CHAPTER 1 Introduction and Literature Review
1.6 Mechanism of action
A drug molecule needs to come in contact with the target cell to exhibit its activity. The first encounter of AMPs takes place with the target cell surface. Bacterial cell surface is negatively charged largely due to the presence of anionic phospholipids that include phosphatidylglycerol, phosphatidylserine, and cardiolipin [26]. In addition to that, Gram- positive bacteria consist of lipoteichoic acid (LTA) as a major cell wall constituent. LTA are polymers of alternating sugar and phosphate groups linked through phosphodiester linkage. The phosphate groups contribute to the negative charge on the surface. The charge, however, can be neutralized if an amino acid modifies the sugars as is the case in type-I LTA. Teichoic acids play an important role in cell adhesion, cell shape determination, virulence, and antimicrobial resistance [116, 117]. The outer membrane of Gram-negative bacteria contains lipopolysachharide (LPS) which is essentially a lipid A
molecule linked to complex carbohydrates that include anionic sugars [118]. The negative charge on LPS contributes to the negative charge on the Gram-negative bacteria.
Eukaryotic membrane, on the other hand, is composed of neutral or zwitterionic lipids such as phosphatidylcholine, phosphatidylethanolamine, cholesterol, and sphingomyelin.
Negatively charged lipids such as phosphatidylserine and phosphoinositides are present only on the inner leaflet on the membrane. [24]. Sterols like ergosterol and cholesterol stabilize the lipid bilayer and neutralize the AMPs [24, 119]. In addition to the difference in the net charge on the membrane, the difference in transmembrane potential between prokaryotes and eukaryotes is believed to contribute to the preferential binding of AMPs to the prokaryotic membrane. A mammalian cell membrane possesses a transmembrane potential of -90 to -110 mV whereas a bacterial cell in exponential growth phase has a transmembrane potential of about -130 to -150 mV [24]. Following membrane-interaction, AMPs can kill microbes in different ways. They can kill either by disrupting their membrane, by gaining access to intracellular targets and disturbing bacterial homeostasis, or both [120]. Membrane-disruption, as discussed above, happens to be the most common mechanism of cationic AMPs. In Gram-negative bacteria, cationic AMPs interact with negatively charged LPS present on the outer membrane. The peptide molecules then displace the divalent cations that support the membrane integrity by bridging the LPS moieties. Displacement of cations leads to destabilization of membrane, allowing AMPs to permeabilize the outer membrane by a process called self-promoted uptake [68]. Once the peptide molecules gain access to the inner membrane (cytoplasmic membrane), their modes of action branch off; membrane-disruptive peptides orient themselves and perturb the cytoplasmic membrane; non-membrane-disruptive peptides, on the other hand, translocate the membrane and target intracellular receptors [120].
1.6.1 Membrane-disruptive peptides
Three models that have been proposed to describe the membrane-disruption by AMPs are discussed below.
1.6.1.1 The barrel stave model
According to this model, the peptide molecules get aligned perpendicular to the membrane surface and form a barrel-like ring surrounding the aqueous pore. The hydrophobic region
of the peptide interacts with the acyl chains of the lipids whereas hydrophilic region lines the aqueous pore [121-123]. Binding to the membrane surface, most likely as monomers, happens to be the first step. Following binding, the peptides undergo a conformational transition. Polar head groups are forced aside, and the hydrophobic region of the peptide interacts with the membrane. As soon as the peptide attains a threshold concentration in membrane, monomeric peptides tend to self-associate and insert deeper into the hydrophobic core of the membrane. Hydrophilic residues are less exposed to the hydrophobic core due to aggregation of peptides. With increasing peptide concentration, the pore size increases allowing the peptide molecules access to inner leaflet [24]. Such a mechanism is proposed for the fungal AMP, alamethicin that forms a channel with 8 peptide helices upon interaction with lipid bilayer [124].
1.6.1.2 The toroidal pore model
According to the toroidal pore model, the amphipathic peptides take up an α-helical conformation upon membrane interaction. The peptide molecules position themselves parallel to the surface of the membrane, and the hydrophobic region of the peptide displaces the polar lipid head group inducing a positive curvature-strain on the membrane creating a pore in the hydrophobic region [125]. As the peptide concentration increases, the membrane weakens and gets destabilized. At threshold concentration, the peptide molecules position themselves perpendicular to the membrane with the hydrophilic amino acids no longer exposed to the hydrophobic lipid chain [24]. This leads to the formation of a dynamic and transient peptide-lipid complex called the toroidal pore (Figure 10) [126].
The unique features of the toroidal pore include limited lifespan, discrete size, ion- selectivity, and requirement of optimum peptide charge. Magainin 2, for example, forms toroidal pores of ~2-3 nm in diameter thereby excluding the larger molecules [127, 128].
Moreover, the high charge on peptide results in intermolecular repulsion between the peptide chains causing destabilization and short life of the pores [119].
Figure 1.8 A diagrammatic representation of the barrel stave (panel B) and the toroidal pore (panel C) models.
1.6.1.3 The carpet model
The carpet model represents a nonspecific mode of AMP action. According to this model, the peptide molecules accumulate and align parallel on the membrane surface interacting with the lipid head groups. As the peptide concentration on the membrane surface increases and reaches a threshold concentration, membrane fluidity is compromised.
Disruption of membrane potential ultimately leads to membrane disintegration [24, 120].
The carpet model is essentially a membrane disruption mechanism very similar to that of detergents [24]. Cecropin P1 is an example of AMP that disrupts the membrane though carpet mechanism [129].
Figure 1.9 A diagrammatic representation of the carpet model. Panel A: binding of the peptides to the membrane, panel B: membrane-disruption.
1.6.2 Non-membrane-disruptive peptides
In the majority of cases, membrane perturbation is the key to antimicrobial peptide action.
However, apart from interacting with the membrane, some AMPs translocate the membrane and target the intracellular processes such as protein and DNA synthesis [130].
PR-39, for example, inhibits protein synthesis. In addition to that, PR-39 induces degradation of some of the proteins essential for DNA replication [131]. Indolicidin, a potent AMP isolated from cytoplasmic granules of bovine neutrophils, is believed to translocate the membrane without disrupting it and targets intracellular receptors for bactericidal activity [132]. Insect AMPs like pyrrhocoricin, drosocin, and apidaecin cause cell death by inhibiting bacterial heat shock protein, DnaK [133]. Buforin II is a derivative of Buforin I, an AMP isolated from stomach tissue of Asian toad Bufo garagriozans. Buforin II is a potent antimicrobial agent against a broad range of organisms [134]. It takes up an amphipathic α-helical conformation in water:trifluoroethanol mixture as determined by NMR spectroscopy[135]. It, however, exerts antimicrobial activity without disrupting the membrane. The peptide is reported to bind to DNA and RNA after translocating the membrane and interrupt the cellular activities leading to cell death [132]. Microcin B17, a
bacteriocin isolated from E. coli exhibit activity by targeting DNA gyrase [136]. Mersacidin, a lantibiotic from Bacillus binds lipid II and inhibits peptidoglycan biosynthesis. [137].
1.7 Selectivity of antimicrobial peptides towards Gram-positive or Gram-