Biophysical Characterization and Mass Spectral Identification of Cationic Antimicrobial Peptides

Abstract

Cationic antimicrobial peptides (CAMPs) are a highly sequence and structurally diverse group of peptides that exert antibacterial, antifungal and antiviral effects. They have recently been of interest due to their therapeutic potential, however the current CAMP library is limited and those that are known have mechanisms that are not fully understood. One of the main objectives of this dissertation was to investigate CAMP- membrane interactions and how it relates to their antimicrobial potency. Chapter 1 examines similarities and differences of short CAMP isomers (L-ATRA-1A and D- ATRA-1A) in their antimicrobial effectiveness and interactions with model membranes. This chapter establishes the ability of these CAMPs to exert differing antimicrobial potencies against varied bacterial strains, as well as, their ability to adopt helical structure and directly interact with model membranes. The results observed here give insights into the ability of D-isomers to be used as viable therapeutic candidates in place of their L-counterparts. In chapter 2, the mechanism by which these truncated CAMPs and their full-length parent peptide (NA-CATH) interact with the bacterial membrane is investigated. Here the ability of these CAMPs to depolarize and disrupt the membranes of different bacteria gives insight into differences in the mechanisms each peptide employs. The results from these studies provides a starting point for developing an understanding how truncating full-length CAMPs effects the antimicrobial mechanism. The second objective of this dissertation was to identify novel antimicrobial peptides to improve the current library of known CAMPs. Chapter 3 describes the development of a new, sample agnostic process for the identification of novel CAMPs, which was applied in the analysis of alligator plasma resulting in the identification of novel peptides that exhibit antimicrobial activity. The process established here has the potential to dramatically impact the way CAMPs and other peptides of interest, such as biomarkers, are discovered in the future. Finally, in chapter 4 the use of LC-MS/MS for de novo sequencing of the novel alligator peptides is further investigated. Initially, known CAMPs exhibiting varying physico-chemical properties were chosen, and mass spectrometry parameters were adjusted in order to yield successful de novo sequences for these peptides. Once these parameters had been established, de novo sequencing of alligator peptides was performed. The results from this study demonstrate the power of LC-MS/MS for de novo sequencing peptides from highly complex samples. Overall, this dissertation provides an improved understanding of the way CAMPs exert their antimicrobial effectiveness, as well as, an improved method for the identification and sequencing of novel peptides, including CAMPs.