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
Graphical abstract
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].
Section snippets
Molecular dynamics simulation
Molecular dynamics (MD) simulations were performed to systematically investigate peptide interaction with lipid bilayer. All simulations were carried out with the GROMACS ver. 4.5.4 software package [17]. A solvated lipid bilayer model was set up with a composition of mixed lipids POPG:POPE (3:1) to mimic the zwitterionic outer membrane of Gram-positive bacteria [18,19], or the pure POPC lipids to represent the neutrally or weakly charged cell membrane of mammals [20]. The force field
Conformational analysis of chikungunya virus fusion domain
The fusion domain of chikungunya virus is an 18-mer polypeptide segment (VT18, 84VYPFMWGGAYCFCDAENT101) located in the structural glycoprotein E1 of viral envelope [36]. In mature virions the E1 forms a heterodimer with E2, which buries the fusion domain [37]. Under the acidic endosomal environment, the E1-E2 heterodimer dissociates and the E1 undergoes a conformational change to β-sheet homotrimer following insertion of the fusion domain into plasma membrane [38,39]. Previous studies have
Conclusions
The membrane permeabilization of Chikungunya virus fusion peptide VT18 and its AMP variants was investigated systematically at molecular level. The permeabilization process can be divided into a fast motion phase in water and a slow diffusion phase in lipid. Difference in peptide permeability between bacterial and mammalian membranes is primarily determined by the first phase, in which the electrostatic property plays a critical role in the membrane–peptide interaction. MD simulations and
Conflict of interest
The authors declare that they have no conflict of interest.
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