University of North Carolina at Chapel Hill Graduate School
Doi
Abstract
The recent increase in multidrug-resistant pathogens and associated morbidity/mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. While natural products are a well-studied source of biologically active small molecules, peptidyl factors contributing to their medicinal properties remain largely unexplored. To expedite the search for this exciting class of compounds this dissertation describes the development, validation, and applications of PepSAVI-MS (Statistically-guided bioactive peptides prioritized via mass spectrometry). This highly versatile pipeline employs whole-cell bioactivity screening coupled with mass spectrometry and bioinformatics to identify bioactive peptides from complex biological extracts (Chapter 2). The development and validation of PepSAVI-MS was originally described using a botanical source through the successful detection and identification of a known antimicrobial peptide, cycloviolacin O2 (cyO2), from Viola odorata (Chapter 3). In addition to pipeline validation, this study widened the known antimicrobial spectrum for V. odorata cyclotides, establishing antibacterial activity of cyO2 against Acinetobacter baumannii, and explored novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast and prostate cancer cell lines.
To demonstrate the versatility and wide applicability of PepSAVI-MS, validation studies were performed using fungal (Chapter 4) and bacterial sources (Chapter 5), with optional experimental modifications to highlight pipeline utility. Identification of the virally-encoded antifungal killer toxin KP4 from Ustilago maydis P4 and the bacteriocin Bac-21 from Enterococcus faecalis pPD1 demonstrates proof-of-principle for bioactive peptide discovery from bacterial and fungal secretomes.
Using the validated pipeline, a variety of natural product sources are probed to prioritize highly active species for downstream analysis. The plant species Amaranthus tricolor is presented as an example of novel antimicrobial activity identified with PepSAVI-MS (Chapter 6). As demonstrated herein, the developed pipeline is powerful and highly versatile; PepSAVI-MS can incorporate material from a variety of natural product sources and is able to accommodate any developed bioactivity screen. The flexible nature of this pipeline presents a valuable tool for diverse natural product exploration, and has the potential to lead to the discovery of a wealth of bioactive peptides.Doctor of Philosoph
