Antimicrobial peptides


Antimicrobial peptides, also called host defence peptides are part of the innate immune response found among all classes of life. Fundamental differences exist between prokaryotic and eukaryotic cells that may represent targets for antimicrobial peptides. These peptides are potent, broad spectrum antimicrobials which demonstrate potential as novel therapeutic agents. Antimicrobial peptides have been demonstrated to kill Gram negative and Gram positive bacteria, enveloped viruses, fungi and even transformed or cancerous cells. Unlike the majority of conventional antibiotics it appears that antimicrobial peptides frequently destabilize biological membranes, can form transmembrane channels, and may also have the ability to enhance immunity by functioning as immunomodulators.

Structure

Antimicrobial peptides are a unique and diverse group of molecules, which are divided into subgroups on the basis of their amino acid composition and structure. Antimicrobial peptides are generally between 12 and 50 amino acids. These peptides include two or more positively charged residues provided by arginine, lysine or, in acidic environments, histidine, and a large proportion α-helical, ii) β-stranded due to the presence of 2 or more disulfide bonds, iii) β-hairpin or loop due to the presence of a single disulfide bond and/or cyclization of the peptide chain, and iv) extended. Many of these peptides are unstructured in free solution, and fold into their final configuration upon partitioning into biological membranes. The peptides contain hydrophilic amino acid residues aligned along one side and hydrophobic amino acid residues aligned along the opposite side of a helical molecule. This amphipathicity of the antimicrobial peptides allows them to partition into the membrane lipid bilayer. The ability to associate with membranes is a definitive feature of antimicrobial peptides, although membrane permeabilization is not necessary. These peptides have a variety of antimicrobial activities ranging from membrane permeabilization to action on a range of cytoplasmic targets.

Activities

The modes of action by which antimicrobial peptides kill microbes are varied, and may differ for different bacterial species. Some antimicrobial peptides kill both bacteria and fungi, e.g., psoriasin kills E. coli and several filamentous fungi. The cytoplasmic membrane is a frequent target, but peptides may also interfere with DNA and protein synthesis, protein folding, and cell wall synthesis. The initial contact between the peptide and the target organism is electrostatic, as most bacterial surfaces are anionic, or hydrophobic, such as in the antimicrobial peptide Piscidin. Their amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Alternately, they may penetrate into the cell to bind intracellular molecules which are crucial to cell living. Intracellular binding models includes inhibition of cell wall synthesis, alteration of the cytoplasmic membrane, activation of autolysin, inhibition of DNA, RNA, and protein synthesis, and inhibition of certain enzymes. In many cases, the exact mechanism of killing is not known. One emerging technique for the study of such mechanisms is dual polarisation interferometry. In contrast to many conventional antibiotics these peptides appear to be bactericidal instead of bacteriostatic. In general the antimicrobial activity of these peptides is determined by measuring the minimal inhibitory concentration, which is the lowest concentration of drug that inhibits bacterial growth.
AMPs can possess multiple activities including anti-gram-positive bacterial, anti-gram-negative bacterial, anti-fungal, anti-viral, anti-parasitic, and anti cancer activities. A big AMP functional analysis indicates that among all AMP activities, amphipathicity and charge, two major properties of AMPs, best distinguish between AMPs with and without anti-gram-negative bacterial activities. This implies that being AMPs with anti-gram-negative bacterial activities may prefer or even require strong amphipathicity and net positive charge.

Immunomodulation

In addition to killing bacteria directly they have been demonstrated to have a number of immunomodulatory functions that may be involved in the clearance of infection, including the ability to alter host gene expression, act as chemokines and/or induce chemokine production, inhibiting lipopolysaccharide induced pro-inflammatory cytokine production, promoting wound healing, and modulating the responses of dendritic cells and cells of the adaptive immune response. Animal models indicate that host defense peptides are crucial for both prevention and clearance of infection. It appears as though many peptides initially isolated as and termed "antimicrobial peptides" have been shown to have more significant alternative functions in vivo. Dusquetide for example is an immunomodulator that acts through p62, a protein involved in toll like receptor based signalling of infection. The peptide is being examined in a Phase III clinical trial by Soligenix to ascertain if it can assist in repair of radiation-induced damage to oral mucosa arising during cancer radiotherapy of the head and neck.

Mechanisms of action

Antimicrobial peptides generally have a net positive charge, allowing them to interact with the negatively charged molecules exposed on bacteria and cancer cell surfaces, such as phospholipid phosphatidylserine, O-glycosylated mucins, sialylated gangliosides, and heparin sulfates. The mechanism of action of these peptides varies widely but can be simplified into two categories: membranolytic and non-membranolytic antimicrobial peptides. The disruption of membranes by membranolytic antimicrobial peptides can be described by four models:
  • Barrel-stave model: The barrel-stave model proposes that AMPs interact with the lipid bilayer of the microbial cell membrane to form transmembrane channels or "barrel staves". These channels are thought to disrupt the membrane's integrity, leading to the death of the microbe.
  • Carpet model: The carpet model proposes that AMPs adsorb onto the lipid bilayer of the microbial cell membrane, forming a dense layer that causes the membrane to become permeabilized. This model suggests that the AMP acts as a "carpet" that covers the surface of the cell, preventing the microbe from functioning properly.
  • Toroidal model: The toroidal model proposes that AMPs interact with the lipid bilayer of the microbial cell membrane to form toroidal structures, which are thought to pinch off sections of the membrane and lead to the formation of vesicles. This process is thought to disrupt the membrane's integrity and cause the death of the microbe.
  • Disordered toroidal-pore model: According to this model, the disordered AMPs wrap around the lipid bilayer and create a pore, which disrupts the membrane's integrity and leads to the death of the microbe. Unlike the toroidal model, which suggests that the AMP creates a stable toroidal structure, the disordered toroidal-pore model suggests that the AMP is flexible and does not form a stable toroidal structure. The peptide-lipid pore complex becomes intrinsically disordered, with the orientation of the peptide not well defined.
Several methods have been used to determine the mechanisms of antimicrobial peptide activity. In particular, solid-state NMR studies have provided an atomic-level resolution explanation of membrane disruption by antimicrobial peptides. In more recent years, X-ray crystallography has been used to delineate in atomic detail how the family of plant defensins rupture membranes by identifying key phospholipids in the cell membranes of the pathogen. Human defensins have been thought to act through a similar mechanism, targeting cell membrane lipids as part of their function. In fact human beta-defensin 2 have now been shown to kill the pathogenic fungi Candida albicans through interactions with specific phospholipids. From the computational point of view, Molecular Dynamics simulations can provide detailed information about the structure and dynamics of the peptide-membrane interactions, including the orientation, conformation, and insertion of the peptide in the membrane, as well as specific peptide interactions with lipids, ions and solvent.
MethodsApplications
Microscopyto visualize the effects of antimicrobial peptides on microbial cells
Atomic emission spectroscopyto detect loss of intracellular potassium
Fluorescent dyesto measure ability of antimicrobial peptides to permeabilize membrane vesicles
Ion channel formationto assess the formation and stability of an antimicrobial-peptide-induced pore
Circular dichroism and orientated circular dichroismto measure the orientation and secondary structure of an antimicrobial peptide bound to a lipid bilayer
Dual polarization interferometryto measure the different mechanisms of antimicrobial peptides
Solid-state NMR spectroscopyto measure the secondary structure, orientation and penetration of antimicrobial peptides into lipid bilayers in the biologically relevant liquid-crystalline state
Neutron and X-ray diffractionto measure the diffraction patterns of peptide-induced pores within membranes in oriented multilayers or liquids
Molecular dynamics simulationsto study the molecular behaviour and search for specific peptide-lipid interactions
Mass spectrometryto measure the proteomic response of microorganisms to antimicrobial peptides

Therapeutic research and use

Antimicrobial peptides have been used as therapeutic agents; their use is generally limited to intravenous administration or topical applications due to their short half-lives. As of January 2018 the following antimicrobial peptides were in clinical use: