Membrane permeabilization design of antimicrobial peptides based on chikungunya virus fusion domain scaffold and its antibacterial activity against gram-positive Streptococcus pneumoniae in respiratory infection

Highlights

• The membrane permeabilization of Chikungunya fusion domain is investigated at molecular level.

• The permeabilization can be divided into a fast motion phase in water and a slow diffusion phase in lipid.

• Electrostatic property plays a critical role in the membrane–peptide interaction.

• Based on the fusion domain a number of linear and cyclic antimicrobial peptides are designed rationally.

Abstract

The structural dynamics of membrane permeabilization are investigated systematically and compared between viral fusion peptides (VFPs) and antimicrobial peptides (AMPs). It is revealed that the permeabilization process can be divided into two phases: a fast motion phase in water (first phase) and a slow diffusion phase in lipid (second phase). Difference in peptide permeability to neutrally or weakly charged mammalian membrane and to negatively charged bacterial membrane is primarily determined by the first phase, which is dominated by the direct electrostatic interaction between peptide and the hydrophilic surface of membranes. With the harvested knowledge we attempt to rationally design anti-Gram-positive AMPs based on the VFP scaffold of Chikungunya virus fusion domain, which is an 18-mer polypeptide segment (VT18, ⁸⁴VYPFMWGGAYCFCDAENT¹⁰¹) located in the structural glycoprotein E1 of viral envelope. Our simulations and previous NMR study suggest that the isolated VT18 peptide can be well structured into a double-stranded β-sheet conformation in water, but would become intrinsically disordering in lipid. Converting the negatively charged VT18 (charge = −2) to two positively charged peptides VT18-KKLV (VYPFMWGGAYCFCKAKLV-NH₂) (charge = +3) and VT18-CAKKLV (VYPFCWGGAYAFCKAKLV-NH₂) (charge = +3) by residue substitution and C-terminal amidation can largely promote peptide approaching to bacterial membrane surface, thus rendering the peptide with a substantially increased antibacterial activity against Gram-positive Streptococcus pneumoniae (MIC changes from >200 to 52–105 and 58–90 μg/ml, respectively). A further cyclization of linear peptide VT18-CAKKLV by adding a disulfide bond across its two strand arms, which results in a cyclic peptide cVT18-CAKKLV (

) (charge = +3), can effectively stabilize the peptide β-sheet conformation in lipid, thus improving its membrane compatibility in second phase and enhancing its antibacterial potency (MIC = 35–67 μg/ml). We also demonstrated that the designed AMPs have no or only a moderate cytotoxic effect and hemolytic activity on human normal cells.

Introduction

Antimicrobial peptides (AMPs) are an evolutionarily conserved component of the innate immune system that defends against invading against bacteria, viruses and fungi [1]. These biologically active oligopeptides or small proteins are usually positively charged and have both a hydrophobic and hydrophilic side that enables the molecule to be soluble in aqueous environments yet also enter lipid-rich membranes [2]. Usually these molecules are composed of 10–50 amino acids and arranged in different groups depending on composition, size and conformation; peptide charge and other characteristics such as size, sequence, structure, hydrophobicity and amphipathicity could be essential for antibacterial activity and mechanism of action [3].

Over the past two decades bacterial resistance to commonly used antibiotics has become a global health problem, causing increased infection cases and mortality rate, for which AMPs have been considered as one of the most potential alternatives to conventional antibiotics against antibiotic-resistant infections [4]. It was found that AMPs exhibited broad-spectrum activity against the drug-resistant strains of Gram-positive and Gram-negative bacteria, which also showed synergy with classical antibiotics, neutralize toxins and are active in animal models [5]. In addition, many AMPs also possess broad spectrum anticancer activities due to the similar anionic feature between the cell membranes of bacteria and tumors [6]. Systematic discovery of new and potent AMPs has been recognized as a new and promising approach to combat multidrug-resistant pathogens in clinical practice [7]. High-throughput techniques as peptide array and SPOT synthesis followed by rapid screening have been developed to identify AMPs [8]. These techniques, however, often miss potent candidates because they are nonspecific and sensitive to physical conditions. Although attempts have been made to overcome such experimental issues by use of bioinformatics algorithms as linguistic model [9] and chemometrics approaches as quantitative structure-activity relationship (QSAR) [10] to determine antibacterial active motifs, given the massive number of amino acid combinations the accuracy of the motifs is still limited by the coverage of all possible peptides.

Several years ago, Joanne and co-workers proposed a new strategy to derive AMPs from viral fusion peptides (VFPs) due to their similarity in membrane activity and membranolytic action [11]. This strategy has later been successfully used to generate antimicrobial agents from the fusion domains of influenza A virus [12,13] and dengue virus [14] via empirical approaches. However, there is a considerable difference between AMPs and VFPs since their targets are distinct in structure and property; the former encounters bacterial outer membranes that are negatively charged and relatively soft, whereas the latter interacts with mammalian cell membranes that are almost neutral and contain a lot of rigid components such as cholesterols [15]. In this respect, the amino acid composition and sequence pattern of AMPs and VFPs should not be consistent, and empirical derivation of AMPs from VFPs is just an accident and has only low success rate. In the current work, we described a rational method to design membrane-permeabilizing AMPs based on the fusion domain scaffold of chikungunya virus. The method employed structural modeling, conformational analysis and dynamics simulation to explore the viral fusion domain in both aquatic and lipidic environments and to investigate its interaction with lipid bilayer. The theoretical study was then successfully applied to guide rational molecular modification of the fusion domain, rendering it with efficient affinity and permeability to bacterial outer membrane. We also performed in vitro susceptibility test to determine the antibacterial activity of newly designed AMPs against two antibiotic-resistant strains and an antibiotic-sensitive strain of Streptococcus pneumoniae (S. pneumoniae), a Gram-positive bacterium responsible for most community-acquired pneumonia, meningitis and otitis media, causing about three million deaths annually in children in developing parts of the world [16].