Mechanism of action of two active antimicrobial peptides derived from aurein 2.2

With the increasing problem of antibiotic resistance, coupled with the limited number of newly developed antibiotics, therapeutic alternatives are urgently required. Host defense peptides (HDPs), also known as antimicrobial peptides (AMPs), are promising candidates as they have multifaceted anti-infective properties as well as a lower likelihood of causing antibiotic resistance. HDPs are ubiquitous in nature and constitute an important part of the innate immune system of almost all life forms. However, only a few peptides are in clinical trials due to issues such as systemic toxicity, proteolytic degradation and short half-life from renal/hepatic clearance. Therefore, it is important to minimize the negative impacts while improving the antimicrobial activity at the same time. Part of this thesis describes the various functions of HDPs including antibacterial, antibiofilm, immunomodulatory activities, along with the associated mechanisms of action (MOAs). The determination of antibacterial and antibiofilm MOAs of active HDPs forms the basis of this thesis, with a description of commonly used methods. From an array of aurein 2.2 analogue peptides, peptide 73 and peptide 77 were chosen for further study as they showed the highest antibacterial activity. Furthermore, both analogue peptides demonstrated better antibiofilm activity than aurein 2.2. Different biophysical techniques were used to investigate the structure – function relationship as well as the antibiofilm MOA of these analogue peptides. Specifically, the binding interaction with the alarmone nucleotide ppGpp was investigated, to determine whether the analogues function by inhibiting the bacterial stringent response. In addition, biological techniques were also employed to characterize their in vitro activity against bacteria. Finally, the antibacterial and antibiofilm activities of several HDPs are compared to attempt to assess whether these anti-infective properties are linked or independent and future experiments are proposed. Overall, the analogue peptides demonstrated higher antibacterial and antibiofilm activities compared to aurein 2.2. In addition, both have a slightly different antibiofilm mechanism of action compared to the antibiofilm peptide, IDR-1018. The results from this thesis will help to establish a foundation for future HDP design so that they can be used as therapeutics to combat antimicrobial resistance (AMR).Science, Faculty ofChemistry, Department ofGraduat