Antibacterial activity and mode of action of the antimicrobial peptide CPF-MW1 against Escherichia coli ATCC 700928

Abstract

Rapidly emerging antimicrobial resistance and subsequent multi-drug resistance are major global health threats, with the available antimicrobials for treatment being limited. The development of new antimicrobials against Gram-negative bacterial infections is especially challenging due to the structural complexity of the bacterial cell wall. Antimicrobial peptides (AMPs) have been identified as important antimicrobial leads for drug development due to their broad-spectrum antimicrobial activity, membrane lytic activity, rapid killing, and the slow rate of resistance emergence. The AMP, CPF-MW1, was derived from the skin secretions of Xenopus muelleri, a West African clawed frog species, and was identified as a promising AMP worthy of further development. The aim of the present study was to further evaluate the antibacterial activity and mode of action of CPF-MW1 against Gram-negative bacteria using Escherichia coli ATCC 700928, a uropathogenic clinical isolate, as a model organism. CPF-MW1 showed activity against Gram-negative (E. coli ATCC 700928) bacteria and was identified to be bactericidal with a minimum inhibitory concentration equal to the minimum bactericidal concentration (MBC) of 5.56 μM. The anti-biofilm activity of the peptide was limited to biofilm prevention with a minimum biofilm prevention concentration of 222 μM, a concentration 40-fold higher than the MBC. Both biofilm inhibition and biofilm prevention were only observed at a concentration 161-fold greater than the MBC (889 μM). Even at the highest concentration evaluated, no significant cytotoxicity was observed for CPF-MW1 against both human erythrocytes (10 ± 4%) and HaCaT cells (25 ± 5%) at 88.9 μM, a concentration 16-fold higher than the MBC. Mode of action studies revealed that CPF-MW1 effectively eradicates planktonic E.coli within 90 minutes, mediated by cytoplasmic membrane permeabilisation. Scanning electron microscopy showed that CPF-MW1 caused membrane blebbing and damage, resulting in cell death. For further application in physiological environments, the stability of the peptide in a proteolytic environment and in fetal calf serum (FCS) was evaluated. At the MBC, CPF-MW1 was degraded by trypsin, and some activity was lost in serum. At 2x MBC, 70 ± 5% activity was retained following pre-exposure to 116.5 μg/mL trypsin. At 2x MBC, 75 ± 5% activity was retained following pre-exposure to 25% FCS. Although preincubation with trypsin and FCS caused some loss in activity at the MBC, significant levels of activity were retained at 2x MBC. Novel findings of this study are that CPF-MW1 is bactericidal, membrane active, and fast killing. Furthermore, it exhibits minimal cytotoxicity and retains some activity in physiological environments. The peptide is active against Gram-negative bacterial pathogens, possessing antimicrobial activity against E. coli ATCC 700928. Although CPF-MW1 possesses antibacterial activity against planktonic cells, further developments are needed to improve its anti-biofilm activity.