Effect of central PxxP motif in amphipathic alpha-helical peptides on antimicrobial activity and mode of action

Abstract

Amphipathic α-helical peptides (AHPs) have shown potential as a therapeutic approach against multi-drug-resistant bacterial infections due to their broad-spectrum antimicrobial activity by disrupting bacterial membranes. However, their nonspecific interactions with membranes often result in cytotoxicity toward mammalian cells. Previous studies have shown that a PxxP motif near the middle of cathelicidin-derived antimicrobial peptides contributes to potent and selective antibacterial activity. In this study, we compared KL18 with KL-PxxP to examine the effects of the central PxxP motif in AHPs on their structure, antibiotic activity, and mode of action. In a membrane-mimetic environment, we observed that KL18 had a much higher helical content compared to KL-PxxP. In aqueous buffer, KL18 adopted a highly ordered α-helical conformation, while KL-PxxP exhibited a disordered conformation. We found that KL-PxxP exhibited 4–16 times higher antibacterial activity than KL18 and significantly reduced the hemolytic activity. These findings suggest that the dynamic conformational behaviors caused by the central PxxP motif conferred the antibacterial selectivity of AHPs. Additionally, KL-PxxP showed strong binding to anionic liposomes and weak binding to zwitterionic liposomes, explaining its selectivity for bacteria over mammalian cells. Despite having a low ability to dissipate the bacterial membrane potential, KL-PxxP translocated efficiently across lipid membranes. Therefore, we propose that the central PxxP motif in AHPs provides dynamic conformational behavior in aqueous and membrane-mimetic environments, enhances binding to anionic membranes, and facilitates translocation across lipid bilayers, resulting in improved antibacterial potency and selectivity. Understanding the unique structural characteristics and functional roles of the PxxP motif in the antimicrobial mechanism of action holds great potential for advancing the development of novel peptide antibiotics.

Introduction

The proliferation of antibiotic-resistant microorganisms has led to global health issues, prompting significant efforts to develop novel antimicrobial agents to combat these infections (Haney et al. 2019; Molton et al. 2013; Zaman et al. 2017). Among the major structural types of antimicrobial peptides, amphipathic α-helical peptides (AHPs) have emerged as a promising class of antimicrobial agents due to their effective activity against a broad range of bacterial pathogens (Mant et al. 2019; Matos et al. 2023; Tossi et al. 2000; Zhang and Gallo 2016; Zhang et al. 2021). While the antimicrobial effectiveness of AHPs is influenced by its multifaceted mode of action beyond mere membrane permeability, comprehending the molecular-level interaction between AHPs and lipid bilayers holds significant importance for the development of antimicrobial drugs.

In summary, our study found that KL-PxxP has significantly enhanced antibacterial activity compared to KL18, while also displaying reduced hemolytic activity. KL-PxxP had a lower helical content than KL18 in a membrane-mimetic environment. We observed that KL-PxxP is distributed randomly while KL18 is self-associated in buffer. Importantly, KL-PxxP had a strong affinity for anionic liposomes and efficiently translocated across lipid bilayers despite its low ability to dissipate the bacterial membrane potential. Therefore, our findings suggest that the central PxxP motif in AHPs provides dynamic conformational behavior in both aqueous and membrane-mimetic environments, enhancing binding to anionic membranes and translocation across lipid bilayers, resulting in improved antibacterial potency and selectivity. This study sheds light on the structural and functional aspects of the central PxxP motif in AHPs, which holds promise as an approach to enhance antimicrobial efficacy while mitigating the cytotoxic effects against mammalian cells. Furthermore, we propose that this motif could be applied to other antimicrobial peptides to facilitate the development of peptides with intracellular targets and the creation of multifunctional antimicrobial peptides.