Fine-Tuning of Alkaline Residues on the Hydrophilic Face Provides a Non-toxic Cationic α-Helical Antimicrobial Peptide Against Antibiotic-Resistant ESKAPE Pathogens.

Xudong Luo,Huanhuan Gao,Zhiwen Zhao,Li Ding,Jue Wang,Weihua Zhong,Wen Zhu,Shan Li,Zongyun Chen,Zhan Shu,Ming Sang,Pengcheng Yi,Xiangdong Ye

doi:10.3389/fmicb.2021.684591

Xudong Luo, Huanhuan Gao + Show 11 more

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https://doi.org/10.3389/fmicb.2021.684591

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Abstract

Antibiotic-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become a serious threat to public health worldwide. Cationic α-helical antimicrobial peptides (CαAMPs) have attracted much attention as promising solutions in post-antibiotic era. However, strong hemolytic activity and in vivo inefficacy have hindered their pharmaceutical development. Here, we attempt to address these obstacles by investigating BmKn2 and BmKn2-7, two scorpion-derived CαAMPs with the same hydrophobic face and a distinct hydrophilic face. Through structural comparison, mutant design and functional analyses, we found that while keeping the hydrophobic face unchanged, increasing the number of alkaline residues (i.e., Lys + Arg residues) on the hydrophilic face of BmKn2 reduces the hemolytic activity and broadens the antimicrobial spectrum. Strikingly, when keeping the total number of alkaline residues constant, increasing the number of Lys residues on the hydrophilic face of BmKn2-7 significantly reduces the hemolytic activity but does not influence the antimicrobial activity. BmKn2-7K, a mutant of BmKn2-7 in which all of the Arg residues on the hydrophilic face were replaced with Lys, showed the lowest hemolytic activity and potent antimicrobial activity against antibiotic-resistant ESKAPE pathogens. Moreover, in vivo experiments indicate that BmKn2-7K displays potent antimicrobial efficacy against both the penicillin-resistant S. aureus and the carbapenem- and multidrug-resistant A. baumannii, and is non-toxic at the antimicrobial dosages. Taken together, our work highlights the significant functional disparity of Lys vs Arg in the scorpion-derived antimicrobial peptide BmKn2-7, and provides a promising lead molecule for drug development against ESKAPE pathogens.

Highlights

Bacterial resistance to traditional antibiotics is a serious threat to human health
By investigating the two scorpion-derived peptides BmKn2 and BmKn2-7, we found that: i. keeping the hydrophobic face of BmKn2 unchanged, and increasing the number of alkaline residues (Lys or Arg) on the hydrophilic face reduces the hemolytic activity of the peptide and broadens its antimicrobial activity; ii. replacing Arg with Lys on the hydrophilic face significantly reduces the hemolytic activity of BmKn2-7 without influencing its antimicrobial activity; and iii: BmKn2-7K, in which all Arg residues were replaced with Lys on the hydrophilic face of BmKn2-7, displays the lowest hemolytic activity
This leads to a negative charge on the bacterial surface that can attract Cationic α-helical antimicrobial peptides (CαAMPs) through electrostatic interactions (Fjell et al, 2011)

Summary

Introduction

Bacterial resistance to traditional antibiotics is a serious threat to human health. The majority of antibiotic-resistant infections are caused by the ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) (Pendleton et al, 2013). CαAMPs can fold into amphiphilic α-helices upon insertion into negatively charged bacterial plasma membranes, where they form various kinds of lipophilic pores that induce membrane disruption, cellular metabolite leakage, and eventually bacterial death (Fjell et al, 2011; Mahlapuu et al, 2016; Ciumac et al, 2019). From this mode of action, CαAMPs kill bacteria rapidly and have a much lower tendency to produce bacterial resistance, which makes CαAMPs attractive alternatives to conventional antibiotics

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