Antibacterial Activity of Ethyl Acetate Extract of Endophytic Fungus (Paraconiothyrium Brasiliense) Through Targeting Pathogenesis Related Dihydropteroate Synthase (DHPS)


 Endophytic fungi are innumerable sources of bioactive metabolites with various biomedical applications. Hence, this study was aimed to isolate and identify the endophytic fungus Paraconiothyrium brasiliense (PB) from the fruit of Capsicum annuum and evaluated their biochemical, antimicrobial, antioxidant, cell viability, and cytotoxicity behavior. Results showed that ethyl acetate extract (EAE) of PE contains flavonoid (31.53 ± 0.9 mg of QE/g of extract) and phenolic (2.59 ± 0.06 mg of GAE/g of extract). The PB-EAE showed significant antioxidant activity in terms of free radicals such as DPPH, ABTS, and Fe3+ scavenging. The antioxidant potential of PB-EAE reflects in normal cell viability and anticancer activity against prostate cancer (PC3) cells. The bacterial inhibitory activity of PB-EAE was equal to commercial antibiotic tetracycline hydrochloride (TCH). Further, GC-MS analysis confirmed the presence of volatile molecules such as o-cymene (CH3C6H4CH(CH3)2), dipentene (C10H16), γ-terpinene (C10H16), 4-carvomenthenol (C10H18O), heneicosane (CH3(CH2)19CH3), 2,5-piperazinedione (C4H6N2O2). Also, molecular docking analysis demonstrated the significant interactions between the compounds of PB-EAE and Dihydropteroate Synthase (Protein ID:5JQ9; DHPS). This study revealed that endophytic P. brasiliense from Capsicum annuum could be a novel source for the isolation of antimicrobials.

derivative compounds showed signi cant anticancer activity when testing with the MCF-7 cell line [18,19]. A study reports that brasilamide K-N (1)(2)(3)(4) and new bergamotane sesquiterpenoids were isolated from the P. brasiliense through the fermentation process [20]. Interestingly, paraconfuranones A-H and I-M were reported from Acrida cinerea gut-associated P. brasiliense [21,22]. A new ketol compound (sporulosol) was isolated from P. sporulosum and found that showed signi cant anticancer activity against different cancer cell lines [23]. The recent study reports that ten new sesquiterpenoids such as paraconiothins A-J were isolated from P. brasiliense ECN258 associated with Cinnamomum camphora [24]. Dihydropteroate synthase (DHPS) is a key enzyme involved in dihydrofolate synthesis in microorganisms and plants. Further, dihydrofolate is required to make the nitrogenous base (purines and pyrimidines) of DNA and RNA. Hence the inhibition of the DHPS enzyme is an important target for achieving antimicrobial activities [25].
Therefore, endophytic fungi could be the key source for the drug discovery and development of several diseases. However, among the large group of endophytic fungi, a limited number of fungal secondary metabolites were reported. Further, isolated novel compounds and their derivatives are not su ciently studied for their biological functions. To the overview of the Paraconiothyrium sp., were reported several new terpenoid derivatives but those compounds are just tested their cytotoxicity and not studied their other biological functions such as antioxidant, antibacterial, antidiabetic, etc. Hence, this study aimed to evaluate biochemical and biological activities of ethyl acetate extract of endophytic fungus P. brasiliense from Capsicum annum. The biochemical studies include the analysis of total phenol and avonoid content and volatile compounds analysis by gas chromatography-mass spectrum (GC-MS) analysis. The biological studies include antioxidant, antibacterial, cytotoxic activities. Also, volatile molecules interaction was tested with DHPS enzyme through molecular docking analysis.s

Sample collection and Isolation of endophytic fungus
The healthy bell pepper (Capsicum annum L.) was purchased from the local former market in Chuncheon, Republic of Korea in May 2020. Then the bell pepper samples were brought to the laboratory and impurities were removed by washing with tap water and distilled water. For the surface sterilization, the bell pepper samples were cut as pieces (approximately 5 × 5 mm) washed with ethanol (95%), sodium hypochlorite (4% chlorine), and ethanol (95 %) for 1 min, 4 min, and 30 s, respectively. Followed by the bacterial cell suspension was prepared by putting the 5 pieces of bell pepper in 10 mL of 0.9 % sterile saline solution and vortexed for 2 min. Then, 50 µL of ten-fold diluted cell suspension was inoculated on potato dextrose agar (PDA) supplemented with tetracycline hydrochloride (20 µg/mL) and incubated at 26 ± 2°C for 72 h. Afterward, about 10 unique single fungal colonies were subculture on PDA plates.
Finally, the unique fungal strain (KNUFCPF01) was selected for further molecular identi cation based on morphological characteristics and antibacterial activities.

Molecular identi cation
Fungal strain (KNUFCPF01) was cultured on PDA plates for 3-5 days. Approximately, 100 mg of fungal mycelia were collected without agar and used for gDNA extraction according to the CTAB method reported elsewhere. The isolated fungal gDNA was used as a template to amplify the marker gene internal transcribed spacer (ITS) using the primers ITS1 and ITS2 [26]. The PCR product separated using the 1% agarose gel electrophoresis and the fragment were isolated and sequenced at macrogen (https://www.macrogen.com/ko/main/index.php). Afterward, the ITS gene sequences data were compared with available ITS data in the NCBI database (https://www.ncbi.nlm.nih.gov/) through Nucleotide BLAST search. The phylogenetic tree was formed to demonstrate the similarity between the existing ITS gene sequence of the same strain along with the out-group of Rhizopus oryzae were obtained from the NCBI GenBank database by the neighbor-joining method using MEGA X software.

Extraction of metabolites
The fungal strain KNUFCPF01 was cultured in the potato dextrose broth (PDB) at 26 ± 2°C under static conditions for 14 days. Afterward, the culture was ltered using Whatman No 1 lter paper. The obtained culture ltrate was mixed with ethyl acetate 5:1 ratio and collected the upper organic layer. Further, the extraction process was repeated 3-4 times and all the extracts pooled together and condensed using a rotary evaporator at 45°C. Finally, the ethyl acetate extract (EAE) was collected and stored at room temperature.

GC-MS analysis
The existence of volatile compounds in PB-EAE was determined using Gas chromatograph/mass selective detector (GC/MSD, Agilent Technologies, USA, 7890A, 5975C) analysis. About 10 mg of EAE was dissolved in 10 mL of methanol. Then 2 µL of sample loaded into the Agilent column (DB-5 ms (30×0.25 mm, 0.25 µm), column ow at 1 mL/min with helium gas (He), and interface temperature at 280°C. The volatile compound was detected by the mass selective detector with the scan range of 40-400 m/z and matched with the electronic library (W8N05ST.L).

Antioxidant and enzyme inhibition assay
Free radical scavenging ability of different concentrations of PB-EAE was determined through DPPH, Ferric reducing and ABTS radical scavenging assay according to the earlier reports [27]. The enzyme (αamylase and α-glucosidase) inhibitory activities of EAE were determined according to the earlier report Cell culture assay The human embryonic kidney cell line (HEK-293 cells), and prostate cancer cell line (PC3 cells) were obtained from the Korea cell line bank. The cells were cultured in a T-25 culture ask using the high glucose DMEM for HEK-293 cells and RPMI media for PC3 cells supplemented with antibiotic penicillin and streptomycin, respectively. Both cells were maintained in a 5 % CO 2 incubator at 37°C. Cell viability of PB-EAE was determined using a WST viability assay kit. Both cells (1x 10 4 ) were seeded into the 96-well plate individually containing the respective cell culture media and incubated for 24 h. Subsequently, 10µL/well of different concentrations of PB-EAE were treated and incubated for 12 h. Then, WST reagent (10 µL/well) was added and continued the incubation for 1 h. Afterward, the 96-well culture plate was read at 450 nm. The cell viability (%) was determined according to the earlier report [29]. Further, the IC 50 of the EAE for PC3 cells was evaluated using different apoptotic uorescent staining assays according to the earlier report [30].

Antibacterial activity
The bacterial inhibition ability of PB-EAE was evaluated by well diffusion assay in both Gram-positive (Bacillus cereus (ATCC 14579), Staphylococcus aureus (ATCC 19095), and Listeria monocytogens (ATCC 19115)) and Gram-negative (Escherichia coli (ATCC 43888) and Salmonella enterica (ATCC 14028) bacterial pathogens. The bacterial pathogens were obtained from the American Type Culture Collection (ATCC), University Blvd, Manassas, US. For the antibacterial assay, each bacterial strain was cultured in nutrient broth at the density of 1 × 10 9 (colony-forming units (CFU)/mL) and it was swapped onto a Muller-Hinton agar (MHA) plate using a cotton swab. Then, wells were made by metal cork-borer, the different concentrations of EAE were added to each well along with the positive control (tetracycline hydrochloride). Afterward, these plates were incubated at 37°C overnight. After incubation, the antibacterial activity was determined in terms of the zone of inhibition. The minimum inhibitory concentration of EAE determined by the broth dilution method was reported elsewhere [31]. In brief, different concentration of 10 µL of EAE of endophytic fungus (1000, 500, 250, 125, 62.5, 31.25, 15.62, 7.8, 3.9 µg/mL) was treated with each bacterial culture (5 × 10 5 CFU/mL) in 100 µL of MHB medium in 96 well plate for 24 h. After incubation, the plates have measured the absorbance at 600 nm for calculation of MIC.
In vivo molecular docking analysis Further, the antibacterial molecular mechanism of volatile molecules in the PB-EAE was demonstrated by molecular docking analysis with Yersinia Pestisis dihydropteroate Synthase (DHPS) enzyme (5JQ9). Firstly, the volatile molecules of EAE were tested and selected depends on their drug-likeness giving to Lipinski's rules [32]. The volatile molecule structure was developed using colonial smiles from NIH PubChem (https://pubchem.ncbi.nlm.nih.gov/) and energy minimalized by UCSF chimera 1.14. Besides, the target bacterial DHPS enzyme (PDB ID: 5JQ9) was obtained from the RCSB protein data bank (https://www.rcsb.org/). The molecular docking analysis was done in Argus Lab (Version 4.0.1), and then the results of interactions were visualized using BIOVIA Discovery Studio Visualizer (v20.1.0.19295).

Statistical analysis
All the experiments were done individually at least thrice, and results were shown as the means with standard deviation (SD). The statistical signi cance was determined using one-way analysis variance (ANOVA). p < 0.05, statistically considered as the signi cance.

Results And Discussion
Isolation and molecular identi cation of strain KNUFCPF01 Among the different fungal isolates, the endophytic fungal strain KNUFCPF01 (Fig. 1A) was selected and identi ed by sequencing of the ITS region (ITS1, 5.8S, and ITS2 of gDNA. The gene sequencing of strain KNUFCPF01 was identi ed as P. brasiliense (MT269522). A study reported that endophytic fungus Paraconiothyrium sp. was isolated from the root of Capsicum annum [33]. Those nding was supported the existence of Paraconiothyrium sp. in Capsicum annum. Further, the phylogenetic relationship among the P. brasiliense strains and other genera was studied and shown in Fig. 1B. The neighbor-joining method of MT269522, a phylogenetic tree constructed using gene sequences of partial ITS1 and ITS2, complete 5.8S rRNA in MEGA X. The phylogenetic tree showed that strain MT269522 was clustered the similarity with Paraconiothyrium strains whereas R. oryzae was used as an outgroup.

Antioxidant activity
Antioxidant molecules are functioning as the substrate to quenching the incomplete oxygen reduction of reactive oxygen species (ROS) formation in mitochondria thereby protects the cells from oxidative damage [50,51]. Also, the increased level of ROS oncogenic signaling molecules initiates uncontrolled cell proliferation [51]. To reduce chronic and degenerative diseases, antioxidant molecules have been used widely from different sources (bacteria, algae, fungi, and plants) [52,53]. The present study showed that EAE of P. brasiliense contains a wide range of antioxidant molecules which are identi ed from biochemicals content and volatile compounds pro le analysis. Hence, EAE of P. brasiliense was tested for inhibition of ROS formation by DPPH and ABTS radical scavenging assay and ferric reducing power assay (Table 1). According to the experimental result, the EAE of P. brasiliense showed the IC50 concentration for DPPH radical scavenging at 383.51 ± 6.97 µg/mL compared to ascorbic acid at 69.11 ± 0.99 µg/mL. Similarly, a previous study reported that EA extract of Paraconiothyrium sp. P83F4/1 showed potential DPPH activity [54]. Also, the EA extract of P. brasiliense showed the IC50 concentration for ABTS radical scavenging at 29.57 ± 1.56 µg/mL compared to ascorbic acid 41.84 ± 1.37 µg/mL. The same way EA extract of endophytic fungus Diaporther sp. showed ABTS radical scavenging activity [55]. In addition, EA extract of P. brasiliense showed the IC50 concentration for ferric reducing power at 358 ± 10.33 µg/mL compared to ascorbic acid 317.6 ± 4.11 µg/mL. Similarly, endophytic fungal (Fritillaria unibracteata var.) extract reported that required a higher concentration for reduction of ferric ion to ferrous [34]. Based on the different antioxidant assays, we concluded that EA extract of P. brasiliense could be the potential source for the isolation of antioxidant molecules.

Cell viability
Cell viability assay is used to determine how the metabolites or drug molecules affect cell proliferation and its biochemical changes. In order to test the cell viability effect, EAE of P. brasiliense, HEK-293 cells, and PC3 cells were used, and cell viability was determined by the WST assay method [56]. The EAE of P. brasiliense treated PC3 cells were showed the IC50 concentration at 187.3 ± 9.29 µg/mL while HEK293 cells did not show any IC50 concentration up to a tested concentration of 1000 µg/mL (Fig. 3). Similarly, EA extract of endophytic Phomopsis sp. did not affect the normal cell (HeLa cells) proliferation while showed the anticancer activity against breast cancer cell line (MCF-7) [57]. Hence, the results revealed that EAE of P. brasiliense showed toxicity against cancer cells but not in a normal cell. Further, EAE of P. brasiliense treated PC3 cell viability and apoptotic changes evaluated using different uorescent staining. The results of nuclear staining (PI and DAPI) demonstrated that more dead cells population in EAE of P. brasiliense treatment compared to untreated control PC3 cells (Fig. 4). The mitochondrial membrane potential of cells was determined using a rhodamine 123 uorescent stain. The results indicated that EAE of P. brasiliense treated cells were lost the mitochondrial membrane potential compared to control. Also, the higher ROS elevation was observed in EAE of P. brasiliense treated cells but not observed in control cells evaluated by DCFH-DA staining. The overall results revealed that EAE of P. brasiliense had the excellent cytotoxic behavior on PC3 cells but less toxic to normal cells. Similarly, the study reported that brasilamide E (1) from P. brasiliense showed the toxicity to cancer cells (HO8910, MCF-7, and HeLa) while non-toxic to normal cell (NIH3T3) [19]. Also, ketal compound sporulosol exhibited considerable cytotoxicity to HeLa, T24, A549, HCT116, and SH-SY5Y cells [23]. But isolated eight furanone compounds (paraconfuranones A-H) from P. brasiliense MZ-1 did not show any toxicity to A549, HepG2, and CaSki cells [22].

Antidiabetic assays
The digestive enzymes play a major role in converting complex sugar molecules into simple sugar molecules through enzymatic digestion [58]. Even if in the case of diabetic patients, blood sugar might be increased than normal people due to the enzymatic action. Hence, the inhibition of enzyme function subsequently decreased the blood sugar level. In the present study, P. brasiliense EA extract did not show considerable digestive enzyme inhibitory activity against α-amylase and α-glucosidase (Data not shown).

Antibacterial activity
Antibacterial agents are used to killing the bacteria (bactericidal) or slow down the bacterial growth (bacteriostatic). Most of the antibacterial agents were initially isolated from natural sources and then they have been synthesized their own or derivative form for the applications [59,60]. However, prolonged use of drugs creates antibiotic-resistant bacterial strains which urge the discovery of novel antibiotics. Hence, EAE of P. brasiliense was evaluated for its antibacterial e cacy with bacterial pathogens such as B. cereus, S. aureus, L. monocytogenes, E. coli, and S. enterica (Fig. 5). The results showed that EAE of P. brasiliense had substantial inhibitory activity against both Gram-positive and Gram-negative bacteria. The inhibitory activity depending on the concentration that means when increasing/decreasing the EA extract concentration, the bacterial inhibition activity was increased/decreased, respectively. Among the different concentrations of EAE of P. brasiliense, 50 µL of maximum tested concentration (1000 µg/mL) showed the zone of inhibition at 17 ± 1.2 mm for B. cereus, 22 ± 1.4 mm for S. aureus, 24 ± 1.4 mm for L. monocytogens, 8 ± 0.4 mm for E. coli, and 21 ± 1.6 mm for S. enterica (Table 3). Also, the positive control 20 µL TCH (1000 µg/mL) exhibited the zone of inhibition 21 [49]. But the antibacterial properties of Paraconiothyrium sp. were studied inadequately the research gap could be lled by future studies.

Molecular docking analysis
Molecular docking analysis was performed to identify the interaction of metabolites with the molecular target of anti-bacterial activity. The inhibition of dihydropteroate synthase (DHPS) is one of the key mechanisms to kill the bacterial or inhibit bacterial growth through pterin-sulfonamide conjugation [61]. According to those strategies, the identi ed volatile compounds from EA extract of P. brasiliense interact with DHPS. The results indicated that out of 12 volatile compounds, 10 compounds completely satis ed Lipinski's rules (Table 4). Further, molecular docking analysis of major volatile compounds was tested with DHPS (5JQ9) was shown in Table 5 and Fig. 6. The docking results showed that 5 volatile compounds such as o-cymene, dipentene, γ-terpinene, heneicosane, and 2,4-di-tert-butylphenol had the higher molecular interaction according to the binding energy. But those volatile compounds had no hydrogen bonding, which indicated that antibacterial mechanism might be done other than inhibition of DHPS mechanisms. Interestingly, one of the volatile compounds 2,5-piperazinedione showed less binding energy (-5.20 kcal/mol) with greater hydrogen bonding (PRO15; THR53; VAL17; GLY51) ( Fig. 5) comparing to other volatiles tested in the present study.

Conclusion
In the present study, endophytic fungus (Paraconiothyrium brasiliense MT269522) was isolated from the fruit of Capsicum annum L. and reported for the rst time. The EAE of P. brasiliense (NCBI accession no. MT269522) contains bioactive volatile compounds such as γ-terpinene, 4-carvomenthenol, heneicosane, 2,5-piperazinedione. Therefore, EAE of P. brasiliense signi cantly inhibits DPPH, ABTS + , and ferric ion free radical formation. In addition, the EAE did not show any toxicity to HEK293 cells, but notable cytotoxicity to cancer cells (PC3). Interestingly, the EAE of P. brasiliense showed broad-spectrum antibacterial activity against human Gram-positive and Gram-negative bacterial pathogens as equal to standard antibiotic tetracycline hydrochloride. Also, molecular docking analysis con rmed 2,5-piperazinedione had the considerable interaction with DHPS enzyme thereby induced antibacterial activity like sulfonamide class of antibiotic. We conclude that EAE of endophytic fungus (P. brasiliense) could be a potential source for the discovery of antioxidant, anticancer, and antimicrobial molecules.

Ethics Approval
This article does not contain any studies with animals or human participants.   Figure 1 The culture morphology of P. brasiliense on PDA medium -A, and phylogenetic tree constructed using neighbour-joining method -B. Δ represent the isolated sequence from Capsicum annuum.