Using metabolomic tools to bioprospect antibiofilm agents from Philippine endolichenic fungi

Rondila, Roberth Riggs L.

Thesis

Using metabolomic tools to bioprospect antibiofilm agents from Philippine endolichenic fungi

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Creator

Rondila, Roberth Riggs L.

Rights statement

Strathclyde Thesis Copyright

Awarding institution

University of Strathclyde

Date of award

2025

Thesis identifier

T17479

Person Identifier (Local)

202284536

Qualification Level

Doctoral (Postgraduate)

Qualification Name

Doctor of Philosophy (PhD)

Department, School or Faculty

Strathclyde Institute of Pharmacy and Biomedical Sciences

Abstract

The rise of drug-resistant pathogenic bacteria is an alarming threat to global public health. One key factor contributing to antibiotic resistance is the formation of bacterial biofilms, which protects microorganisms from antimicrobial agents and causes genetic mutations. Thus, the discovery of compounds that can inhibit or disrupt biofilm formation is an urgent and promising research direction. This PhD study investigated endolichenic fungi (ELF) as a novel source of antibiofilm secondary metabolites. ELF are mutualists within the lichen thallus and are known producers of a wide array of bioactive metabolites. A total of ten ELF collected from the Philippines were screened for their antibacterial and antibiofilm activities against methicillin-resistant Staphylococcus aureus (MRSA; ATCC 29213) and P. aeruginosa (ATCC 43300). Metabolomics-based chemical profiling using liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance spectroscopy (NMR) were employed. Three ELF stood out both with antimicrobial effects and distinct chemical profiles and thus were prioritised for subsequent investigations. These ELF were identified by molecular DNA sequencing as Daldinia eschscholtzii, Hypoxylon lateripigmentum, and Xylaria cubensis. To enhance metabolite production, different culture media (four types) and incubation durations (15 and 30 days) were tested, along with the supplementation of chemical elicitors (5-azacytidine and suberoyl bishydroxamic acid). Optimised culture conditions were established and applied for scaled-up fermentation. For X. cubensis, rice medium with a 30-day incubation was optimal. Chromatographic isolation led to the identification of mellein, a possible mixture of hydroxylated mellein analogues, phloroglucinol (first reported from a fungus), and monolinolein. Mellein inhibited MRSA biofilm formation by 77.83% (prebiofilm) and 71.82% (postbiofilm) at100 μg/mL. Phloroglucinol showed 93.8% antibacterial and 98.69% pre-biofilm inhibition at the same concentration. Both compounds exhibited no cytotoxicity to HEK293 cells. H. lateripigmentum was found to have enriched production of metabolites in potato dextrose broth (PDB) for 15 days and malt extract broth for 30 days. Applying the isolation strategy described above, TAN-1612, 3-hydroxypropionic acid, and 2,3-didehydropalitantin were isolated and identified from the PDB culture while TAN-1612, daldinones A, B, and E were obtained from MEB cultures. These compounds were reported here for the first time in this species. TAN-1612 exhibited potent antibacterial activity (MIC = 13.83 μM) and biofilm inhibition (BPC = 12.50 μM), with low cytotoxicity (IC50>200 μM). D. eschscholtzii produced 2(3H)-benzofuranone, regiolone, tyrosol, and niacin in rice medium, though these exhibited negligible bioactivity (<40% prebiofilm inhibition at 100 μg/mL) which suggested possible synergistic and/or additive effects in crude extracts or parent fractions. Supplementation with chemical elicitors in its PDB culture led to the enhanced production of cytochalasin derivatives and BNT. These compounds, along with daldinone B, showed strong antibiofilm activity in MRSA (>70% prebiofilm inhibition at 100 μg/mL) but with weak cytotoxicity (IC50 range 95-115 μM), warranting further structure-activity relationship (SAR) studies as proposed. In conclusion, herein this study demonstrated the utility of metabolomics in natural product bioprospecting, from extract prioritisation, culture optimisation, and compound isolation. A total of 25 compounds were isolated and identified from the three Philippine ELF strains. This study identified compounds that have potential to be developed as antibiofilm drugs such as TAN-1612, BNT, and cytochalasins. This work positions Philippine ELF as promising sources of compounds targeting drug-resistant biofilm-forming pathogens.

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