Metabolomics of Endophytic Fungal Isolate Grown with and without Antibiotics to Identify Bioactive Metabolites and their In-silico Studies

Abstract

Endophytes are the microbes resides inside plant without any apparent harm to the host. These microbes are potential source of new therapeutic compounds against many diseases especially against microbial infections. Resistance to antibiotics by bacterial pathogens is growing concern at present, antibiotics are becoming increasingly ineffective as drug-resistance spreads globally leading to more difficult to treat infections. According to WHO currently, at least 700,000 people die each year due to drug-resistant diseases. Therefore, the need of new potent compounds with broad spectrum antibacterial activity is of high time. Natural products have been as widely used as chemical drugs against clinical diseases. Studies revealed that currently, approximately 60 % of approved small molecule medicines are related to natural products, and 69 % of all antibacterial agents originate from natural products. In the search towards discovery of novel drugs, endophytic fungi have appeared as an outstanding source of high metabolic versatility. This study describes the metabolic profiling, isolation, structure elucidation, bioactivity, and In-silico studies of the bioactive compounds obtained from the fungal isolates of Taxus fauna of West Himalayan region of Pakistan. The fungal isolates were grown in culture medium with and without addition of different antibiotics. The presence of antibiotics in culture medium may induce certain bioactive metabolites or the antibiotic themselves can be biotransformed into a different chemical structure comprising improved antibacterial activities. The aim of the study was to find potentially new compounds with antibacterial activities. Seven endophytic fungal isolates of Taxus fauna including Epicoccum sp. NFW1, Mucor hiemalis NFW6, Epicoccum nigrum NFW7, Chaetomium sp. NFW8, Fusarium oxysporum NFW16, Penicillium milleri NFL1 and Paraconiothyrium sp. NFL6 were cultivated on Sabouraud dextrose medium with and without the addition of antibiotics Clarithromycin, Moxifloxacin, Ciprofloxacin, Cephradine and Cefixime. After incubation, the fermentation broth was extracted with ethyl acetate and the crude extracts were evaluated for antibacterial activities. The crude extracts of Epicoccum sp. NFW1, Chaetomium sp. NFW8 and Fusarium oxysporum NFW16 showed enhanced antibacterial effect when media was amended with clarithromycin and moxifloxacin, so these three fungal isolates were selected for metabolomics profiling and bioactive xviii compounds isolation under the stress of clarithromycin and moxifloxacin in the medium. Metabolic profiling of the endophytic fungal isolates with and without antibiotics were carried out by screening the crude extracts with LC-ESIHRMS and data was analyzed and dereplicated by using global natural product social molecular networking (GNPS). Molecular networks were created using the online workflow at GNPS (http://gnps.ucsd.edu). The molecular networking revealed that the antibiotic in the medium changed the metabolic profiles of the fungal isolates by inducing certain metabolites as well as by overwhelming the production of few other metabolites which were produced without the antibiotic stress. The dereplication studies showed that several known bioactive metabolites were produced by these endophytic fungal isolates while many of the metabolites in molecular network were were putatively new or novel metabolites. Epicoccum sp. NFW1 among them showed strong metabolic profile in terms of unknown compounds. The crude extracts were initially purified by normal phase silica gel column chromatography. The active fractions obtained from the column were pooled up and subjected to preparative HPLC for purification. The purified bioactive compounds were structurally elucidated by 1D, 2D NMR and HRESI-MS. A novel dimeric compound (8E,16E)-5,7,10,15,18,20-hexahydroxy-3,22-dimethoxy-5,20-dimethyl-1,24- diphenyltetracosa-8,16-dien-1,2,4,6,19,21,23,24-octaone with chemical formula C40H46O16 was isolated and characterized from the Epicoccum sp. NFW1 grown in the medium under clarithromycin stress. Another new compound (1R,2R,3R,4S,5S,6R)-1- ((1R,2S,3S,4R,5R,7R,Z)-6-(1-aminoethylidene)-8-(cyclopenta-2,4-dien-1-ylmethyl)- 1,3,4,5,7-pentahydroxy-2 isopropoxynon-8-en-1-yl)cyclohexane-1,2,3,4,5,6-hexaol with chemical formula C26H43NO12 was also obtained from the same fungal isolate when grown with the addition of moxifloxacin to the medium. Similarly, one novel compound 11-(tert-butyl)-11-(hydroxymethyl)-13,13-dimethyl-2,5,8-trioxa-1(1,4)- benzenacyclotridecaphan-10-ol (C22H36O5) was isolated from Chaetomium sp. NFW8 grown in medium containing clarithromycin while a previously known compound 4-N butylphthalic acid (C12H14O4) was first time isolated from any fungal source in this study from Chaetomium sp. NFW8 when grown in SDB supplemented with moxifloxacin. A novel compound (S)-1,2,3,6-tetrahydrocyclopenta[d]azepine-6- xix carboxylic acid (C10H11NO2) was isolated from Fusarium oxysporum NFW16 grown in the presence of clarithromycin in SDB medium. These compounds showed moderate to strong antibacterial activities against test bacterial strains. The structurally characterized compounds were tested for antibacterial activity but to get a better picture without losing the compound in-silico studies (docking, molecular dynamics simulation ad binding energies) were performed. A random screening was performed to check the binding affinity of reported compounds to antibacterial targets. It was unraveled that MurF enzyme of bacterial cell wall machinery might be the target to which these compounds bind with good affinity. It was found that the affinity is the attribute of both hydrophobic and hydrophilic interactions and binding was seen both at the ATP and UDP binding substrate of the MurF. All the reported compounds form stable complex with MurF protein. This study showed that these endophytic fungal isolates can grow on medium added with high concentrations of antibiotics. The addition of antibiotics to medium will change the metabolic profile of endophytic fungal isolates. Metabolomics and dereplication studies revealed that the endophytic fungal isolates are reservoirs of many known and new therapeutically important compounds. Antibacterial activity and In silico studies suggest that these compounds have significant inhibitory effect against several disease-causing bacteria.