Gaspar, A.R.M. (Anabella Regina Marques)2025-01-152025-01-152025-04-302024-09-16*A2025http://hdl.handle.net/2263/100045Dissertation (MSc (Biochemistry))--University of Pretoria, 2024.A major challenge associated with antimicrobial resistance (AMR) is the lack of new drugs especially for the treatment of infections caused by Gram-negative bacteria. Antimicrobial peptides (AMPs) present an alternate solution to the crisis of AMR, as AMPs are novel antimicrobials with unique modes of action and potentially reduced risk of developing antibiotic resistance. Previous studies have identified the antimicrobial potential of the frog skin-derived AMP, the caerulein-precursor fragment-P2 (CPF-P2). To advance the development of this AMP for therapeutic applications, the aim of this study was to further evaluate the antimicrobial activity, mode of action, cytotoxicity and stability against Escherichia coli ATCC 700928. Against E. coli ATCC 700928, the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of CPF-P2 was 16 ± 0.7 μg/mL (6.08 ± 0.27 μM), with an MIC/MBC ratio of 1 indicating that this AMP is bactericidal. Against E. coli biofilms, the minimum biofilm prevention concentration (MBPC) of CPF-P2 was 640 μg/mL (243.2 μM), whereas both the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) were >2560 μg/mL (> 972.8 μM). This implies that although some biofilm prevention occurred, against established biofilms CPF-P2 was ineffective. Only at 128 μg/mL (48.64 ± 2.64 μM) and 256 μg/mL (97.28 ± 2.25 μM) CPF-P2, did human erythrocyte haemolysis occur while some cytotoxicity was observed against HaCaT cells at 256 μg/mL (97.28 ± 4.81 μM) after 24 hours exposure. The mode of action studies included the determination of the killing kinetics, kinetics of membrane permeabilisation and the subsequent effects on bacteria ultrastructure. At the MBC of CPF-P2 (i.e. 16 μg/mL) the killing time was 120 minutes, mediated by membrane permeabilisation which was time- and concentration-dependent. Ultrastructural studies with scanning electron microscopy, confirmed membrane targeting with structural changes to cell shape, ruptured cell membranes and leakage of cellular content with evidence of blebbing. The stability of CPF-P2 in physiological environments was evaluated. CPF-P2 retained activity in 5% foetal bovine serum (FBS) but lost activity in 25% FBS. Pre-incubation with trypsin also resulted in complete loss of CPF-P2 activity. In conclusion, CPF-P2 was identified as bactericidal, selective for planktonic E. coli with a killing time of 120 minutes at its MBC. The bactericidal activity is mediated by membrane permeabilisation leading to associated changes in cell morphology. Its activity against biofilms was limited and was compromised in physiological environments. Nonetheless, this study identified CPF-P2, as a promising template for the further development of analogues for topical infections.en© 2023 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria.UCTDSustainable Development Goals (SDGs)Antimicrobial resistanceAntimicrobial peptidesBiofilmsEscherichia coliMembrane permeabilisationDissertationu18303880https://doi.org/10.25403/UPresearchdata.27961263