Fungal endophytes as source to combat bacterial infections By Jaimie Green An Undergraduate Thesis Submitted to Oregon State University In partial fulfilment of the requirements for the degree of Baccalaureate of Science in BioResource Research, Biotechnology, Plant Growth and Development Options May 21, 2019 APPROVED: _____________________________________________________ ______________ Jeffery Stone, Botany and Plant Pathology Date _____________________________________________________ ______________ Sandra Loesgen, Chemistry Date _____________________________________________________ ______________ Katharine G. Field, BRR Director Date © Copyright by Jaimie Green, May 21, 2019 I understand that my project will become part of the permanent collection of the Oregon State University Library, and will become part of the Scholars Archive collection for BioResource Research. My signature below authorizes release of my project and thesis to any reader upon request. _______________________________________________________ ____________ Jaimie Green Date Fungal endophytes as source to combat bacterial infections Endophytes, foliage inhabiting fungi, are an unexplored source of fungal biodiversity and a potential resource for the production of bioactive natural products. In this research, leaf tissues from the species Arbutus menziesii, Rhododendron macrophyllum and the genus, Ilex and Salix were collected for the isolation of endophytic fungi. Fungal extracts were prepared from liquid cultures of selected fungi and tested for activity against methicillin-resistant Staphylococcus aureus (MRSA) . Bioactive extracts from isolate JG-74P showed high activity against methicillin-resistant Staphylococcus aureus and yielded the molecule, biruloquinone . Keywords: methicillin-resistant Staphylococcus aureus, Leaf Endophytes, Fungal Natural Products, Antibiotics Introduction Endophytic fungi are single cell organisms that occupy the intracellular space of plants (Stone el al 2004). This internal colonization is generally viewed as non-pathogenic, since endophytes are capable of asymptomatic colonization (Stone et al. 2004). This means that part or all of the fungus’s life cycle does not cause harm to the plant. The fungi are often in mutualistic relationships with plant hosts. Benefits include fitness factors, such as drought resistance, and even protection from pathogens (Saikkonen et al. 1998). Many endophytes grow as saprobes (feeding off dead tissue) and this blurs the line between pathogenic (disease causing) and non-pathogenic (Stone et al. 2004). Endophytes often co-evolve with the host and form symbioses with the plant host, and the plants can gain benefits from the fungi (Stone et al. 2004). Endophytes live in diverse environments; not only can they be found in a plethora of plant phyla, but even different types of tissue can harbor different endophytes (Petrini et al. 1991). These factors mean that each plant could be a potential source for a wide diversity of fungal species. Antibiotics have been a valuable resource to humans since the discovery of penicillin in 1928 (Earnst et al. 1974). They are used in both medicine and agriculture (Hillary et al. 2016). Bacterial infections that were once life threatening were widely treated with penicillin through the 1940s (O’Neil et al. 2016). However, an increasing concern is the evolution of drug resistance in human pathogenic bacteria. Current clinical and agricultural practices provide strong selection for evolution of drug resistance (O’Neil et al. 2016). Bacteria will naturally evolve and overcome antimicrobials through natural selection (O’Neil et al. 2016). A prime, relevant, example is Staphylococcus aureus, which is capable of producing a new generation every 24 hours with the potential to generate resistant genes every generation (Hilary et al. 2016). Antimicrobial resistance is predicted to kill 10 million people worldwide by the year 2050, which is more than the expected deaths due to cancer (O’Neil et al. 2016). With the rise of resistance within community and hospital bacterial strains, the need for new antibiotic classes is urgent. The objective of this research was to explore endophytic fungi as a source of new compounds bioactive against MRSA. Over the course of this research, 78 fungi were isolated. Host plants included Ilex (Holly), Arbutus menziesii (Madrone), Rhododendron , and Salix (Willow). Materials and methods Chemicals used Table 1. Chemicals and nutrients used throughout the course of the research CHEMICALS FUNGAL NUTRIENTS Sodium Hypochlorite Agar Ethanol Malt Extract Ethyl Acetate DMSO Acetonitrile Dichloromethane Methanol Plants collections Leaf tissue samples were taken from Corvallis, Benton County, OR ( Ilex, Rhododendron), Lewisburg Saddle, Benton County, OR ( Arbutus), and Peoria, Linn County, OR ( Salix) . Figure 1 shows locations, and the genus of plant collected. Figure 1. Map of Corvallis, OR and the surrounding area. All plants samples were taken within 20 miles of the city limits. (Google) Lewisburg Saddle Collected Arbutus menzeisii Area is high elevation Peoria, Oregon Cordley Hall, OSU Collected Salix Collected Ilex Wetland Area Urban Area Avery Park, Corvallis Collected Rhododendron Public Park Collected plants were taken back to the lab for processing. Leaf tissue was surface- sterilized to prevent non-endophytic contaminants from growing. Plants with a thick waxy cuticle, like Arbutus menziesii, Rhododendron, and Ilex, required longer sterilization times (Schulz et al. 1993). Using a protocol described by Schulz, the leaf tissue from different plants were soaked in 70% ethanol for 1 minute, followed by a soaking in 2.5% sodium hypochlorite solution, and a final rinse for 1 minute in 70% ethanol. A preliminary experiment was performed for each plant host with varying duration of hypochlorite treatment to determine optimum disinfection times. Tissue was disinfected for 1-5 minutes, cut into 1cm squares and placed on 1.5% water agar plates in order to isolate endophytic fungi. The plates were monitored four times a week for two weeks, and any fungal growth was transferred onto 1.5% malt-based agar. To determine what isolates were epiphytic or endophytic, we used a modified procedure was performed as described by Schulz et al.. (1993). For each isolate, a small piece of fungus and agar was sterilized in bleach for 1-5 minutes. Any isolates surviving the secondary wash were considered to be epiphytes (living outside the cellular matrix) and therefore discarded. We found that 2 minutes was a sufficient amount of time to disinfect tissue in sodium hypochlorite, and this was used for the rest of the experiment. In total, 74 endophytic fungi (see supplementary data) were isolated. Five isolates were selected for further chemical and bioactivity analysis. Isolates include JG-37 ( Arbutus menziesii ), JG-45 ( Arbutus menziesii ), Genus Penicillium (JG-49, identified through morphological traits) ( Arbutus menziesii ), JG-74P ( Ilex ) and Genus Cladosporium (JG- 74W, identified through morphological traits) (Ilex) . Extraction process Duplicates of 50 mL sterile 1.5% malt media were inoculated with each fungal isolate in baffled flasks and grown at 28 °C on an orbital shaker at 110 rpm for seven days. After seven days, 10 mL of these cultures were then transferred into 1 L of 1.5% malt media. A clean streak was performed on small scale cultures to confirm single species. Large-scale cultures were left to grow at 28°C on an orbital shaker at 110 rpm for seven to fourteen days. After seven to fourteen days, the cultures were then adjusted to a pH of 6.0 ± 0.1 with HCl or NaOH, followed by the addition of equal parts ethyl acetate (EtOAc) left to stir overnight and the organic layer separated. The culture broth was subjected to two more extractions using equal volumes of EtOAc. The combined organic layers were collected, dried over anhydrous MgSO 4, and concentrated under vacuum. Bioassays Bioassays were performed using a protocol from the Loesgen lab (2016). Aliquots of extracts were prepared in DMSO at 10 mg/ml. Testing was done with methicillin- resistant Staphylococcus aureus (MRSA, ATCC BAA-41), for the purpose of new drug discovery. The microbroth assay was performed in 96 well plates and sample wells were dosed with 1.25 µg of extract. Positive control was 25 µg vancomycin and negative control was 1.25% DMSO. Further MRSA tests were done on fractions as well. Assay plates were left to incubate at 37°C for 18 hours. After 18 hours, optical density measurements at 600 nm were used to determine the cell density. Chemical analysis and strain identification While the extract was being tested in bioassays, 60-120 mg of fungal extracts were separated via vacuum liquid column chromatography (VLCC). VLCC uses silica gel and various solvent systems to separate mixtures by polarity. The dry extracts were mixed with 10x the mass in silica, and enough acetone to dissolve. The solvent was removed using a rotary vacuum evaporator. Columns were packed with 3 cm of silica, the silica/extract mixture, and then laboratory grade sand on top. The column was attached to an extraction manifold and connected to a vacuum system. A pre-weighed vial was placed at the collection point and solvent was poured into the column. Table 2 shows the gradients of solvent used for each fraction. Table 2. Dichloromethane:Methanol (DCM:MeOH) ratios used in VLCC FRACTION DCM:M EOH 1 1:0 2 30:1 3 15:1 4 9:1 5 3:1 6 1:1 7 0:1 Bioassays were performed on seven fractions that showed activity