0
6	C14H16N2O3	Cyclo(Phe-4-hydroxy-Pro)	261.123	3.86
7	C28H45N5O7	Destruxin A2	564.339	0
8	C28H45N5O8	Destruxin E2	580.334	3.47
9	C22H43NO	13-Docosenamide, (Z)	338.341	2.98
10	C18H35NO	9-Octadecenamide, (Z)	282.279	3.57
11	C11H15N5O4S	5′-Deoxy-5′-(methylsulfinyl)adenosine	314.091	3.21
12	C19H24N2O3S	Fusaperazine E	361.158	0
13	C10H30O5Si5	Cyclopentasiloxane decamethyl	371.101	2.63
14	C13H14N2O4S2	Gliotoxin	327.047	0
15	C47H95N2O6P	N-Tetracosanoyl-4-sphingenyl-1-O-phosphorylcholine	815.699	4.94
16	C10H13N5O3	2′-Deoxyadenosine	252.109	0
17	C10H13N5O4	Adenosine	268.104	0
18	C55H74N4O5	Pheophytin A	871.573	2.31
19	C15H20N2O4S2	Bisdethiobis(methylthio)gliotoxin	357.093	2.82
20	C14H16N2O3	Cyclo(tyrosyl-prolyl)	261.124	3.86

Bioautography and preparation of the anti-MRSA compound of EA-BLR24

EA-BLR24 was subjected to TLC analysis and showed several bands with different migration rates through either iodine vapor or ultraviolet light for detection (Fig. 8A, B). As the bioautography result showed, the spot at R_f = 0.64 revealed the activity against MRSA with significant inhibition zones (30.94 ± 0.78 mm) (Fig. 8C, D).

Fig. 8.

TLC analysis of EA-BLR24 and bioautography. Revealed with 365 nm UV light (A) and iodine vapor (B). Zones of inhibition by the bioactive fraction on TLC plate (C) and dye by MTT (D)

From the preparative TLC plate, the individual band (Fr.1, R_f = 0.64) was collected and performed with gradient analytical HPLC to ensure the best solvent system (0.05% trifluoroacetic acid in water: ACN = 13.5:86.5, v/v; monitored at 269 nm wavelength) (Fig. S6). Further purification was conducted, collecting the target fraction with a retention time of 10.89 min, and obtaining sufficient antibacterial agents (Fr.1.1) (Fig. 9, Figs. S7-8).

Fig. 9.

HPLC analysis of Fr.1

Structure elucidation of the anti-MRSA compound (Fr.1.1)

Compound Fr.1.1 was obtained as a white needle, and its molecular formula was determined as C₁₃H₁₄N₂O₄S₂ from the ESI-MS at m/z 327.24 [M+H]⁺: 1H NMR (600 MHz, CDCl₃), δ 4.44 (1H, dd, J=12.8, 5.8 Hz, H-3a1), 4.28 (1H, dd, J=12.8, 9.7 Hz, H-3a2), 3.67 (1H, dd, J=9.8, 5.8 Hz, 3a-OH), 4.84 (2H, s,H-5a, H-6), 5.71 (1H, s, 6-OH), 5.80 (1H, d, J=9.6 Hz, H-7), 5.96 (1H, dd, J=9.6, 4.7 Hz, H-8), 6.02 (1H, d, J=3.2 Hz, H-9), 3.77 (1H, dd, J=17.8, 2.4 Hz, H-10a), 2.98 (1H, d, J=17.9 Hz, H-10b), 3.22 (3H, s, Me-2). 13C NMR (151 MHz, CDCl₃), δ 166.03 (C-1), 77.15 (C-3), 60.66 (C-3a), 165.31 (C-4), 69.81 (C-5a), 73.15 (C-6), 129.99 (C-7), 123.34 (C-8), 120.25 (C-9), 130.70 (C-9a), 36.58 (C-10), 75.58(C-10a), and 27.49 (2-Me). Based on the above NMR data (Figs. S9-10) and the comparison with those reported in literatures [31, 32], compound Fr.1.1 was confirmed as gliotoxin.

Discussion

Compounds with medicinal properties derived from various endophytic fungi have contributed to human health, energy, agriculture, etc., and performed various biological activities, especially in antimicrobial, antioxidant, and anti-cancer activities [33]. Numerous metabolites obtained from endophytic fungi exhibit unique structures, and several of them hold potential as valuable drug precursors [34]. In this study, phytochemical investigations reveal that EA-BLR24 was rich in alkaloids, phenols, and flavonoids, which were considered to have the major biological activities of cytotoxicity, antiviral, anti-inflammatory, and antimicrobial [35]. Thus, it was significant to investigate the bioactivity of EA-BLR24.

Some investigators had isolated a certain percentage of MRSA-resistant fungal endophytes, and the extract of endophytic fungi demonstrated antimicrobial activity against MRSA with MIC values ranging from 26 to 256 μg/mL [36–38]. In this study, we obtained two fungi strains (22.22%) with excellent antibacterial activity among the isolates, and the MIC of EA-BLR24 was 25 μg/mL, displaying the best activity. The SEM result indicated that EA-BLR24 could directly damage the cells and harm cell morphology. This finding is consistent with the results of a study by Ibrahim et al. [39], in which treatment with the extract of Nigrospora sphaerica CL-OP 30 resulted in the formation of cavities and disintegration of MRSA cells. Chowdhury et al. [37] also reported similar results.

Compared to planktonic growth, MRSA favored the biofilm mode of growth in natural environments, which led to highly resistant to the antibiotic. MRSA biofilms often cause infections associated with medical implants and increased medical burden [40]. The present study revealed that EA-BLR24 effectively inhibited the formation of MRSA biofilm in a dose-dependent manner. There are two possible effects of EA-BLR24 on biofilm inhibition on the surface of the coverslip. EA-BLR24 could kill the cell before it was attached to the surface or modify the surface to dispute bacterial growth, adherence, and colonization [41]. Similar to the research in a previous study [42], the results of transcription levels of the biofilm-related genes (polysaccharide intercellular adhesion locus gene icaA, staphylococcal accessory regulator locus gene sarA, and quorum-sensing gene agrA) were downregulated at the initial stage (6 h) after treatment. It indicated that EA-BLR24 prevented biofilm formation mainly by repressing biofilm-related gene expression at the initial infection stage, limiting the intercellular adhesion, and reflecting the complex mechanism of action of EA-BLR24 in preventing biofilm formation.

In the quest to discover the bioactive compounds from endophytic fungi BLR24, metabolomic tools based on LC-MS/MS were used. The GNPS platform is a valuable tool for rapid and automated comparison of fragmentation patterns based on the metabolomic data repository MassIVE so that it effectively dereplicates [43, 44]. This dereplication process was significant in identifying novel compounds or known substances, guiding the isolation of target compounds. Among GNPS annotated compounds, diketopiperazines were the main classes in EA-BLR24, which have been previously reported with antimicrobial activity, including cyclo(proline-leucine), cyclo-(L-Val-L-Pro), gliotoxin, bisdethiobis(methylthio)gliotoxin, and cyclo(tyrosyl-prolyl) [45–48]. Thus, we hypothesized that these components in EA-BLR24 dominate the anti-MRSA activity.

Additionally, our previous study found that EA-BLR24 also had significant antiproliferative effects on cancer cell lines and eased lipopolysaccharide (LPS)-stimulated inflammation in RAW264.7 cells (data not shown). Among the results of GNPS annotations, compounds destruxin A2 and destruxin E2 had previously been reported to exhibit cytotoxicity activity [49], and pheophytin A was observed, which has been reported to exert anti-inflammatory effects by reducing proinflammatory cytokine [50]. Natural products from endophytic fungi BLR24 could be potential anti-MRSA, anti-cancer, and anti-inflammatory drugs.

TLC bioautography is an effective method that combines chromatographic separation and in situ biological activity determination, particularly in natural antimicrobial preliminary screening and bioactivity-directed isolation [51]. Based on the molecular network results, our current work designed a suitable elution system and then directly isolated an anti-MRSA compound, identified as gliotoxin, a known compound with strong antimicrobial activity [52]. LC-MS/MS data also observed the specific fragments of gliotoxin (Fig. 10). To our knowledge, gliotoxin is the first epidithiodioxopiperazine (ETP) class fungal toxin reported, catalyzed by the non-ribosomal peptide synthetase (NRPS) GliP, which has been widely reported as a broad-spectrum antimicrobial, immunosuppressive agent, and antitumor drug at the correct dose [53]. Studies investigating the mechanism of action of gliotoxin have made some progress, revealing that it has the potential to induce apoptosis and inhibit proteasome activity, which in turn can lead to the inhibition of NF-κB activity and reduction in immune response, inhibit NADPH oxidase assembly, and generate ROS, resulting in an imbalance in redox homeostasis and severe oxidative stress [53, 54]. The anti-MRSA, cytotoxic, and anti-inflammatory activities of EA-BLR24 may be the result of the interaction of various secondary metabolites.

Fig. 10.

A GNPS network of gliotoxin. B The specific MS/MS peaks of gliotoxin in EA-BLR24

The results illustrated that endophytic fungi were indeed a treasure, containing abundant active secondary metabolites. Our study proved the feasibility of combining molecular networks and bioautography for targeted compound isolation and also provided us with new ideas to continue the study. There are still many unannotated compounds by GNPS that can be subsequently purified by designing suitable technical routes, which may lead to the isolation of novel compounds. Such findings hold promise for the development of novel drugs derived from endophytic fungi.

Conclusion

In this study, a fungal endophyte BLR24 was isolated from the medical plant Ampelopsis japonica roots and identified as Trichoderma virens. The crude ethyl acetate extract of fermentation supernatant of strain BLR24 (EA-BLR24) showed good anti-MRSA activity, down-regulation in expression levels of biofilm formation-related genes, and notably inhibited the formation of MRSA biofilm. EA-BLR24 was abundant in biologically active compounds. A compound gliotoxin was isolated by bioassay guided with anti-MRSA biological activity. The results indicated that endophytic Trichoderma virens BLR24 could be a promising source of natural anti-MRSA agents. Endophytic fungi of medicinal plants harbor a profusion of natural active products, bestowing upon them a distinct and noteworthy application value, thereby warranting further investigation.

Author contribution

Jianbin Li: conceptualization, methodology, investigation, data analysis, project administration, and writing—original draft. Siyun Xie: methodology, investigation, and data analysis. Qing Gao: methodology, investigation, and data analysis. Zujun Deng: conceptualization, supervision, project administration, writing—review and editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 31971384).

Data availability

The ITS rDNA sequence has been submitted to the NCBI GenBank and assigned the accession number ON927121.1. The metabolite data file was deposited in MassIVE Dataset with ID: MSV000090625. Data will be made available on request.

Declarations

Ethics approval

Not applicable.

Consent to participate

All authors declared their consent to participate.

Consent for publication

All authors declare their consent to publish their work.

Competing interests

The authors declare no competing interests.