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 during testing of the original extract. Liquid chromatography-mass spectroscopy was used to determine what compounds were shared between the different fractions (HP 1100 LCMS). This was done by comparing molecular weights and UV patterns of peaks found in fractions.

Thin layer chromatography was also used to visualize chemical differences between fractions.

Results

Five fungal strains were selected for bioassay testing. These strains were selected due to interesting morphology or color, indicating potential secondary metabolites of interest.

JG-74P showed high activity against Methicillin-resistant Staphylococcus aureus, with only 8.15% cell survival (Table 3). JG-74W had even more activity, but JG-74P was chosen due to its color and interesting phenotype.

Figure 2 Antibiotic testing against methicillin-resistant Staphylococcus aureus

(ATCC BAA-41). Extracts at 1.25 μg.

JG-74P

Extract of strain JG-74P (40 mg) was fractionated using vacuum liquid column chromatography. Fractions 5-7 showed low cell survival of MRSA (Figure 3. LCMS chromatography indicated that fractions 5-7 share a similar absorption peak at 20 minutes (Figure 4), indicating a shared molecule. The target molecule was most abundant in fraction 6, with 73% purity (7.1 mg). UV maxima for this compound were found at 240, 320, 400, and 540 nm (Figure 5), and the molecular weight was determined to be 326 g/mol (m/z: 327.1; M+H +) (Figure 6). The compound is a dark blue powder. Figure 3 Antibiotic testing against methicillin-resistant Staphylococcus aureus (ATCC BAA-41). JG-74P Fractions were tested at 1.25 μg.

Figure 4 360 nm UV absorption of JG-74P fractions. LCMS comparison of molecules present in fractionated extracts of JG-74P.

Figure 5 UV absorption of the metabolite at 20 minutes in fraction 6.

Figure 6 Mass spectroscopic data of the metabolite at 20 minutes in fraction 6.

The molecule isolated from JG-74P is biruloquinone (Figure 7), a purple crystalline powder isolated from Cladonia macilenta (Leu Heng et al. 2012). Biruloquinone has a matching molecular weight of 326.1 and the same UV pattern of 240, 320, 400, 540 nm as well as matching 1H NMR data with chemical shifts of 2.87, 4.03, 6.96, 7.02, 12.50, and 12.66 δ/ppm. Figure 7 Biruloquinone

Discussion Currently, MRSA is resistant to drugs in the penicillin class, as well as many drugs from the cephalosporin class. Vancomycin is a final option for treating MRSA, but the emergence of multidrug-resistant Staphylococcus aureus might limit treatment options in the future (Baddour et al. 2010). Molecules with unique architectures and derived from underexplored resources might stand a higher chance of being effective against

MRSA. Endophytes, especially those producing pigments, have a high probability of producing complex molecules (Oudar et al. 1977) and therefore unique molecules.

Discovering a bioactive molecule could not only provide a possible treatment for

MRSA, but could also give insight on molecule types and features that may be effective as drug leads.

In this study, we isolated fungi from five genera of plants and selected unique colonies of endophytes. Metabolites from these colonies were extracted and tested against

MRSA. JG-74P was found to be highly toxic to MRSA cells (Figure II). These extracts were fractionated and tested against MRSA, using an approach called bioactivity-guided fractionation (Figure III). Figure V shows the LCMS data for all seven fractions, showing a peak at 20 minutes that was shared only between the three active fractions. We hypothesized that the compound eluting at 20 minutes is most likely the bioactive compound, and we selected it for further study.

One limitation of this study was the growing of the cultures. Metabolites produced can vary due to time the culture was grown, amount of light, temperature, and variation in media composition. Another limiting factor was that the plants selected were chosen due to metabolites (such as grayanotoxin in Rhododendron ) that may impact or select the endophytes found. Arbutus menziesii (Madrone) was selected for having medicinal properties (Kabadi et al. 1963) as well as Salix (Willow) (Asgarpanah et al. 2012) and

Ilex (Holly) (Lu et al. 2018). Fungal cultures do not always continue to produce the same compounds or quantities of small molecules when taken out of the intracellular environment (Schulz et al. 2002). Lastly, when examining a plant for endophytes, our methods are unable to detect and cultivate obligate biotrophs.

Future work includes purifying more of the biruloquinone found in JG-74P by chromatographic means. Next, DNA identification of JG-74P would also be useful to add taxonomic data to the documentation of natural products produced. Additionally, mammalian cell culture testing would be valuable to determine the cytotoxicity of the molecule to be useful as an antibiotic. Special thanks to the E.R. Jackman Internship Support Program for undergraduate research funding.

References: A MRSA-terious enemy among us. (2011). Nature Medicine , 17 (2), 168. Retrieved from https://link-galegroup- com.ezproxy.proxy.library.oregonstate.edu/apps/doc/A249223117/AONE?u=s8 405248&sid=AONE&xid=32f7f769 Asgarpanah, J. (2012). Phytopharmacology and medicinal properties of Salix aegyptiaca L. African Journal of Biotechnology, 11(28), 7145–7150. https://doi.org/10.5897/AJB12.418 Baddour, M. M. (2010). MRSA (methicillin resistant Staphylococcus aureus) infections and treatment. New York: Nova Science Publishers. Ernst Chain. (1974). Penicillin. Nature, 249(5458), 608. https://doi.org/10.1038/249608c0 [Google Maps view of Corvallis, OR]. (20 April 2019). Google Maps. Google. Retrieved from https://www.google.com/maps/place/Corvallis,+OR/@44.562951,- 123.3535767,17788m/data=!3m2!1e3!4b1!4m5!3m4!1s0x54c0409daa14d77d:0 xd70d808f22bdc0be!8m2!3d44.5645659!4d-123.2620435 Hikino, H., Shoji, N., Koriyama, S., Ohta, T., Hikino, Y., & Takemoto, T. (1970). Stereostructure of rhodojaponin. IV. Toxin of Rhododendron japonicum , and of grayanotoxin. v, VI, and VII, toxins of Leucothoe grayana'. Chemical &, Nov (11), 2357-2359. Hillary D, Marston, H. D., Dixon, D. M., Knisely, J. M., Palmore, T. N., & Fauci, A. S. (2016). Antimicrobial Resistance. JAMA, 316(11), 1193–1204. https://doi.org/10.1001/jama.2016.11764 Stone, Jeffrey (2004). Endophytic Fungi in Mueller, Gregory M.. Biodiversity of Fungi : Inventory and Monitoring Methods, edited by Gerald F. Bills, and Mercedes S. Foster, Elsevier Science & Technology. Kabadi, B., & Hammarlund, E. R. (1963). Preliminary Identificaiton of the Antibacterial Principles of “Madronin” from the Leaves of Arbutus menziesii. Journal of Pharmaceutical Sciences, 52, 1154–1159. Loesgen, Sandra. “Preparation and Maintenance of Culture of Pathogenic Bacteria and Fungi for Antimicrobial Assays.” Department of Chemistry Oregon State University, Oct. 2016 Luo, H., Jiangsu, Li, C.T., Jilin Agricultural, kim, J.C., Korea Research Institute of Chemical Technology, Daejeon, Republic of Korea, Liu, Y.P., Jiangsu, Jung, J.S., Sunchon National, Koh, Y.J., Sunchon National, & Hur, J.S., Sunchon National. (2013). Biruloquinone, an Acetylcholinesterase Inhibitor Produced by Lichen-Forming Fungus Cladonia macilenta. Journal of Microbiology and Biotechnology, (2), 161–166. Lu, X., Luo, C., Xing, J., Han, Z., Li, T., Wu, W., … Chen, W. (2018). Optimization of Storage Conditions of the Medicinal Herb Ilex asprella against the Sterigmatocystin Producer Aspergillus versicolor Using Response Surface Methodology. Toxins, 10(12). https://doi.org/10.3390/toxins10120499 O'Neill, Jim. “Tackling Drug-Resistant Infections Globally: Final Report and Recommendations.” Amr-Reveiw , 2016, amr review.org/sites/default/files/160518 Oudar, J. L. (1977). Optical Nonlinearities of Conjugated Molecules. Stilbene Derivatives and Highly Polar Aromatic Compounds. The Journal of Chemical Physics, 67(2), 446–457. https://doi.org/10.1063/1.434888 Pischon, H., Petrick, A., Müller, M., Köster, N., Pietsch, J., & Mundhenk, L. (2018). Grayanotoxin I Intoxication in Pet Pigs. Veterinary Pathology , 55 (6), 896–899. https://doi.org/10.1177/0300985818789482 Saikkonen, K., S.H. Faeth, M. Helander, and T.J. Sullivan. 1998. Fungal Endophytes: A Continuum of Interactions with Host Plants. Annual Review of Ecology and Systematics. 29:319– 343.

Supplementary Material

Table VIII Details of all isolated strains with morphological description

Reference Genus of Plant Location Minutes Description Number JG-## Collected From Collected Sterilized in Bleach

1 Rhododendron Avery 0 Brown/ Oily Park

2 Rhododendron Avery 0 Brown/ Fuzzy Park

3 Rhododendron Avery 0 Black Park

4 Rhododendron Avery 0 Filamentous/ Park Green Speckling/ Branched Ascus Keyed out dichotomopilus

5 Rhododendron Avery 0 Dark Green/Black Park

6 Rhododendron Avery 1 White/ Fuzzy/ Flat Park

7 Rhododendron Avery 1 White/ Flat/ Dark Park Sclerotia

8 Rhododendron Avery 1 White/ Flat/ Dark Park Sclerotia

9 Rhododendron Avery 1 Brown/Black/ Oily Park

10 Rhododendron Avery 1 Brown/ Black/ Park Dark Sclerotia

11 Rhododendron Avery 2 Light Brown/ Park Fuzzy

12 Rhododendron Avery 2 Brown/ Fuzzy/ Park Splotchy

13 Rhododendron Avery 2 Olive/ Park Downy/Filiform

14 Rhododendron Avery 2 Olive/ Park Downy/Filiform 15 Rhododendron Avery 2 Olive/ Park Downy/Filiform

16 Rhododendron Avery 2 Olive/ Park Downy/Filiform

17 Rhododendron Avery 2 Olive/ Park Downy/Filiform

18 Rhododendron Avery 2 Dark Brown, Media Park Dark as Well

19 Rhododendron Avery 2 White/ Flat/ Dark Park Sclerotia

20 Rhododendron Avery 2 Olive/ Park Downy/Filiform

21 Rhododendron Avery 2 Olive/ Park Downy/Filiform

22 Rhododendron Avery 2 Dark Green/ Park Sclerotia/ Filamentous

23 Rhododendron Avery 2 Olive/ Park Downy/Filiform

24 Rhododendron Avery 2 Olive/ Park Downy/Filiform

25 Rhododendron Avery 2 Olive/ Park Downy/Filiform

26 Rhododendron Avery 2 Olive/ Park Downy/Filiform

27 Rhododendron Avery 2 Olive/ Park Downy/Filiform

28 Rhododendron Avery 2 White/ Flat/ Park Somewhat Transparent/ Lots of Spores

29 Rhododendron Avery 2 Olive/ Park Downy/Filiform

30 Rhododendron Avery 2 Dark Green/ Green Park Sclerotia

31 Rhododendron Avery 2 Darkly Pigmented Park Filamentous Hyphea/ Pigment Formed a Broken Pattern 32 Rhododendron Avery 2 White/ Somewhat Park Transparent

33 Rhododendron Avery 2 Dark/ Park Undulate/White on Top

34 Rhododendron Avery 2 White/Lots of Park Spores

35 Rhododendron Avery 3 White/ Flat/ Park Somewhat Transparent

36 Rhododendron Avery 4 White/ Flat/ Park Somewhat Transparent

37 Madrone Lewisburg 0 Orange-Pink colony Saddle with White Fuzz/ Media Turned Orange

38 Madrone Lewisburg 0 Brown/ Patches of Saddle Fuzz

39 Madrone Lewisburg 1 Brown/ Downy/ Saddle Matted

40 Madrone Lewisburg 2 Brown/ Downy/ Saddle Matted

41 Madrone Lewisburg 2 Brown/ Fuzzy/ Saddle Splotchy

42 Madrone Lewisburg 2 Olive Saddle Colored/Downy

43 Madrone Lewisburg 2 Brown/ Slightly Saddle Fuzzy

44 Madrone Lewisburg 2 White/ Flat/ Dark Saddle Sclerotia

45 Madrone Lewisburg 2 Bright Blue Saddle Colony/Forms White Growths Over Time

46 Madrone Lewisburg 2 Dark Brown Colony Saddle with a White Crust

47 Madrone Lewisburg 2 Dark/Oily Saddle 48 Madrone Lewisburg 2 Olive Saddle Colored/Downy

49 Madrone Lewisburg 2 Light Green/ Lots Saddle of Spores/ Turned Agar Orange

50 Madrone Lewisburg 2 White/ Flat/ Dark Saddle Sclerotia

51 Madrone Lewisburg 5 Dark/ Saddle Filamentous/ Turned Agar Dark

52 Juniperus OSU 0 Brown/Black Campus Cladosporium

53 Salix Peoria 5 Black/Oily Oregon

54 Crataegus Peoria 0 Brown/ Fuzzy/ Oregon Splotchy

55 Crataegus Peoria 0 Brown/ Fuzzy/ Oregon Splotchy

56 Crataegus Peoria 0 Light Brown/Fuzzy Oregon

57 Crataegus Peoria 0 White/Flat/Slightly Oregon Transparent

58 Crataegus Peoria 1 White/Flat/Slightly Oregon Transparent

59 Crataegus Peoria 1 Brown/Fuzzy Oregon

60 Crataegus Peoria 2 Brown/Fuzzy Oregon

61 Crataegus Peoria 2 Dark/ Oily Oregon

62 Crataegus Peoria 2 Very Dark/Slimy Oregon

63 Crataegus Peoria 2 Reddish Brown Oregon

64 Crataegus Peoria 2 Dark Brown/ Oily/ Oregon Filamentous

65 Crataegus Peoria 2 Dark/Oily Oregon 66 Crataegus Peoria 2 Dark Brown/Fuzzy Oregon

67 Crataegus Peoria 2 Dark Brown/Fuzzy Oregon

68 Ilex OSU 2 Green Campus Colony/White Fuzz on Top

69 Ilex OSU 2 Green Campus Colony/White Fuzz on Top

70 Ilex OSU 2 Green Campus Colony/White Fuzz on Top

71 Ilex OSU 2 Dark Brown/ Oily/ Campus Flat

72 Ilex OSU 2 Dark Brown Campus Colony/White Fuzz on Top

73 Ilex OSU 2 Dark Brown Campus Colony/White Fuzz on Top

74* Ilex OSU 2 Dark Purple Colony Campus with White on Top