Breaking or following the membrane-targeting mechanism: Exploring the antibacterial mechanism of host defense peptide mimicking poly(2-oxazoline)s

doi:10.1016/j.jmst.2020.06.006

Abstract

Abstract:

Peptides exert important biological functions but their application is hindered by their susceptibility to proteolysis and poor stability in vivo. Thus, functional peptide mimics have drawn a great deal of attention to address this challenge. Poly(2-oxazoline)s, a class of biocompatible and proteolysis-resistant polymer, can work as host defense peptide mimics without following the general membrane-targeting mechanism as shown in our previous work. This observation encouraged us to figure out if poly(2-oxazoline)s are special and break the general membrane-targeting mechanism of host defense peptides and their mimics. In this study, we aimed at the connection between structure and antibacterial mechanism of poly(2-oxazoline)s. A new γ-aminobutyric acid (GABA)-pendent poly(2-oxazoline) was synthesized and investigated to compare with glycine-pendent poly(2-oxazoline) in our previous study, with the former polymer has two extra CH₂ groups in the sidechain to increase the hydrophobicity and amphiphilicity. Membrane depolarization assay suggested that incorporating two more CH₂ groups into the sidechain of poly(2-oxazoline) resulted in a mechanism switch from DNA-targeting to membrane-targeting, which was supported by the slow time-kill kinetics and slightly distorted and sunken membrane morphology. Besides, GABA-pendent poly(2-oxazoline) showed potent activity against methicillin-resistant S. aureus and low hemolysis on human red blood cells. Moreover, repeated use of the antimicrobial poly(2-oxazoline) did not stimulate bacteria to obtain resistance, which was an obvious advantage of membrane-targeting antimicrobial agents.

Key words: Poly(2-oxazoline), Host defense peptide, Antibacterial mechanism, Membrane-targeting, Antimicrobial resistance

Chengzhi Dai, Min Zhou, Weinan Jiang, Ximian Xiao, Jingcheng Zou, Yuxin Qian, Zihao Cong, Zhemin Ji, Longqiang Liu, Jiayang Xie, Zhongqian Qiao, Runhui Liu. Breaking or following the membrane-targeting mechanism: Exploring the antibacterial mechanism of host defense peptide mimicking poly(2-oxazoline)s[J]. J. Mater. Sci. Technol., 2020, 59: 220-226.

Figures/Tables 9

Fig. 1. Amphiphilicity induced antibacterial mechanism switch from DNA-targeting to membrane-targeting: (a) similar backbone structure allowed poly(2-oxazoline) to exert as functional mimic of peptide. (b) Gly-POX20 quickly entered into the cytoplasm, bound to DNA to generate ROS that damaged the membrane and killed the bacteria. (c) GABA-POX20 mildly interacted with the bacterial membrane, following a pore-forming and slow cytoplasm-releasing mechanism.

Fig. 2. (a) Synthesis of ProNHBocOx; (b) Synthesis of GABA-POX20; (c) Characterization of GABA-POX20. The average degree of polymerization of GABA-POX20 was calculated by GPC analysis of N-Boc-GABA-POX20 and was calibrated by 1H NMR characterization of fully deprotected GABA-POX20. GPC analysis was conducted using N, N-dimethylformamide (DMF) as the mobile phase at a flow rate of 1 mL/min. D, dispersity index; Mn, relative number-average molecular weight; DP, degree of polymerization. 1H NMR was collected using D2O as the solvent.

Fig. 3. Cytoplasmic membrane depolarization of S. aureus USA 300 induced by GABA-POX20 at 2 × MBC and Gly-POX20 at 2 × MBC.

Fig. 4. In vitro killing kinetics of GABA-POX20 against MRSA at 2 × MBC and 5 × MBC respectively. Data of Gly-POX20 was reported previously [17] and was included here for comparison with GABA-POX20 to analyze their difference in antibacterial mechanism. S. aureus USA 300 was used in this study.

Fig. 5. SEM characterization of untreated S. aureus, S. aureus treated with Gly-POX20 for 5 min at 1 × MBC, and S. aureus treated with GABA-POX20 for 20 min at 1 × MBC respectively. S. aureus USA 300 was used in this study.

Table 1 MIC values (μg/mL) of GABA-POX20 and Gly-POX20 against gram-positive bacteria.

Bacterial strains	GABA-POX₂₀	Gly-POX₂₀^a	Magainin II	NorHCl
S. aureus ATCC 6538	12.5	12.5	100	0.39
S. aureus USA 300	6.25	12.5	>200	6.25
S. aureus USA 300 LAC	6.25	12.5	>200	12.5
S. aureus USA 400	6.25	12.5	>200	0.39
S. aureus Mu 50	6.25	12.5	>200	12.5
S. aureus Newman	12.5	12.5	>200	0.39
S. epidermidis ATCC 49134	6.25	12.5	>200	12.5

Table 2 MBC values (μg/mL) of GABA-POX20 and Gly-POX20 against gram-positive bacteria.

Bacterial strains	GABA-POX₂₀	Gly-POX₂₀^a	Magainin II	NorHCl
S. aureus ATCC 6538	25	25	>200	0.39
S. aureus USA 300	12.5	12.5	>200	12.5
S. aureus USA 300 LAC	12.5	12.5	>200	>12.5
S. aureus USA 400	12.5	12.5	>200	0.39
S. aureus Mu 50	12.5	25	>200	>12.5
S. aureus Newman	12.5	12.5	>200	0.39
S. epidermidis ATCC 49134	12.5	12.5	>200	>12.5

Fig. 6. Selectivity index of GABA-POX20 towards human red blood cells, calculated from HC50/MIC. The zigzag line on the top of histogram means the value of selectivity index exceeds the up-limit of the hemolysis assay.

Fig. 7. Antimicrobial resistance test on GABA-POX20 and methicillin against S. aureus ATCC 6538.

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