teixeira2012

Progress in Lipid Research 51 (2012) 149–177
Contents lists available at SciVerse ScienceDirect
Progress in Lipid Research
journal homepage: www.elsevier.com/locate/plipres
Review
Role of lipids in the interaction of antimicrobial peptides with membranes
Vitor Teixeira a,b,⇑, Maria J. Feio b, Margarida Bastos a
a
b
CIQ(UP), Departamento de Química e Bioquímica, FCUP, R. do Campo Alegre, Porto 4169-007, Portugal
REQUIMTE, Departamento de Química e Bioquímica, FCUP, R. do Campo Alegre, Porto 4169-007, Portugal
a r t i c l e
i n f o
Article history:
Available online 8 January 2012
Keywords:
Antimicrobial peptide
Immunity
Structure–activity relationship
Peptide design
Target activity
Phospholipids
Mechanism of action
Membrane permeabilization
Membrane topology
Intracellular targets
Cell death
Resistance
Therapeutic agents
a b s t r a c t
Antimicrobial peptides (AMPs) take part in the immune system by mounting a first line of defense against
pathogens. Recurrent structural and functional aspects are observed among peptides from different
sources, particularly the net cationicity and amphipathicity. However, the membrane seems to be the
key determinant of their action, either as the main target of the peptide action or by forming a barrier
that must be crossed by peptides to target core metabolic pathways. More importantly, the specificity
exhibited by antimicrobial peptides relies on the different lipid composition between pathogen and host
cells, likely contributing to their spectrum of activity.
Several mechanisms of action have been reported, which may involve membrane permeabilization
through the formation of pores, membrane thinning or micellization in a detergent-like way. AMPs
may also target intracellular components, such as DNA, enzymes and even organelles. More recently,
these peptides have been shown to produce membrane perturbation by formation of specific lipid–peptide domains, lateral phase segregation of zwitterionic from anionic phospholipids and even the formation of non-lamellar lipid phases. To countermeasure their activity, some pathogens were successful in
developing effective mechanisms of resistance to decrease their susceptibility to AMPs. The functional
and integral knowledge of such interactions and the clarification of the complex interplay between
molecular determinants of peptides, the pathogen versus host cells dichotomy and the specific microenvironment in which all these elements convene will contribute to an understanding of some elusive
aspects of their action and to rationally design novel therapeutic agents to overcome the current antibiotic resistance issue.
Ó 2012 Elsevier Ltd. All rights reserved.
Contents
1.
2.
3.
4.
5.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Antimicrobial peptide structural diversity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.
Classic small peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.1.
Amphipathic a-helical peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.2.
Amphipathic b-sheet peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1.3.
Other and specific residue-rich peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2.
Larger antimicrobial proteins and polypeptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Biological significance of membrane composition as a measure of affinity to antimicrobial peptides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Molecular determinants of antimicrobial peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1.
Sequence and Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2.
Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.
Amphipathicity and hydrophobic moment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4.
Hydrophobicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5.
Polar angle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Antimicrobial peptide’s mechanisms of action: an overall perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1.
Adsorption and binding to membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.2.
Threshold concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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⇑ Corresponding author at: REQUIMTE, Departamento de Química e Bioquímica, FCUP, R. do Campo Alegre, Porto 4169-007, Portugal. Tel.: +351 22 0402511; fax: +351 22
0402659.
E-mail address: [email protected] (V. Teixeira).
0163-7827/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plipres.2011.12.005
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5.3.
5.4.
6.
7.
8.
9.
Conformational transition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Peptide insertion and membrane permeability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.1.
Membranolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4.2.
Non-membranolytic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Intracellular targets of antimicrobial peptides: an alternative mechanism of action? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mechanisms of antimicrobial peptide resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1.
Membrane electrostatics and structural modifications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2.
Membrane electrical potential . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.
Sensor-transducer response systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4.
Proteases and peptidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.
Efflux-dependent resistance mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Development of antimicrobial peptides for clinical applications: a novel advance in therapeutics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1.
A promising future for antimicrobial peptides as reliable antibiotics? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Concluding remarks and future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
The integrity of any eukaryotic organism depends not only on
the proper expression of its genes but also on its ability to resist
the action from invading microorganisms. It demands a structured
and a functional immune system in constant interaction with the
dynamic surrounding environment which in turn determines the
ability of the host to prevent an infection.
In order to establish an infection, a pathogen must first overcome many surface barriers, such as physical and mechanical elements like skin, mucous membranes and the epithelia of the
respiratory system or the gastrointestinal and genitourinary tracts
[1]. In addition, there are also important chemical mediators of the
immune system that constitute an effective arsenal to overcome
the harmful action of microorganisms. Some of these chemicals
and molecules include gastric juices, salivary glycoproteins, lysozyme, antimicrobial peptides, the complement system, cytokines
and acute-phase proteins which possess antiviral, antifungal, antitumoral and immunomodulatory activities [1–3].
Antimicrobial peptides (AMPs) are a universal feature of the defense systems of virtually all forms of life, with representatives
found in organisms ranging from bacteria to plants, fish, amphibians, insects, mammals, and even viruses [1,4–15]. They take part in
an ancient, nonspecific innate immune system, which is the main
defense mechanism for the majority of living organisms during
the initial stages of an infection [1,16,17]. These peptides usually
display a broad range activity as they act on bacteria, fungi, metazoans and other parasites, viruses and even cancer cells [18,19].
The importance of these mechanisms to host defense may vary between different sites (skin, oral cavity, gastrointestinal tract, respiratory system) within a particular organism and even between
different organisms [1].
Antimicrobial peptides may be expressed constitutively or can
be inducibly expressed in response to exposure to foreign microorganisms. Indeed, they may be expressed systemically, as observed
for cecropins isolated from the hemolymph of bacteria-challenged
moths and flies of the Lepidoptera and Diptera orders. They can
even be localized in specific cell or tissue types in the body, most
susceptible to an infection from a particular set of pathogens. For
instance, histatins are the main representatives on major salivary
glands in humans with bactericidal and fungicidal activities that
contribute to the innate defense of the oral cavity and more recently as oral wound healing factors [20–22]. Plant defensins are
more abundant on the epidermal cell layer and leaf primordial of
the potato tuber, which is consistent with a role in first-line
defense of vulnerable tissues [23]. Such compartmentalization
has proved to be important according to the type and specificity
of the invading organism, which naturally implies not only
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cell-specific regulation of expression but also a polarity in their distribution on the organism.
Antimicrobial peptides have typically 12–50 amino acids, a
molecular mass less than 10 kDa, and possess 2–9 positively
charged lysine or arginine residues that confer their overall positive net charge at physiological pH. Only a small number of acidic
residues (aspartate and glutamate) are usually found in these peptides, presumably contributing to the increase of amphipathicity
when present on the polar face [7,10,14,24]. Moreover, these
immunity peptide mediators usually present up to 50% hydrophobic amino acids, contributing to the common amphipatic conformation that they tend to assume at the lipid membrane interface
when interacting with the target cell [1,11,24–27].
Although these peptides are mostly recognized by their antibacterial activity, many studies have pointed out an increasingly recognized function as modulators of the innate immune response
in higher organisms (Fig. 1) [1,2,28–30]. In fact, a number of properties that modulate the immune response have been attributed to
these host defense peptides in many organisms, particularly in
mammals. These include epithelial cell proliferation, enhanced
wound healing, angiogenesis and the stimulation of chemokine
production, regulation of the production of pro-inflammatory cytokines and direct chemotaxis of expression of many types of leukocytes [1] (Fig. 1). It has been known that many cationic peptides,
Fig. 1. Biological functions of antimicrobial peptides in immunity. Antimicrobial
peptides are mostly recognized by their antibacterial activity (mainly mediated by
membrane permeabilization and cell death) but many studies have provided
evidence of their immunomodulatory functions in infection, such as chemotaxis,
angiogenesis and regulation of the magnitude of the adaptive immune response by
modulating the Th cell polarization.
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
including human cathelicidin LL-37 [31] and CP26 [28], are able to
neutralize endotoxins (e.g. LPS), both in vitro (inhibiting LPS-induced TNF-a production in macrophages) and in vivo, by protecting animals against endotoxemia. Cationic host defence peptides
derived from a range of other sources, including the bovine cathelicidin BMAP-27 [32], indolicidin [33], insect and bee-derived
cecropin–melittin hybrid peptides [34], and other synthetic cationic peptides [35], have been shown to contribute to a significant
reduction of endotoxin-induced inflammatory responses. This
apparent convergence of action, though from different sources,
suggests that the anti-endotoxin activity exhibited by these peptides may be conserved across species.
It is clear that host defence peptides have the potential to modulate the innate immune response through a wide variety of mechanisms, but recent results suggest that these peptides may also
play a role in modulating and bridging the adaptive immune response elements. Some studies have demonstrated that different
defense effector molecules working in synergy with cationic defense peptides results in an increased clearance of invading pathogens [36]. In vitro studies with human defensins have
demonstrated that they can enhance cellular proliferation and
cytokine responses of CD4+ T cells through IFN-c and IL-10 induction, as well as modulation of the expression of co-stimulatory
molecules [37]. This provides evidence for peptide involvement
in humoral (Th2-dependent) responses, and suggests that host defense peptides may possess adjuvant-like properties [38]. Similarly, human a-defensins and murine b-defensins can stimulate
Th1-dependent cellular responses mainly represented by antigen-specific cytotoxic T lymphocytes, and enhance anti-tumoral
and anti-viral immunity [39]. Indeed, such antiviral action by antimicrobial peptides cecropin, melittin and indolicidin has already
been demonstrated for a few viruses, such as murine leukaemia
viruses, feline immunodeficiency viruses and HIV-1 virus [9,13].
Taken together, current data provides evidence that antimicrobial peptides play a vital role in immunity, either by direct antibacterial action or acting on both innate and adaptive branches of
immune responses. They are involved in signaling events influencing initiation, development, polarization, magnitude and amplification of the mechanisms that provide an efficient immunity to
the host. The focus on understanding the extent and mechanisms
of action of cationic host defense peptides has intensified in the
last decade, leading to the description of a plethora of novel biological determinants governing their action towards pathogens
(Fig. 1).
This review describes and integrates advances in understanding
the action of antimicrobial peptides during the last years and focuses on the role of these peptides in immunity and their potential
in therapeutics, with biophysical and biochemical perspectives.
Previous reviews should be consulted for topics that are not extensively described here and for more detailed coverage of areas only
briefly mentioned in this review [3,15,19,27,40–48].
2. Antimicrobial peptide structural diversity
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2.1.1. Amphipathic a-helical peptides
The largest group of the antimicrobial peptides known so far is
the linear cationic a-helical peptides where more than 300 different members have been described so far. The most important representatives are cecropins, magainins, cathelicidins and melittin
[9,50,51]. These linear peptides have been discovered from numerous sources, including the extracellular fluids of invertebrates, insects, nematodes, teleost fish, frogs and mammalian neutrophils
[24]. These peptides are typically 12–37 residues in length, and
may have a kink or a central hinge region, as observed for dermaseptins [52] and caerin 1.1 [53].
One of the most studied cationic a-helical peptides belongs to
the cecropin family. Cecropins A, B and D are close homologues
with 35–40 residues that were firstly isolated from the pupae of
the cecropia moth. A mammalian homologue, cecropin P1, was
also found in the upper part of the pig small intestine [54]. In
the presence of membrane-mimicking environments, Holak et al.
have demonstrated that cecropin A adopts an amphipathic helical
conformation [55]. Silvestro et al. [56] have further studied the
folding of cecropin A in HFP–D2O (HFP – hexafluoro-2-propanol)
and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) monoand multibilayers (MBLs) and it was shown that the folded structure of cecropin A is characterized by a pair of connected helical
segments and terminates in short random sequences. The N-terminal a-helix (5–21) has a continuous distribution of basic
residues and forms an amphipathic structure that is connected
by a flexible hinge region to the more hydrophobic C-terminal
helix (24–37) [57].
Another well-characterized group of a-helical membrane-active peptides are magainins, which comprise a family of immunogenic peptides that are expressed in the skin and intestine of frogs.
Magainins are 23-residue peptides that exhibit a broad-spectrum
antibacterial, antifungal, and tumoricidal activities. Magainin
adopts an a-helical secondary-structure upon binding to phospholipid membranes, as determined by several spectroscopic techniques, namely CD (circular dichroism) [58], Raman [59] and
solid-state NMR (nuclear magnetic resonance) [60].
Melittin is found in bee venom and exhibits potent broad-spectrum antibacterial activity, but its derivatives are usually highly
hemolytic. These 26-residue peptides, like cecropins, are amidated
and also appear to structure into two a-helical regions separated
by a non-alpha-helical segment at residues 11 and 12, but the polarity is reversed as the N-terminus is hydrophobic and the C-terminus is basic when compared to cecropins [61,62]. An early study
by Frey et al. [63] on the orientation of melittin in phospholipid
bilayers by polarized attenuated total reflection infrared spectroscopy (ATR-IR) has shown that the peptide folds into an amphipathic a-helix in phospholipid multibilayers of 1,2-dipalmitoylsn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and a 4:1 mixture of POPC and 1-palmitoyl-2-oleoyl-phosphatidylglycerol (POPG), as well as when
bound to supported planar lipid bilayer (SPBs) of POPC:POPG
(4:1). Cornut et al. have further demonstrated that melittin is
mainly a-helical and presents some disordered domains in DMPC
model membranes [64].
2.1. Classic small peptides
The promising applications of AMPs on the treatment of infections and immunomodulation have provided clear evidence of
the therapeutical potential of this class of peptides. One of the
most intriguing aspects of antimicrobial peptides is the limited sequence homology and the wide range of secondary structures, despite their similar general physical properties. The most prominent
structures found are amphipathic a-helices, amphipathic peptides
with two to four b-strands and loops and randomized structures
[1,11,24,27,48,49].
2.1.2. Amphipathic b-sheet peptides
The b-sheet antimicrobial peptides present a well defined number of b-strands, with relatively few or no helical domains, organized in the common amphipathic pattern. Most peptides are
constrained either by disulfide bonds, as in the case of the tachyplesins, protegrins and lactoferricin, or by cyclization of the peptide backbone, as in the case of gramicidin S, polymyxin B or the
tyrocidines.
The cysteine-containing b-sheet peptides represent a highly diverse group of molecules mainly represented by defensins. These
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peptides are frequently formed by several antiparallel b-strands,
and are stabilized by a series of up to six disulfide bonds. All defensins are cationic and contain 6–8 highly conserved cysteines that
pair in three or four disulfide bonds [65]. The structure of the
two main defensin subfamilies, a and b-defensins, has been solved
by two-dimensional NMR and X-ray crystallography and both consist of a triple-stranded b-sheet with a distinctive defensin fold.
Recent data have demonstrated the existence of a common motif that integrates all of the above classes of cysteine-stabilized
antimicrobial peptides [65]. This motif, termed the c-core, is composed of two antiparallel b-sheets, with basic residues concentrated along its axis.
2.1.3. Other and specific residue-rich peptides
There are many examples of antimicrobial peptides that do not
present a specific archetype or motif; instead, they are defined by
the predominant presence of a particular aminoacid that imposes
particular constraints to their structure [27]. This group of peptides
includes those with relatively high amounts of tryptophan (indolicidin) [66], proline and arginine (PR-39), and histidine residues
(histatins) [21]. Although some of these peptides are not so recurrent and consequently are less studied, available data shows that
the structures of the known peptides with enriched amino acid
composition tend to differ from prototypic a-helical or b-sheet
structures. Some studies have shown that tryptophan-rich indolicidin [67] seems to adopt a wedge-shaped structure in membranemimetic environments and that some proline–arginine rich peptides, such as PR-39, folds into polyproline helix type-II structures.
In particular, the later motifs are specifically bound by SH3 domains and this binding may be relevant for protein–protein interactions [68].
2.2. Larger antimicrobial proteins and polypeptides
Recently, polypeptides and proteins considerably larger than
classical short antimicrobial peptides have been shown to have
unambiguous antimicrobial activity. Interestingly, some of the
smaller peptides have been shown to result from enzymatic proteolytic cleavage from these larger precursors [69].
Peptidoglycan recognition proteins (PGRPs) are an example of
this class of proteins. They were first discovered in insects, and
comprise a family of variable sized proteins (20–120 kDa) that specifically bind peptidoglycan (the main component of cell wall of
bacteria, particularly on Gram-positive bacteria) [70]. In a first approach, insect PGRPs were characterized as pattern recognition
receptors (PRR) that instigate downstream immunomodulatory
signaling cascades after interaction with immune effector cells
through receptor-linked Toll or Imd pathways. Some homologues
have already been found in vertebrates, especially in mammals,
such as the human PGRP-1, -3, and -4 family and bovine oligosaccharide binding protein (bOBP49) with similar antimicrobial profiles, as described in studies performed with Bacillus (B.) subtilis
and Staphylococcus (S.) aureus strains [71].
Lactoferrin is another well known case, being an 80-kDa ironbinding glycoprotein member of the transferrin family that is
found in mammalian milk and other fluid secretions such as tears,
saliva and seminal fluid [72–74]. It is also found in the secondary
granules of polymorphonuclear leukocytes (neutrophils) where it
is released during inflammatory responses [72]. This glycoprotein
is considered to be a part of the innate immune system. Due to
its strategic position on the mucosal surface, lactoferrin represents
one of the first defense systems against microorganisms invading
the organism. Somehow lactoferrin may support the proliferation,
differentiation, and activation of immune system cells and
strengthen the immune response [72]. Moreover, lactoferrin affects
the growth and proliferation of a variety of infectious agents
including both Gram-positive and Gram-negative bacteria, viruses,
protozoa, and fungi both by iron deprivation [74] and by a
porin-dependent mechanism, as more recently reported for
Escherichia coli [75–77].
A number of reports suggest that peptides derived by acidic
hydrolysis of lactoferrin have also direct antimicrobial functions
[78], for example, lactoferricins (LFcins) that are cleaved from the
N-terminus of lactoferrin by pepsin under acidic conditions. It is
thus likely that such process occurs naturally in biologically-relevant contexts, such as in the stomach after food ingestion [73,79].
Bovine lactoferricin (LFcin B) and human lactoferricin (LFcin H)
are representative cationic peptides constituted by 50 amino acids
with one to two disulfide bonds, and some studies have shown that
they may present improved antimicrobial properties when compared to the parental protein [78–80]. The effect of LFcin B is initiated by rapid binding to the bacterial and fungal cells in a pHdependent manner. This binding is modulated by divalent cations
such as Mg2+ and Ca2+, suggesting the importance of electrostatic
interactions in the mechanism of action [78]. LFcin B also causes
rapid K+ release and H+ internalization by Candida albicans cells, reduced uptake of proline by E. coli and B. subtilis and reduced uptake
of glucose by Trichophyton rubrum [78]. Some human derivatives,
such as LFcin H (20–30), induce loss of membrane potential and
cell lysis [78], which is also observed for the majority of lactoferrin-derived peptides [69,80–83]. In order to elucidate the mechanism of action, recent studies have demonstrated that many
lactoferrin derivatives have a preferential interaction with negatively charged membranes [69,80,81,83,84] and suggest that they
perturb the lipid bilayer through membrane permeabilization, possibly by surface insertion followed by membrane thinning
[82,85,86]. Other mechanisms have also been described in the literature for other lactoferrin derivatives. Some human lactoferrin
derivatives have also been suggested to be capable of binding to
unmethylated nucleotides and, therefore, of restraining exacerbated pro-inflammatory events by regulating the activation of immune cells [73]. Moreover, some derivatives have been shown to
target organelles in eukaryotic cells, as demonstrated for the N-terminal peptide of human lactoferrin hLF (1–11) [87].
3. Biological significance of membrane composition as a
measure of affinity to antimicrobial peptides
The cell membrane is regarded as a defined bilayer of phospholipids that regulate the flux of metabolites between the external
environment and the intracellular content. The physicochemical
nature of membrane lipids is the crucial basis for the lipid assembly into structural and functional membranes. This physical organization allows the membrane to function as a permeability
barrier and limits the occurrence of chemical reactions for the purposes of biochemical energetic efficiency to a particular cellular
microenvironment, leading to chemical compartmentalization.
The conjugation of lipids with proteins in supramolecular complexes is known to be paramount for many biological processes,
namely cellular membrane biosynthesis, cell homeostasis and regulation of membrane fluidity in response to environmental
challenges.
In the case of pathogens, the membrane is the main target of
most antimicrobial peptides. Upon peptide interaction with the
membrane, it is known that several factors modulate the peptide’s
activity and the extent of its partition to the membrane. The most
prominent is the membrane electrical potential, which is dictated
by electrostatic interactions between the lipid headgroups and
the cationic peptide. The curvature strain of the membrane is
mainly dictated by the nature of the lipids. Unsaturated type II lipid phosphatidylethanolamine (PE) naturally promotes a negative
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higher negative electrical potential ( 130 to 150 mV) and a lack
of rigidifying lipids like cholesterol as compared to host cell membranes seem to be key molecular determinants of the selective
activity of AMPs towards these organisms [27].
As for other lipids, sphingomyelin (SM) and phosphatidylcholine (PC) are neutrally charged and they are mostly present in
eukaryotic cell membranes, as observed in mammalian erythrocytes (Table 1) [4,89]. Therefore, bilayers enriched in the zwitterionic phospholipids PC and SM are regarded as a reliable model
of the erythrocyte membrane, to assess the toxicity of AMPs for
host mammalian cells [10,11,13,91,95,97]. It should be noted that
at odds with bacteria, phosphatidylcholine is also found in fungi
(eukaryotic), in variable proportions (Table 1), which may in part
explain their lower susceptibility to AMPs as compared to some
bacteria [41,98].
Sterols such as cholesterol (mammalian) and ergosterol (fungi),
found in eukaryotic but rarely in prokaryotic membranes, are also
neutral and they are very important to regulate membrane fluidity,
the formation of specific lipid domains and the antimicrobial peptide’s activity (Table 1) [10,12,99]. The condensing and acyl chain
ordering effects of cholesterol and ergosterol on phospholipids in
their liquid disordered (ld) state is well established, and are assigned to the rigid ring structure of the sterol limiting trans ?
gauche isomerization of vicinal phospholipid acyl chains. Since
the volume of the membrane’s hydrocarbon region is approximately conserved, a decrease in surface area leads to an increase
in membrane thickness. As a result, less peptide is expected to bind
to the membrane and the membrane thinning effect induced by
AMPs is reduced, leading to a decrease of their potency. This aspect
is of major importance since it is believed that toxicity to host cells
is partially inhibited by the presence of these lipid components, as
demonstrated in many studies. For instance, Raghuraman et al.
have shown that the presence of cholesterol inhibits melittin-induced calcein leakage of DOPC lipid vesicles and the extent of inhibition appears to be dependent on the concentration of membrane
cholesterol [99]. A similar inhibition by cholesterol has been reported for melittin-induced leakage from PC vesicles [100].
It is interesting to note that PS is also present in the inner leaflet
of the membrane of mammalian cells, but does not impart a significant attraction to AMPs since it is not exposed. This may partly explain why AMPs are usually non-cytotoxic despite the presence of
this lipid. However, cancer cells contain a small amount of this
curvature strain in monolayers due to its inverted cone geometry,
in contrast to PC and PG lipids, that do not have that propensity,
due to their cylindrical molecular shape. In addition, the hydrophobic interactions between the hydrocarbon acyl chain and the
amphipathic peptide (mainly mediated by van der Waals and
hydrophobic interactions) can induce lipid packing frustration
(e.g. membrane thinning) and have an enormous impact on membrane structure, either by membrane disruption and permeabilization or even induction of non-lamellar phases to relieve the strain
stress and the acyl chain packing frustration. This aspect will be
described in detail later.
A crucial pre-requisite for the development of AMPs or any
other therapeutic agent is the selective toxicity, acting only on foreign microorganisms while retaining the structural and functional
integrity of host cells. Since they are active over a wide range of
pathogens, these peptides have taken advantage of biochemical
divergence and evolution inherent to cell membrane composition
to act preferentially on pathogens and be harmless to the host. This
difference may also account for some of the varying levels of effectiveness that antimicrobial peptides exhibit against different types
of cells. Membranes of prokaryotic and eukaryotic cells differ considerably in lipid composition and this aspect is particularly important for the AMPs action, since it is thought to be the basis of
specificity of antimicrobial peptides towards the target cell
[24,88,89]. This trait is also very important in the definition of
more realistic mimetic membranes as a means of deferring important information on the potency of antimicrobial peptides and the
possible toxicity for host cells [88,89].
In Gram-negative bacteria, PE is the most abundant lipid, as observed for E. coli (Table 1), [88–93]. This lipid is usually used as a
zwitterionic lipid on bacterial model membranes in many biophysical studies [94–96]. In contrast, hydroxylated phospholipids such
as phosphatidylglycerol (PG), cardiolipin (CL) and phosphatidylserine (PS) present a negative net charge at physiological pH and are
abundant on pathogens membrane (Table 1). These lipids are commonly used in model membranes of bacterial and fungal microorganisms (usually mixed with zwitterionic lipids in a defined
stoichiometry) [24,89,91]. As cell membranes composed predominantly of PG, CL, or PS tend to be highly electronegative, this trait
partly explains the specificity of cationic antimicrobial peptides towards these organisms [10,11,24,93]. Therefore, the higher proportion of anionic lipids at their membrane surface, a significantly
Table 1
Major components of membrane lipid composition of representative species of Gram-negative and Gram-positive bacteria [262,263] (and references therein) and mammalian
host cells (erythrocytes) [264]. The lipid content and composition in fungal membranes, as exemplified by the yeast Saccharomyces cerevisiae, is extremely complex and highly
dependent on the growth conditions [262] (and references therein).
Microorganism
Lipid (%)
Cardiolipin (CL) a.k.a. diphosphatidylglycerol
(DPG)
Phosphatidylglycerol (PG)
Lysylphosphatidylglycerol (LPG)
Phosphatidylethanolamine (PE)
Phosphatidylcholine (PC)
Sphingomyelin (SM)
Phosphatidylserine (PS)
Phosphatidic acid (PA)
Phosphatidylinositol (PI)
Sterol
Staphylococcus aureus
(Gram-positive
bacteria)
Escherichia coli
(Gram-negative
bacteria)
OM
CM
Total
lipid
6
12
3
5*
3
–
90
–
–
–
–
–
–
6
–
82
–
–
–
–
–
–
19
–
74
–
–
–
<1
–
–
57
38
–
–
–
–
–
–
–
Saccharomyces
cerevisiae
(Fungi)
**
**
U
Ergosterol
Erythrocyte
Outer
leaflet
Inner
leaflet
–
–
–
U
20
40
40
–
–
U
Cholesterol
–
U
40
20
10
30
–
U
Present in trace or undetermined amounts.
*
Lipid composition for whole cells in exponential growth phase. Cardiolipin is a minor component during this growth stage but accumulates towards the stationary phase
[265].
**
Major lipids of yeast cell membrane in variable proportions.
U
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V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
lipid (3–9% of the total membrane phospholipids) in the outer leaflet of the membrane, which presumably turn these cells more susceptible to AMPs. This observation is consistent with some studies
that demonstrate that AMPs are more toxic to cancer cells than benign cells [18,101,102].
Despite the higher content of PS in the outer leaflet of these
cells, this feature only partly explains their increased susceptibility. The membranes of many cancer cells also present a higher
expression of O-glycosylated mucines, which creates an additional
negative charge on the cancer cell’s surface [18]. Another plausible
explanation for the different susceptibilities of normal and cancer
cells to AMPs is membrane fluidity. The membrane fluidity of some
cancer cell lines is lower than that of normal cells, which may decrease their cytotoxicity. This hypothesis arises from observations
that the decrease of membrane fluidity usually shields the membrane from the action of these peptides, as described for the effect
of sterols on peptide activity. Indeed, it has been shown that some
breast and prostate cancer cell lines exhibited increased levels of
cholesterol-rich lipid rafts, which may decrease their susceptibility
to killing by AMPs [103]. In addition, tumorigenic cells exhibit a
higher cell surface area than normal cells due to a higher number
of microvilli, presumably increasing the available membrane surface area to bind a larger amount of peptide [18].
4. Molecular determinants of antimicrobial peptides
Structure–activity relationships (SAR) have become a useful tool
to study the molecular determinants governing the biological
activity of AMPs towards pathogens. Several comparative analyses
have demonstrated that antimicrobial peptides have common motifs that may be used to correlate specific parameters with their
biological activity. Several approaches have been used, such as sequence modification methods, minimalist approaches, synthetic
combinatorial libraries and template-assisted methods. All these
methods are based on the comparative analysis and systematic
alteration of peptide’s properties such as sequence, charge, hydrophobicity, amphipathicity, degree of structuring (a-helix, b-sheet
and random coil content), size and the balance between the hydrophobic and polar regions (the angles subtended by hydrophobic
and polar faces in the structured peptide) in order to obtain representative and valuable information on the relative contribution of
each property to the peptide’s biological activity. Therefore, a systematic comparison of common structural parameters of these
peptides will be important for a more comprehensive understanding on the way peptides act on killing pathogens and their cytotoxicity [42].
4.1. Sequence and Structure
The evidence collected so far has proven that most AMPs have
quite different peptide sequences and despite the absence of evident sequence homology, they tend to adopt similar conformational patterns upon interaction with the membrane [41,42].
One important feature are the capping interactions that are observed at the N-terminus of the peptide, independently of the following residues. Tossi et al. have reported a statistical analysis of
residue distribution in the N-terminal region of a-helical AMPs
from different sources, showing that glycine in position 1 is highly
conserved. This is mainly attributed to the ability to act as a good
capping residue and to provide resistance to proteolytic cleavage
by aminopeptidases [41,42]. Another important structural aspect
is peptide amidation, which is the most common post-translational
modification in a wide variety of peptides, such as cecropins, melittin, dermaseptins, PGLa, prophenin and polyphemusin. It is
known that amidation prevents the activity of carboxypeptidases
on peptides and provides one additional hydrogen bond that may
account to the energetics of the folding of a-helical peptides [104].
Lysine and arginine are the most representative amino acids and
they are usually concentrated in one stretch of the peptide sequence. This is consistent with the cationic nature of AMPs and accounts for the electrostatic interactions that these residues establish
with the negatively charged pathogen membrane, as reported before [41,48]. It is important to realize that a strong electrostatic
attraction promoted by these basic residues on the hydrophilic face
of the peptide is not per se a prerequisite for interaction, as it also occurs between AMPs and neutral membranes, as shown for cecropin–
melittin hybrid peptides [95,105,106] and other peptides [107,108].
Nevertheless, it seems to be important in the initial approach to the
membrane as demonstrated by the magnitude of the partition –
most AMPs have partition constants that are orders of magnitude
higher to negatively charged membranes when compared to zwitterionic ones [95,106,109]. In addition, aromatic residues (mainly
tryptophan) are considered to have a pivotal function on the partition, by anchoring the peptide to the membrane, particularly at the
headgroup level, as recently reviewed [42,73].
A well-known conserved pattern is their amphipathic conformation, as a result of the polarity and concentration of hydrophobic residues facing one side of the helix and polar residues residing
on the other face. This trend is paramount for antimicrobial peptides as the cationic polar domain is particularly important for
the initial interaction with the membrane surface whereas the
hydrophobic patch will drive the peptide insertion into the hydrocarbon chain membrane core, mostly mediated by hydrophobic
and van der Waals interactions.
Another important aspect is the degree of structuring, which is
best documented for a-helical and b-sheet AMPs, and its impact on
peptide’s activity. Many biophysical studies aiming at characterizing the thermodynamics of partition to the membrane have demonstrated that partitioning of polypeptide chains into membranes
is usually accompanied by peptide secondary structure formation
by the well known partitioning–folding coupling process [110–
113]. This trait will be further explored later in this review.
Increasing evidence is showing that structuring (helicity) is a
requirement for hemolytic but not antimicrobial activity [114].
Oren et al. [115] have studied diastereomers of melittin and investigated their structure and cytolytic activity towards bacterial and
mammalian cells. It was shown that melittin diastereomers lost
their a-helical structure, which abrogated their hemolytic activity
toward human erythrocytes while retaining their antibacterial
activity against Gram-positive and Gram-negative bacteria, as revealed by transmission electron microscopy studies. Similar results
were obtained with the diastereomers of the less cytolytic peptide
pardaxin [116].
Therefore, this apparent dissociation between peptide helical
content and antibacterial activity may indicate that a complex
interplay with other molecular determinants must be taken into
consideration to elucidate the influence of degree of structuring
and the biological activity exhibited by AMPs. Most likely the relative contribution of the positively charged and hydrophobic domains is very important, as suggested by Shai and co-workers
[116]. In this study, a series of diastereomeric peptides composed
of varying ratios of lysine and leucine residues were investigated.
The authors have reported that the highly hydrophobic diastereomers exhibited lytic activity against both negatively charged and
zwitterionic phospholipid vesicles and showed both antibacterial
and hemolytic activities. In marked contrast, the highly positively
charged diastereomers induced leakage only from vesicles of acidic
phospholipids and exhibited activity only against bacteria [116].
This particular feature was also highlighted by Dathe et al. for KLAL
peptide analogs and a double D-amino acid replacement peptide
set [117]. The adsorption and concentration of cationic KLAL pep-
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
tides at the membrane and the membrane-disturbing activity on
bilayers of high negative surface charge were found to be dominated by charge interactions, independently of any structural propensity. In contrast, the hydrophobic helix domain was particularly
decisive for binding and permeabilization of the membranes of low
negative surface charge, as it promotes the insertion of the amphipathic peptide into the hydrophobic core, thus disturbing the lipid
arrangement and causing local disruption [117].
155
are essential for antimicrobial activity. Their architectures range
from the a-helical peptides of some amphibians to the cyclic cystine knot structures observed in some plant proteins. Some peptides appear to use metal ions to form cationic salt bridges with
negatively charged components of microbial membranes, which
in turn favours the interaction with their target organisms. In
many cases the role in innate immunity and mechanisms underlying the antimicrobial action of these peptides are not yet fully
understood [48,119,120].
4.2. Charge
4.3. Amphipathicity and hydrophobic moment
Most cationic amphipatic peptides present a net positive charge
ranging from + 2 to + 9. Upon interaction with the membrane, they
are expected to cluster at the lipid-peptide interface, establishing
strong electrostatic interactions with the negatively charged
phospholipid membranes of pathogens. This represents actually
the main driving force for the folding of peptide at the lipid-peptide interface [24]. Indeed, bacterial cytoplasmic membranes
(CM) are rich in the acidic phospholipids PG and CL, which are
responsible for their overall negative charge (Table 1). The existence of other components, such as the LPS in Gram-negative and
teichoic or teichuronic acids in Gram-positive bacteria confers an
additional negative charge to the surfaces of these organisms
[24]. Similarly, phosphomannans or related constituents, chitin
chains and the presence of a layer of b-1,3-glucan also confer a
highly negative charge to fungal cell walls. Charge thus seems a
critical parameter in antimicrobial peptide’s mechanism of action
and the significant difference in membrane chemiosmotic potential and lipid composition between prokaryotes and eukaryotes
seems to play a pivotal role on their selective toxicity [15].
However, this association between charge and biological activity is not linear and there are examples of direct, indirect and even
inverse relationships between these variables [118]. Giangaspero
et al. have performed experiments using a sequence modification
approach, by varying several properties on a template peptide,
P19 [42]. Studies with several analogs, in which the mean hydrophobicity, amphipathicity and degree of structuring were maintained, have shown that a reduction of charge (ranging from + 6
to + 1) had considerably reduced the peptides’ potency against bacteria. However, it was shown that peptides with increasing cationicity above a threshold displayed increasing hemolytic activity. On
the other hand, an analogue with a formal net charge of + 8, presented an improved activity towards yeasts but a reduced activity
against bacteria, such as Bacillus megaterium (BM 11 strain) and S.
aureus (710 A) [42]. This may be related to a steric impediment for
helix formation due to close proximity and repulsive electrostatic
interactions between basic residues in the packed structure. Furthermore, this electrical repulsion is likely to decrease the lifetime
of the pore, which hinders the membranolytic effect of AMPs. Jiang
et al. [14], using the amphipathic a-helical antimicrobial peptide LV13 K as the framework to study the variation of some molecular
determinants on the peptide’s activity, have shown a similar pattern. A similar correlation was observed between the increase in
the number of positively charged residues and the antibacterial
activity, but also here a threshold was found, as a further increase
in cationicity resulted in an increased hemolytic activity rather
than improved antibacterial activity.
More recently, some antimicrobial properties have been described for anionic peptides, such as maximin H5 from amphibians,
small anionic peptides rich in glutamic and aspartic acids from
sheep, cattle and humans and dermcidin from humans. Anionic
antimicrobial peptides were first reported in the early 1980s and
they present a similar broad spectrum of action as their cationic
counterparts. Their structural characterization has demonstrated
that these peptides have a net charge ranging from 1 to 7,
and for some of these peptides, post-translational modifications
These parameters are crucial for the action of all AMPs since
most of them fold into amphipathic structures upon interaction
with target membranes. Amphipathicity is conventionally defined
as the relative proportion and distribution of hydrophobic and
hydrophilic residues or domains within a peptide. One quantitative
measure of amphipathicity is the hydrophobic moment, calculated
as the vectorial sum of individual amino acid hydrophobicity vectors, normalized to an ideal helix [49,121]. The amphipathic a-helix is the most common conformation, having a periodicity of three
to four residues per turn, which is optimal for interaction with biomembranes and folding of monomeric a-helices. In fact, it is well
accepted that the amphipathicity of AMPs is essential for their
mechanism of action because the positively charged polar face will
drive the initial electrostatic attraction to the negatively charged
components of the membrane, and then the nonpolar face of the
peptides will insert into the membrane through hydrophobic and
van der Waals interactions, causing increased permeability and
loss of the barrier function [15].
Nevertheless, there is still some controversy regarding the influence of amphipathicity on the biological activity of AMPs. Giangaspero et al. reported that the ability to fold into a helical
structure without an amphipathic conformation restricts the potency and range of activity of AMPs [42]. A recent paper by Jiang
et al. reports the absence of a linear correlation between amphipathicity and biological activity (both microbiological and hemolytic profiles) on a series of analogs of L-V13 K [14]. In a study
with magainin 2 analogs, Wieprecht et al. [122] have demonstrated
that peptides with slightly increased hydrophobic moment (while
retaining other structural parameters) were considerably more active in permeabilizing vesicles mainly composed of zwitterionic
lipids and had no effect on Large Unilamellar Vesicles (LUVs) entirely composed of anionic phospholipids. Nevertheless, both the
antibacterial and hemolytic activities of these analogs were enhanced. On this basis, it was proposed that the correlation between
permeabilization and binding was related to an increased membrane affinity or to an enhanced permeabilizing efficiency of the
membrane-bound peptide.
Therefore, the increase of the amphipathicity, quantitatively
measured by an increase of the hydrophobic moment, seems to
be important for both antibacterial (desirable) and hemolytic activities (undesirable effects). It is interesting to observe that amphipathicity appears to have a negligible effect on peptide–lipid
interactions on purely-negatively charged membranes, which
may have important consequences on the mechanism of action towards Gram-positive bacteria with no zwitterionic lipids, such as S.
aureus. On the other hand, for zwitterionic membranes, where
electrostatic interactions are not expected to play the main role,
the variation of amphipathicity may have important implications
on host cell cytotoxicity [27]. In fact, Kondejewski et al. [123] have
provided evidence for the correlation between amphipathicity and
hemolytic activity. This group has modulated this parameter using
the framework of b-sheet-containing tetradecameric cyclic peptide, GS14, showing that a decrease on amphipathicity resulted
in a decrease on the hemolytic profile.
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Interestingly, Stark et al. [124] have now reported a new category of non-amphipathic hydrophobic antimicrobial peptides to
be active against Staphylococcus epidermidis, Corynebacterium xerosis, and E. coli. It was shown that the activity was mostly related to
the threshold hydrophobicity required for insertion of the core
hydrophobic segment, which suggests that the biological activity
of some AMPs is better justified by this threshold value for amphipathicity than by its absolute value.
4.4. Hydrophobicity
Peptide hydrophobicity, defined as the percentage of hydrophobic residues within a peptide, is approximately 50% for most antimicrobial peptides [41]. It is an important parameter for peptide’s
biological activity as it determines the extent of partition of the
peptide into the membrane hydrophobic core [24,41]. The importance of hydrophobicity has been extensively studied, although
many authors have demonstrated that, in general, hydrophobicity
of a-helical amphipathic peptides appears to have a higher impact
on host cell toxicity than on antibacterial action. In fact, highly
hydrophobic peptides beyond a threshold are related to higher
hemolysis and a remarkable reduction on antimicrobial activity
and thus to a decrease in discrimination between host cytotoxicity
(hemolytic profile) and antimicrobial activity [8,14].
The relationship between peptide hydrophobicity and membrane permeabilization was examined in an interesting study by
Chen et al. [125] resorting to Ala to Leu substitutions on the nonpolar face of the template peptide L-V13 K. Notably, it was shown
that the increase in hydrophobicity led to an increased hemolytic
activity, as the triple Leu analog (A12L/A20L/A23L) was 62.5-fold
more active than L-V13 K against human erythrocytes. A very
strong correlation between dimerization and hemolysis was also
observed. Interestingly, a relationship between hydrophobicity
and antibacterial activity was more difficult to establish. Although
the antimicrobial activity of more hydrophobic versions of the
parental peptide, A12L and A20L, was improved, with a 2.0- and
3.6-fold increase compared to L-V13 K, the most hydrophobic analog (triple Leu version) had essentially no antimicrobial activity
against resistant strains of Pseudomonas (P.) aeruginosa.
In another study, Giangaspero et al. [42] have shown that the
absence of large aliphatic residues, responsible for the decrease
of mean hydrophobicity of the peptide P19(5/U), completely abolished the antimicrobial activity against Gram-negative and Grampositive bacteria. Chou et al. have studied the effect of some
parameters, including hydrophobicity, on a series of cationic AMPs
of 20 amino acids and it was shown that high hydrophobicity was
correlated with increased hemolytic activity, whilst antimicrobial
activity was found to be less dependent on this property [126].
Altogether, these studies suggest that optimal activity may be
achieved by moderately hydrophobic peptides, as increasing levels
of hydrophobicity beyond a threshold level may increase the propensity to dimerize or oligomerize that may cancel the desired
antimicrobial activity. From a thermodynamic view point, highly
hydrophobic peptides in an aqueous environment are energetically
more stable in aggregates than in monomeric form, as this allows
shielding of the hydrophobic moieties from the aqueous media.
The higher aqueous stability could prevent the partition to the
membrane as a higher energetic cost for the partition of peptide
aggregates is expected rather than on a monomeric form and
therefore the aggregates might display weaker membrane interactions. Moreover, it may become energetically more costly for the
peptide units in the aggregate to reorganize and assume the correct folding and orientation upon the partition to form peptide
supramolecular assemblies, such as pore-like structures.
An interesting interplay between hydrophobicity and amphipathicity was observed by Wieprecht et al. [122]. Although a
reduction of the hydrophobicity of magainin 2 amide peptide from
0.036 to 0.096 (L2R11A20 M2a) substantially decreased activity
at POPC-rich membranes, the activity could be completely restored, even at this low hydrophobicity, by enhancing the hydrophobic moment (a measure of amphipathicity) from 0.287 to
0.332 (I6R11R14W16 M2a). Conceptually, related observations in
systematic studies of the cyclic peptide antibiotic gramicidin S
indicate that a balance between peptide hydrophobicity and
amphipathicity for therapeutic effectiveness is a key factor for this
peptide to be used as a therapeutic agent [127,128].
Another important relationship was established between
hydrophobicity and charge. It was reported that charge increase
led to a striking increase of hemolytic effect of magainin derivatives and the low hemolytic activity was restored by decrease of
hydrophobicity of hydrophobic helix surface [129]. In this case,
an increase in net positive charge must enhance accumulation of
peptide at lipid head groups (phosphate groups) of erythrocyte
membrane whereas a decrease in hydrophobicity may decrease
the ability of peptide interaction with the membrane hydrophobic
core, thus reestablishing the lower hemolytic profile of the peptide.
4.5. Polar angle
Polar angle is a parameter that measures the relative proportion
of polar and nonpolar faces when peptides adopt an amphipathic
helix conformation. As a reference, an optimal amphipathic a-helix
in which one face is mainly constituted by hydrophobic residues
and the other one mostly composed of charged or polar residues
presents a polar angle of 180o [27]. Therefore, alterations on the
predominance or segregation of one type of residue are expected
to change this parameter. Many studies have suggested that a
smaller polar angle (and therefore a greater nonpolar domain) is
related with increased membrane permeabilization [130,131].
The polar angle has been shown to correlate with the overall stability of peptide-induced membrane pores since a study by Uematsu
and Matsuzaki [130] has shown a correlation of polar angle on
membrane permeabilization and pore formation. Two model peptides with polar angles of 100o and 180o were shown to fold into a
typical native a-helical amphipathic peptide and to form pores, but
overall data has provided evidence that peptides with smaller polar angles induced greater translocation and pore formation rates
[130]. Since a higher stability of pores formed by peptides with larger polar angles is correlated with the formation of larger charged
surfaces, and/or more peptide molecules per channel, the ability to
disrupt pathogen membranes is necessarily linked to polar angle of
AMPs [27].
In summary, these data clearly shows that a subtle interplay between all these parameters plays a pivotal role in defining the
range of activity of AMPs and that these features are not independent, since modification of one parameter often leads to compensatory adjustments in others, clearly demonstrating that these interrelationships are key determinants to unravel the mechanism of
action and the biological activity of AMPs.
5. Antimicrobial peptide’s mechanisms of action: an overall
perspective
It is widely accepted that the main mechanism by which antimicrobial peptides exert their action against pathogens is membrane permeabilization. As a consequence, the dissipation of the
electrochemical potential, lipid asymmetry and loss of important
metabolites and cellular components usually culminate in cell
shrinkage and ultimately cell death [27]. Currently, many studies
have provided consistent evidence of additional mechanisms, as
an increase on membrane permeability alone may not be sufficient
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
to cause cell death. Data supporting this well-accepted concept
arises from a distinction between membrane perturbation and cell
death as the latter may occur in the absence of significant perturbation in membrane structure. In fact, some authors have reported
the existence of mechanisms in which downstream, intracellular
sites are regarded as the main targets of antimicrobial peptides
[43,132] and receptor-mediated signaling activities of some peptides have also been reported [133]. Direct contact with or affinity
for heterotrimeric G proteins has been proposed as a basis for cationic peptide binding to mammalian cells and the process would
involve translocation of peptides across the plasma membrane.
This mechanism has been referred to when describing the exposure of mast cells to KLA analogue cationic peptides [134].
5.1. Adsorption and binding to membranes
AMPs are thought to be unstructured in solution and to fold into
their final amphipathic conformation upon interaction with biological membranes. These peptides must first be attracted to the pathogen cell wall and there is widespread acceptance that the initial
mechanism by which antimicrobial peptides target membranes is
mediated by electrostatic interaction [4,8,10,13,14,135] (Fig. 2A).
This interaction mainly occurs between the cationic charged AMPs
and the anionic components of the membrane [8,10–
13,132,136,137], a view supported by the conservation of basic
residues within many antimicrobial peptides isolated from different organisms.
The precise mechanism by which electrostatic attraction drives
peptide–membrane interaction has been extensively studied and
many authors emphasize the composition and architecture of
157
Gram-negative and Gram-positive cell wall as the basis for the different susceptibility of pathogens to AMPs. Apart from the anionic
phospholipids, the lipid A core of lipopolysaccharides (LPS) of
Gram-negative bacteria and the teichoic and teichuronic acids on
the surface of Gram-positive bacteria must also play an important
role in this context, showing that not only phospholipids are
important for peptide-mediated killing action [11,135]. In the case
of Gram-negative organisms, Hancock has proposed a mechanism
of peptide interaction with membranes termed self-promoted uptake [138], similar to the one described for cationic polymyxin B
[139]. According to this model, the initial action of the peptide involves a competitive displacement of LPS-associated divalent cations (Mg2+ and Ca2+), from which peptides destabilize this
supramolecular assembly and gain access to both outer and inner
membranes [138,139]. For Gram-positive organisms, however, a
different approach must be considered as they do not possess
LPS or an outer membrane, but instead, a thicker peptidoglycan
layer and the negatively charged teichoic and teichuronic acids,
which are successfully explored by peptides to interact with the
bacteria.
A general mechanism by which peptides may permeabilize or
rearrange microbial membranes is still under intense debate. However, some aspects are shared by most peptides. In brief, once close
to the microbial surface, peptides must cross capsular polysaccharides and other components of the cell wall before they can interact with the outer membrane in Gram-negative bacteria or the
cytoplasmic
membrane
in
Gram-positive
bacteria
[11,49,135,140]. This fact is often disregarded in most mechanistic
studies derived from model membranes. The initial interaction
with the cell wall usually does not involve any specific receptor,
Fig. 2. General mechanism of action of membrane-permeabilizing antimicrobial peptides. After interaction with the membrane through electrostatic interaction with anionic
components of the bacterial membrane (such as LPS, CL and PG) (A), peptides fold into a secondary structure (mostly a-helix), concentrate and usually lie parallel at the
interface. (B) Reaching the threshold concentration, the peptide adopts an orientation perpendicular to the axis defined by the water/membrane interface or remains on a
‘tilted state’, (C) inserts into the hydrophobic core of the membrane and (D) promote the formation of pore-like structures, which are responsible for the membrane
permeabilization. (Adapted with permission from [257]. Copyright 2007 National Academy of Sciences, U.S.A). note: In the original reference, this mechanism is proposed for
HIV-Tat, but it presents the general features of the partition and the mechanism of action of AMPs.
158
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
as enantiomers of lytic peptides, composed solely by D-amino
acids, maintain a biological activity comparable to that of their Lcounterparts. The parallelism generally found between antibiotic
activity and the ability to alter the permeability of lipid membranes (achiral molecules) rather than interaction with specific
receptors also supports this notion [14,41].
However, some studies have provided evidence of some exceptions to this generalization. The initial work of Sahl’s group and
others that followed showed that nisin actually acts by a receptor-mediated mechanism, which involves lipid II as the receptor.
This is crucial in the initial step of the action of nisin and similar
bacteriocins [141,142]. This interaction inhibits peptidoglycan synthesis and forms highly specific pores that result in the depletion of
intracellular components. As a result, the combination of highaffinity binding to Lipid II and permeabilization of the plasma
membrane potentiates the antibiotic activity and contributes to
the high potency exhibited by the peptide in nanomolar minimum
inhibitory concentration (MIC) values [141]. Another study has
demonstrated that sensitivity of Listeria monocytogenes to the bacteriocin mesentericin Y105 was dependent on ManR, a new sigma(54)-associated activator, and Ell(t)(Man), a new sigma(54)dependent PTS permease of the mannose family. Such PTS permeases are involved in sensitivity of different target strains to mesentericin Y105 and could be potential receptors for the subclass IIa
bacteriocins [143]. More recently, Destoumieux-Garzón et al. have
demonstrated that the iron-siderophore transporter FhuA is the
receptor for the antimicrobial peptide microcin J25 [144]. These
studies suggest that receptor-mediated interactions are important
for the mechanism of action of some antimicrobial peptides in targeting specific components of the pathogen cell wall, accounting,
at least partially, for their mechanism of action.
5.2. Threshold concentration
After initial membrane binding, peptides must locally concentrate in order to exert their antimicrobial activity, until they reach
a concentration that enables productive action [4,26]. Such feature
is commonly described as the threshold concentration concept [4].
At this concentration, peptides begin to rearrange and alter pathogen permeability via current accepted mechanisms, which will be
described later. However, a concentration threshold, characteristic
for each peptide/membrane composition, seems to be a necessary
requisite for their biological activity and it is independent of the
subsequent mechanism of action triggered by the peptide [26,106].
Conceptually, the threshold concentration is described as the
minimum peptide concentration [or the peptide-to-lipid ratio
(P:L) in an experimental perspective] necessary at the target surface to promote its biological effects [26]. At low peptide-to-lipid
ratios, the peptide tends to interact and concentrate at the level
of the lipid headgroup. At this stage, peptides fold and remain adsorbed parallel to the lipid bilayer (Fig. 2B), probably at the interface of head groups and fatty acyl chains and may already display a
significant content in secondary structure [4,105,145,146]. As the
P:L ratio increases, peptides begin to orient perpendicular (or in
a ’tilted state’ [147]) to the membrane, inserting and partitioning
into the hydrophobic core of the bilayer (Fig. 2C). Accordingly,
the Huang-Matzusaki-Shai’s two-state model establishes that the
threshold value (P:L)⁄ is the peptide concentration at which the energy levels of the S state (the surface adsorbed, parallel state observed at low P:L ratios) and the L state (the pore-forming state,
inserted and non-parallel to the surface, above the threshold concentration P:L⁄) are equal. In this perspective, the (P:L)⁄ threshold
value is a function of the adsorption binding energy, the elastic
constants of the bilayer, and the energy level of the pore state [4].
The most important aspects governing the partition are peptide
concentration, propensity to self-assemble or multimerize (both in
bulk solution or at the water/membrane interface), the membrane
composition, fluidity, and headgroup chemistry and size, transmembrane electric potential and pH, the latter influencing also
the charge state of peptides and ionic lipids [4,11,146,148–150].
Threshold concentration and peptide parallel-to-transmembrane
surface orientation are also considerably affected by trans-negative
membrane potential of many bacterial membranes, since it is the
connecting factor between peptides and membrane. At a peptideto-lipid ratio above the threshold value (P:L)⁄, the peptide may
promote alterations in conformation, both in-depth membrane
localization and association state, as well as indirect changes in bilayer topology, such as pore formation or disintegration (Fig. 2D).
Recently, Melo et al. have established that extremely high peptide concentrations are expected at the membrane surface and that
those can reach (or be close to) bilayer saturation [26,106]. The saturation (P:L) ratio estimates based on biophysical parameters (partition coefficients, Kp) reproduce observed MIC values, which may
imply that such high threshold concentrations are expected at the
membrane level in order to produce the biological effects in vivo.
Similar considerations had been previously addressed by Tossi
et al. [41].
5.3. Conformational transition
One of the most important processes occurring after membrane
binding is the rearrangement of the conformation of the peptide at
the lipid–water interface, a process to date better documented for
a-helical AMPs.
Although the precise mechanism by which conformational transition occurs is not fully understood, it is likely that fundamental
thermodynamics contributes to such conformational changes.
The thermodynamics of the a-helix coil transition of antimicrobial
peptides in a membrane environment and the formal implications
for the peptide–membrane equilibrium are well characterized and
have provided some consistent evidence that have been crucial for
the development of thermodynamic models and the clarification of
the steps involved in the peptide–lipid interaction mechanism
[110–112,151]. It is interesting to observe that this process has
evolved to take advantage of thermodynamic equilibrium states
by regulating the heights of barriers separating such states. Indeed,
such modulation proceeds in a very subtle way since it seems to be
adjusted through a refined balance between peptide properties,
their interactions with the aqueous media and the membrane
environment.
The thermodynamics of folding of amphipathic peptides in
water and in organic solvents has been extensively investigated
[152]. The interaction of peptides with the lipid membrane can
be divided into four thermodynamic steps-partitioning, folding,
insertion and association, as described by White and Wimley
[111]. After electrostatic adsorption and correct orientation
according to the plane of binding, the partitioning of the peptide
to the membrane and conformational transition are the key stages
of the process. Kaiser et al. firstly demonstrated that the partitioning of peptides to membranes can be described by a partitioning–
folding coupling process, as the formation of secondary structure
makes the partition of the structured peptide energetically less
costly [153]. Wimley and White proposed that the formation of
peptide bonds upon folding to a structured conformation would reduce the peptide’s partitioning Gibbs energy, contributing to the
partitioning pathway [154]. Based on a hydrophobicity scale, it
was estimated that the change in Gibbs energy for the process
amounts to DGhelix = 0.2 to 0.5 kcal/mol per residue, which is
ascribed essentially to hydrogen bond formation. Wieprecht et al.
have shown that helix formation was accompanied by a change
in Gibbs energy of DGhelix = 0.14 kcal/mol per residue. Calorimetric measurements and non-calorimetric estimates showed that a-
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
helix formation is driven by a negative enthalpy, DGhelix, and opposed by entropy, DShelix. Enthalpy values of DHhelix = 0.7 to
1.3 kcal/mol per residue were reported for helix formation in
water, as derived from different systems [110,155–158]. The enthalpy of helix formation was found to be largely independent of
the peptide sequence and was mainly attributed to the formation
of intra-molecular CO–NH hydrogen bonds [155,156]. Helix formation is opposed by entropy with typical entropy values ranging
from DShelix = 2.5 to 4.6 cal/mol.K per residue [110,158]. In
M2a and its diastereomers, the entropy change upon interaction
with the POPC/POPG (3:1) small unilamellar vesicles (SUVs) was
found to be DShelix 1.9 cal/mol.K per residue [110]. This is less
negative than reported for the coil–helix transition of peptides in
water. The smaller negative entropy change of membrane-induced
helix formation may reflect an already decreased conformational
freedom of the still unfolded but membrane associated peptide.
As a result, helix formation can be considered an important driving
force for partitioning–folding coupling arising from the Gibbs energy reduction associated with H-bonding.
Another contribution to the overall reduction in Gibbs energy
comes from the shielding of peptide bonds into intra-molecular
H-bonds in a-helices and b-sheets when the peptide is already inserted on the hydrophobic core of the membrane [159]. This feature was addressed by White and Wimley, that reported that the
main source of the peptide stability in a non-polar environment
arises from the high energetic cost of breaking H-bonds compared
to an aqueous environment – the authors have emphasized that
the unfolding of a helix of 20 amino acids within a non-polar environment would require approximately 100 kcal/mol [159].
Although helix formation is indeed important for the conformational transition, the per-residue reduction in Gibbs energy,
DGresidue, that drives secondary structure formation in the membrane interface cannot be ascribed to this sole effect, and other
interactions must contribute as well, including the effects of folding/assembly entropy, side-chain packing, relative exposure of
side-chains to membrane and water, and the depth of membrane
penetration of secondary structure units. Perhaps the most striking
aspect of the conformational transition is possibly the lack of
discrimination between microorganisms, even belonging to different kingdoms. In fact, the simple existence of a bioactive negative
surface appears to be a key condition for AMPs to fold from an
unfolded peptide into a structured conformation [27].
In comparison, b-sheet AMPs are typically much more ordered
in aqueous solution and membrane environments, due to constraints imposed by disulfide bonds and the intrinsic rigid structure of this type of secondary structure as compared to the ahelix. Tam et al. [160] have designed b-sheet cyclic peptides based
on the antimicrobial peptide tachyplesin-1 (anti-parallel b-sheet
structure connected by a type I b-turn) and they have shown that
the design of a highly rigid peptide template may be useful for further enabling to dissociate antimicrobial activity from cytotoxicity,
anticipating that conformation transition is also a key step to modulate the biological activity displayed by the peptide. In contrast to
a-helical peptides, the common dimerization of b-sheet peptides
could also increase antimicrobial activity, by promoting a deeper
positioning into the hydrophobic membrane core than would be
allowed to a monomer. Besides, the assembly of small peptide
aggregates may facilitate the formation of transmembrane pores
or channels, as proposed in studies for peptides such as cecropins
[161].
Peptide–membrane interactions can also depend on the conformational dynamics of the peptide in solution both at the water–lipid interface or at the target membrane. For instance, the
antimicrobial peptide trichogin GA IV acts through a complex equilibrium involving two different peptide species, monomers and
small aggregates [5], and it was shown that only the aggregates
159
promote membrane permeability [5,162]. More recently, it was reported that many AMPs, such as temporin B and L, LL-37, plantaricin A, magainin-2, sakacin P and melittin form amyloid–like
structures in the presence of acidic phospholipids, which seem
important for their mechanism of action [163,164]. Based on the
observation of protofibrilliar structures on the folding/aggregation
process, the cytotoxic action of these amyloid-forming peptides
resembles the supramolecular protein–lipid amyloid-like fibers
formed upon binding to negatively charged phospholipid-containing membranes, as demonstrated for Ab, prion, a-synuclein, and
IAPP [163]. Within this framework, it was demonstrated that these
AMPs form fibrilliar protofilaments at the membrane, preceding
the formation of inert and non-toxic mature amyloid fibers, after
which the membrane permeability is compromised, demonstrating
that conformational dynamics (aggregation/protofilament formation) is indeed important for the free energy landscape and specificity of action exhibited by some AMPs.
5.4. Peptide insertion and membrane permeability
5.4.1. Membranolytic activity
Many authors have explored this feature using different biochemical and biophysical approaches, such as NMR, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction. As a result,
several models have been proposed to explain how, following initial attachment, antibacterial peptides insert into the bacterial
membrane to form transmembrane pores (membrane permeabilization and lysis) [13,165]. On the other hand, other processes by
which transient topological alterations on membrane structure occur have been recently described as a means to gain access to intracellular targets [5,9,26,48,132,166]. Here, we provide a
comprehensive perspective of the current knowledge on the mechanism of action of AMPs and incorporate some novel aspects that
have been unraveled in the past few years.
Several mechanisms of action for AMPs have been recently reported, and include membrane permeabilization through the formation of stable pores (either barrel-stave or toroidal pore
models), membrane thinning (molecular electroporation or sinking
rafts models) or micellization on a detergent-like way (carpet model). Following access to the intracellular space, the sequential leakage of ions and other metabolites, loss of cytoplasmic components,
dissipation of electrochemical potentials and ultimately cell death
may explain the peptide’s lytic action over pathogens.
In the ‘barrel-stave model’, peptide helices form a bundle in the
membrane with a central lumen, much like a barrel composed of
helical peptides as the staves. This type of transmembrane pore
is unique and is induced by quite hydrophobic peptides such as
peptaibols alamethicin and zervamicin [165,167,168]. In this
mechanism, the hydrophobic domains of a-helical or b-sheet peptides face inwards and interact with acyl chains of the membrane
core, whereas the hydrophilic face forms the pore lining (Fig. 3A).
The initial step in barrel-stave pore formation involves the initial binding and attachment process, which seems to be accomplished by monomeric but not aggregated forms. After binding,
the peptide may undergo a conformational phase transition, forcing polar-phospholipid head groups aside to induce localized
membrane thinning. At this point, the hydrophobic portion of the
peptide is inserted into the membrane to an extent corresponding
to the hydrophobic part of the membrane’s outer leaflet since most
peptides do not present sufficient length to transverse all membrane. When the bound peptide reaches the threshold concentration, peptide monomers self-aggregate and insert deeper into the
hydrophobic membrane core [15]. Continuous association of peptide monomers may result in further expansion of the membrane
pore and upon phospholipid translocation or relaxation of the pore,
peptides are transported to the inner membrane leaflet due to the
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Fig. 3. Antimicrobial peptide mechanisms of action. In this figure, various models are shown, illustrating advances in proposed mechanisms of antimicrobial peptide action.
(A) In the barrel-stave model, the peptides span the membrane and form a pore with the hydrophilic portion lining the pore. (B) The carpet model is characterized by the
spanning of the membrane by the peptide followed by a detergent-like action that disrupts the membrane structure. (C) The toroidal model differs from the barrel–stave
mechanism as the hydrophilic portion of the peptide (in its amphipathic conformation) is associated with the lipid headgroup. (D) In the molecular electroporation model, the
interaction of the cationic peptide with the pathogen membrane promotes an electrical potential difference across the membrane. When this potential reaches 0.2 V, a pore is
believed to be created by molecular electroporation. (E) The sinking raft mechanism proposes a mass imbalance between the two leaflets of the membrane induced by the
peptide. By creating a curvature gradient along the membrane and by self-association, peptides sink into the membrane and form transient pores that are thought to promote
a transitory increase on membrane’s permeability and leakage of intracellular contents. After membrane relaxation, peptides will reside on both leaflets of the membrane.
(Adapted with permission from [73]. Copyright 2006 Elsevier, UK).
concentration gradient of surface-bound peptide, the tension exerted by peptides locally as well as the trans-negative electrochemical potential [169].
In the ’carpet model’, peptides adsorb and span the bilayer surface. This model explains the activity of antimicrobial peptides
such as ovispirin [170], dermaseptin natural analogues, cecropins,
caerin 1.1, trichogin GA IV [169] and some magainins [171]. Peptides are electrostatically bound to the anionic phospholipid head
groups at numerous points, covering the surface of the membrane
in a carpet-like manner (Fig. 3B). At the critical threshold concentration, the peptides might form toroidal transient holes in the
membrane, allowing additional peptides to access the membrane,
from which the membrane disintegrates and forms micelles after
disruption of the bilayer curvature [27]. From this perspective,
membrane structure disruption occurs in a dispersive-like manner
rather than channel formation, and peptides do not necessarily insert into the hydrophobic membrane core, as observed for cecropin
P1 [172].
In the ’toroidal-pore mechanism’, antimicrobial peptides partition into the membrane and continuously induce a bending in
membrane leaflets through the pore so that the water core is lined
by both the inserted peptides and the lipid head groups [27]. The
constitution of a toroidal pore implies that the polar faces of the
peptides associate with the polar headgroups of the lipids due
to bending and therefore, the lipids tilt from the lamellar normal
and connect the two leaflets of the membrane, forming a continuous bend from the top to the bottom in a toroidal pore manner
(Fig. 3C) [27]. This creates an unfavorable elastic tension that may
culminate in the formation of transient defects and ultimately
pore disintegration. Yeaman et al. suggest that some peptides
may cross through the membrane and act on intracellular targets
[27]. Molecular dynamics simulations have also shown that peptide aggregation, either prior or after binding to the membrane
surface, seems to be a prerequisite to pore formation although a
stable secondary structure is not required [173]. This type of
transmembrane pore is induced by some magainins, protegrins
and melittin.
The toroidal model differs from the barrel-stave model as the
peptides are always associated with the lipid head groups even
when they are perpendicularly inserted in the lipid bilayer
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
(Fig. 3). Indeed, this seems to be of major importance since the
presence of several peptide monomers in the pore would result
in a thermodynamically unfavorable Coulomb energy due to an
excessive density of charges lining the pore. Therefore, the association with the anionic headgroup of the lipids partly cancels the
positive net charge of peptides and favors the peptide aggregation
process leading to pore formation.
Both the toroidal and carpet models predict that killing activity
of antimicrobial peptides occurs with concomitant dissipation of
the membrane potential, due to the collapse in membrane integrity. However, the peptides elicit different biological responses
and a large set of studies have provided evidence that ion channels,
transmembrane pores and extensive membrane rupture followed
by leakage of metabolites and intracellular content in general do
not proceed through three completely different modes of action,
but instead are a continuous gradation between them. This concept
corroborates the observation that the formation of peptide induced
ultrastructural lesions lags behind the loss of cell viability. Initially,
the transmembrane potential and pH gradient are impaired, the
osmotic regulation is affected and membrane integrity is then
compromised in a concentration-dependent manner. For instance,
cecropin A dissipates ion gradients in synthetic lipid vesicles at a
concentration much lower than that required to release encapsulated calcein, indicating that the activity of cecropin A at low concentrations is primarily due to the dissipation of transmembrane
electrochemical ion gradients rather than membrane permeabilization by pore formation [174]. Interestingly, cecropin A dissipated
ion gradients even in the absence of anionic lipids, although their
presence dramatically increased peptide binding and moderately
increased the release of calcein. In Gram-negative bacteria, cecropin A was considerably bactericidal at similar concentrations
which promoted ion conductance changes, but much higher concentrations were required to cause the release of cytoplasmic
contents.
5.4.2. Non-membranolytic activity
Although permeabilization of the cell membrane seems fundamental to the antimicrobial effect of AMPs, several studies indicate that permeabilization alone may not be enough to explain
antimicrobial activity [43]. In fact, antimicrobial peptides may
also affect cell membrane topology by creating transient defects
on its structure or modulate particular core metabolic pathways
after crossing the membrane and gaining access to intracellular
targets, such as DNA, RNA, enzymes and even organelles such as
mitochondria.
5.4.2.1. Aggregate channel model. Wu et al. have demonstrated that
individual peptides present significant differences on their ability
to depolarize the cytoplasmic membrane potential of E. coli, such
as the loop peptide bactenecin and the a-helical peptide CP26
being unable to cause depolarization at the MIC [175]. This implies
that membrane depolarization by itself is not necessarily the crucial event in the killing of microorganisms by these peptides. On
this basis, the aggregate channel model was proposed [15]. After
binding to the phospholipid head groups, the peptides insert into
the membrane and then cluster into unstructured aggregates that
cover the membrane. These aggregates are proposed to be associated to water molecules providing channels for the leakage of ions
and possibly larger molecules through the membrane. This model
essentially differs from the other models as only short-lived transmembrane clusters of an undefined nature are formed, which allow the peptides to transiently cross the membrane without
causing significant membrane depolarization and membrane
structure disruption. Once inside, the peptides may home to their
intracellular targets to exert their lethal activities by acting on
polyanions, such as DNA or RNA [15].
161
5.4.2.2. Molecular electroporation model. Another mechanism that
has been proposed for some antimicrobial peptides is the molecular electroporation model (Fig. 3D). This mechanism establishes
that the formation of pores in membranes occurs under the influence of an external electric field. When short pulses are used, a
voltage of about 1 V across the membrane is required for pore formation. The threshold falls to about 0.2 V for electric fields applied
over long time periods (0.1 ms) [176]. Molecular electroporation
only occurs when the peptides present a sufficient charge density
to generate an electric field and is triggered during the period of
time in which the electrostatic potential is at least 0.2 V.
This model has been proposed to explain membrane pore formation by annexin V [177]. Pore sizes of 2–4 nm diameter have
been reported by conventional electroporation and by at least
two other cationic peptides, polymyxin B and melittin [176]. These
membrane pores present comparable sizes to the NK-lysin–membrane interface area with a potential 0.2 V greater [176,178]. This
mechanism is particularly important to describe the action of those
peptides that present antimicrobial activity without apparent formation of transmembrane pores, providing new insight for the
means by which peptides increase membrane permeability without necessarily causing its disruption.
5.4.2.3. Sinking-raft model. More recently, a novel mechanism has
been reported according to which, the biological activity displayed
by some AMPs is a result of imbalance of mass ratio for preference
of binding to a particular lipid domain, locally producing a mass
disproportion that directs the peptide translocation through increase in local membrane curvature (Fig. 3E). This mechanism,
commonly named the sinking raft model, is responsible for the formation of transient pores after the dissipation of the peptide-induced membrane leaflet mass imbalance [179,180]. Pokorny
et al. [181] have reported that d-Lysin, an a-helical amphipathic
peptide, binds more efficiently to the outer leaflet of the mammalian cell membrane, which are enriched in sphingomyelin, cholesterol and unsaturated phosphatidylcholine. Mixtures including
these lipids have been shown to exhibit phase segregation between
liquid-disordered (ld) and liquid-ordered (lo) domains, the last one
particularly rich in sphingomyelin and cholesterol (lipid rafts). In
such systems, Pokorny et al. established that the peptide preferentially binds to the ld domains, producing a local concentration of dLysin and enhancing peptide aggregation in these domains, which,
in turn, creates the mass imbalance [181]. Therefore, the curvature
strain is relieved as the peptides bind to membranes, sink into the
bilayer, and translocate to the cytoplasmatic leaflet of the
membrane in a process that perturbs the membrane and causes
the efflux of intracellular metabolites [180]. In this case, the
equilibrium across the membrane is attained after peptide translocation between the two leaflets, which concomitantly ends the
transient ion leakage and small metabolites release [180]. The
same model has also been proposed for polyphemusin [182].
5.4.2.4. Peptide-induced lipid segregation mechanism. Some peptides
have been shown to produce significant membrane perturbation
by formation of specific lipid–peptide domains, lateral phase segregation of zwitterionic from anionic phospholipids and even the
induction of non-lamellar phases at physiologically-relevant conditions. These observations lead to the proposal of new models
for the mechanism of action of some AMPs.
One of the most intriguing models arises from current observations of peptide-induced lipid segregation of anionic components
from zwitterionic lipids [95,96,137] (Fig. 4) and even de-mixing
of anionic phospholipids in Gram-positive model membranes
[183]. Such feature was observed by Arouri et al. in a study on
the influence of linear and cyclic arginine- and tryptophan-rich
antimicrobial peptide analogues (RRWWRF) on the thermotropic
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V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
Fig. 4. Representation of lipid rearrangement upon binding of cationic antimicrobial peptides (blue) according to the lateral lipid segregation mechanism. Clustering
of anionic lipids (red) into separate peptide–lipid domains and segregation of
zwitterionic lipids (yellow) occurs as a consequence of binding of the antimicrobial
peptide, causing a rearrangement of lipids on the membrane with possible
significant consequences on cell viability and survival.
phase transitions of lipid membranes [96]. It was shown that the
presence of both peptides led to the appearance of two peaks in
the DSC curves, assigned to a DPPG peptide-enriched and a
DPPE-enriched domain, indicating thus induced de-mixing in
DPPG/DPPE bilayers. Based on these results, this group proposed
that peptide induced lipid segregation in PG/PE membranes could
be a further specific effect of the antimicrobial peptides operating
only on bacterial membranes, as they contain appreciable amounts
of these two lipids (Table 1). In addition, membranes composed of
PG and PE do not mix homogeneously, which further favours lipid
demixing [88].
Epand et al. have also recently proposed that antimicrobial peptides with a high positive net charge, conformational flexibility and
sufficient hydrophobicity facilitate preferential interaction with
anionic lipids and the promotion of lipid lateral segregation
[137]. It was also hypothesized that peptides that fulfill these
requirements are more active towards bacteria composed of zwitterionic and anionic lipids, due to the lipid segregation mechanism
proposed. This is supported by a study by Epand et al. [93] using
OAK C12 K-7a8, which shows that Bacillus cereus (a Gram-positive
bacterium with a high content of the zwitterionic PE) has a considerably lower MIC value than the one found for Gram-positive bacteria whose membrane is mainly constituted by anionic lipids, as
observed on S. aureus. In this later species, the much lower percentage of zwitterionic lipids [137], implies that it must be able
to counteract more effectively the segregation mechanism because
there is no significant proportion of neutral lipids to segregate from
the anionic ones. Additionally, the presence of higher content of
negatively-charged lipids, such as cardiolipin (CL) and PG, is likely
to require a higher peptide concentration to neutralize the mem-
brane. These observations are consistent with the higher MIC usually observed for these types of bacteria [137].
A similar trend was observed for a series of monomethylated
derivatives of cecropin A-mellitin hybrid peptide CA(1–7)M(2–9).
Calorimetric studies [95] have provided evidence of a lipid lateral
segregation mechanism in DMPC:DMPG (3:1) and POPE/POPG
(3:1) bacterial lipid model systems, and microbiological studies
have demonstrated higher MIC50 values for S. aureus (mostly anionic phospholipids) than for the Gram-negative bacterium Acinetobacter baumannii (which has zwitterionic lipids in its membrane
composition) [184]. Further evidence was provided by other studies performed by Epand et al. [185] on LL-37 derivatives which
indicated that the peptide fragments with the ability to induce lipid phase segregation of anionic lipids away from zwitterionic lipids were active/selective toward bacteria containing zwitterionic
and anionic lipids in their cytoplasmic membranes, but presented
no activity towards species with only anionic lipids. Overall, these
studies suggest that some antimicrobial peptides are able to induce
lipid segregation on species with membranes composed of zwitterionic and anionic lipids and that pathogens with such membrane
constitution appear to be more susceptible than organisms mostly
composed of anionic lipids.
Some biophysical and biochemical implications of the action of
these peptides on the bacteria based on this mechanism will be addressed now. The occurrence of lateral lipid segregation promoted
by the peptides may create membrane line defects that are responsible for the increased permeability of the membrane. This is in
agreement with the results for SYTOX leakage of liposomes reported by Fernández-Reyes et al. for the monotrimethylated derivatives of CA(1–7)M(2–9) reported above [184]. In a biological
context, this would cause primarily the loss of the permeability
barrier property, which would lead to unregulated diffusion of ions
and metabolites. The effect of lipid segregation was already reported by den Hertog et al. in Candida albicans where LL-37 and
its truncated variants were able to induce this phenomenon on
the cytoplasmic membrane [186]. Additionally, the perturbation
of the membrane by a coupled mechanism involving pore formation and lipid segregation would amplify this effect, further contributing to the increase of the membrane permeability and
impairment of the cell metabolism, as proposed by Teixeira et al.
for the CA(1–7)M(2–9) peptide on the Gram-negative mimicking
membrane system, POPE:POPG (3:1) [95].
However, the biophysical alterations to the membrane due to lipid segregation can have other implications, namely in terms of
curvature strain whose importance in bacterial metabolic processes has been extensively reviewed. A study by Iwamoto et al.
[187] has revealed that phosphatidylethanolamine on the yeast
plasma membrane is implicated in cell polarity, since the treatment of Saccharomyces cerevisiae cells with a biotinylated probe,
which specifically binds to PE, resulted in aberrant F-actin accumulation, implying that limited surface exposure of PE is involved in
the polarized organization of the actin cytoskeleton. Similar PErich domains were observed in the septal regions of the cells of
many Bacillus species [188]. The same lipid is also involved in cytokinesis as shown by studies with a mutant cell line with a specific
decrease in the cellular PE level. The cultures present limited cell
growth because the contractile ring remains in the cleavage furrow, promoting the arrest of the cell in intermediate states of cell
division [189]. Recent studies have also highlighted the importance
of monolayer and bilayer curvature for the budding and fission of
biological membranes [190].
Overall it is likely that the activity of antimicrobial peptides
may affect the curvature strain and modulate similar relevant biological processes. It would introduce a high destabilization of the
membrane beyond the capacity of the bacteria to rearrange and
accommodate this change in its organization [96] with relevant
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
consequences on biological processes dependent on the curvature
strain of the membrane, such as cell division and sporulation, as
stated before [191].
One particular property of lipids that prevails is their phase
behavior. In cells, lipids can adopt various fluid and liquid-ordered
phases, which are characterized by a different spatial arrangement
and motional freedom of each lipid with respect to its neighbors.
Biophysical approaches have defined the principles of coexistence
of two fluid phases (with different physical characteristics within a
single membrane plane) that are delimited by a phase boundary,
and the consequences on membrane organization have been
pointed out, such as lateral phase segregation or domain formation
[90]. Indeed, membrane lipids can occur in various phases depending on their composition, structure and environment [88]. These
phases have specific properties that determine the orientation,
packing and mobility of membrane lipids and proteins and the
interactions between them, which in turn have an important impact on many biological processes. In this context, apart from lateral lipid segregation, it is likely that the peptide-mediated phase
segregation by the formation of PE- and PG-enriched domains
(with different physical states) would have an enormous impact
on membrane fluidity since it would diminish the lipids’ diffusion
rate, the lateral movement of proteins and particularly the required fluidity of the membrane for cell viability [192].
Recently, the existence of specific ’functional’ membrane domains has been extensively studied. These domains recruit both
lipids and proteins from the cytosol that assemble into specialized
microdomains with important functions on the cell dynamics and
signaling. The best example are the lipid rafts, which comprise
small, dynamic assemblies with high concentrations of cholesterol,
sphingolipids and saturated phospholipids that create a local area
of increased order and, potentially, bilayer thickness, with important implications on membrane fluidity and organization. Certain
proteins seem to be localized preferentially in raft lipid regions,
and it has been suggested this may contribute towards the clustering of specific proteins for processes such as signal transduction
and even pathogenesis, such as Alzheimer’s disease [193].
In E. coli, similar ’functional’ membrane domains are proposed
to have an important role in cell cycle events, such as timing of
DNA replication and as space marker for cell division, as demonstrated by Norris et al. [194], as well as in chromosome segregation
[195]. Therefore, the functionality of these domains is likely to be
modulated by AMPs. From this perspective, an alternative mechanism would involve the loss of the physiological role of these natural-occurring domains due to phase boundary defects resulting
from the peptide-induced reorganization and de-mixing of lipids
in the membrane. The occurrence of lipid segregation could promote the dissolution of these peptide–lipid complexes and turn
them non-functional for cells.
5.4.2.5. ’Leaky-slit’ mechanism. The formation of specific peptide–lipid interactions has been recently reported as well. Zhao et al. have
provided evidence of an additional mechanism of action based on a
possible connection between fibril formation and the toxicity displayed by some AMPs (Figs. 5 and 6), in line with the mechanism
that has been described for lytic bacterial toxins, such as cytolysin
[196] and other cytotoxic proteins [197].
According to this model, the lipid-bound peptides adopt a linear, amphipathic array with the hydrophobic side facing the lipid
bilayer to promote membrane insertion. In this case, the toxicity
would be caused by the hydrophilic domain of these aggregates,
which allows self-association of the peptides by hydrogen bonding
in the target cell membrane, leading to initial aggregation and formation of fibrilliar and toxic oligomers (Fig. 5). As a consequence,
lipids would be forced to adopt a highly positive curvature and
transient toxic oligomers (‘leaky slit’) would increase the mem-
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Fig. 5. Illustration of the ‘leaky slit’ model as a possible mechanism of action of
antimicrobial peptides. The peptide adopting an amphipathic alpha-helix is
depicted in blue and the lipid headgroups and chains are shown in bright grey
and black, respectively. (Adapted with permission from [164]. Copyright 2006
Elsevier, UK).
Fig. 6. Microscopy images of fibrilliar structures formed upon interaction of
plantaricin (plA) with DOPC/brain PS-containing liposomes. (A) Fibers stained with
Congo red and viewed under a polarizing microscope. (B) Fluorescence microscopy
image of fibers incubated in the presence the fluorescent phospholipid analogue
NBD-PG, which was additionally included in the PC/PS liposomes. The analogous
formation of fibrous aggregates consistent with supramolecular protein–lipid fibers
formed by several other cytotoxic proteins and peptides are highlighted. The
observed fiber formation promoted by the presence of anionic phospholipids is
thought to be directly related to its cytotoxicity, similarly to the toxicity of fibrils
formed by the paradigm of amyloid-forming peptides. (Adapted with permission
from [164]. Copyright 2006 Elsevier, UK).
brane permeability to metabolites. At the end, the oligomers would
convert into inert amyloid-like fibers, detoxifying the peptides.
The authors have stated that the requirements to promote these
effects on target cells are fibrilliar organization and amphipathic
nature of the fibril, spanning the bilayer. Factors like lipid composition and the molecular determinants of the peptide, mainly conformational flexibility, amphipathic nature, and propensity to fold
into amyloid-like structures are likely to greatly influence the peptide activity at the membrane level. In fact, plantaricin A and other
similar peptides tend to fold into amyloid-like fibers mostly in the
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presence of negatively charged liposomes causing vesicle aggregation and leakage of vesicle content in membranes containing both
zwitterionic and anionic lipids (Fig. 6) [164].
In addition, the fibril could vary in length and could be constituted by a-helical as well as b-sheet structures and the peptides
may be long enough to span the bilayer or require dimer formation
[97]. These fibrils could further incorporate lipids by orienting the
lipid head groups to face the hydrophilic surface of the fiber, while
the acyl chains would protrude from the hydrophobic surface and
extend into the bilayer, as described by the ‘toroidal-pore model’.
Accordingly, the ‘leaky slit’ model readily complies with the profound toxicity of fibrils. The authors have cautiously referred that
other related mechanisms, both ‘carpet’ and the ‘toroidal pore’
models could not be excluded to explain the interaction of these
peptides. Alternatively, it was suggested that the ‘carpet’ and
‘toroidal pore’ models and the proposed ‘leaky slit’ mechanism,
could somehow overlap and contribute to membrane perturbation
by antimicrobial peptides. Although the ‘leaky slit’ mechanism
could be regarded as a refinement of these two models, this model
provides new aspects, in particular the importance of the amphipathic conformation and the fibrilliar nature exhibited by some
AMPs for their toxicity towards target cells. In addition, the model
can also predict how these fibrilliar protofilaments can induce positive curvature in membranes, in a different manner to what is
proposed on the ‘toroidal pore’ model and account for their action
on the membrane.
Even though this model provides new insights on bridging antimicrobial peptide biological activity and the cytotoxicity shown by
other amyloid-forming peptides, it is important to emphasize that
more detailed molecular aspects of the process of amyloid-like fiber formation by AMPs and the importance of structural features
of peptides and membrane composition at different stages of the
aggregation/folding landscape are needed in order to obtain a more
comprehensive picture of the membrane association and cytotoxicity displayed by these peptides. This would contribute to understand elusive aspects of the very striking degree of similarity
between the action of AMPs and the disease-associated amyloid
forming peptides on membranes.
5.4.2.6. Peptide-mediated non-lamellar phase formation mechanism. An exciting research field has emerged from studies showing a possible correlation between non-lamellar phase formation
and the activity of cationic amphipathic peptides. Membrane lipids can self-assemble into numerous different phases in aqueous
solution, including micellar, lamellar, hexagonal and cubic phases
(Figs. 7 and 8). The tendency to acquire a particular membrane
organization arises from the well recognized shape-structure concept, as reviewed by Haney et al. [182]. Mixtures of lipids that
present similar lipid headgroup and acyl chain cross sections tend
to form a planar bilayer structure, such as PG + PC. Lipids that are
cone-shaped like PE (due to a higher acyl chain volume-to-lipid
headgroup proportion) tend to favor negative mean curvature
so that the polar/apolar interface curves towards the polar region.
These lipids prefer inverted micelles and inverted hexagonal lipid
phases or regions with high negative membrane curvature strain.
Most biologically-relevant lipids are mostly bilayer-forming
molecules, but can form either lamellar or non-lamellar phases
under specific conditions [182]. However, the phase properties
of biological membranes are far more complex, since the biological membranes are constituted by a wide variety and assembly
of lipids and proteins that overall influence the membrane
dynamics.
In biological systems, the membrane is mostly depicted as a flat
structure in the lamellar phase. Due to environmental factors or lipid lateral stress induced by proteins or other lipids, the membrane
may alter its structure to form a phase possessing curved interfaces
with conformationally preferred curvature strain and thickness,
such as hexagonal, cubic or even micellar phases. According to Kirk
et al., the total free energy of the lipid–water assembly is mainly
determined by the conjugation of four factors, namely the membrane curvature elasticity, gC; the packing of the hydrocarbon
chains, gP; the hydration force; and the electrostatic contribution
[198]. Membrane dynamics derives both from inherent factors
(such as the length, packing density and geometry of acyl chains
of component lipids, the size of lipid headgroup and the resulting
spontaneous curvature profile) [13,93] as well as from all external
forces that modulate membrane shape and structure
[88,89,93,146]. Therefore, it is important to understand how membrane properties governing membrane dynamics are affected by
changes induced by the interaction with AMPs.
In the absence of stress factors, the stored curvature elastic energy translates into the bilayer expanding laterally and across the
headgroup area, increasing above its preferred value in order to release the lateral stress and lipid packing frustration and adopting
the thermodynamic most favorable membrane organization. However, this effect can only be tolerated up to a threshold as small
alterations of membrane conformation may involve a very high
energetic cost due to the exposure of lipid acyl chains to water
(commonly known as the hydrophobic effect). Upon stress, transition to an inverse phase may occur. In this case, the interface can
bend inwards the aqueous compartment, allowing the headgroup
area to decrease and the chains to expose to the solvent hence,
releasing the stored lateral stress. A similar feature was observed
for some integral membrane proteins, which may release stored
curvature elastic stress locally during insertion into the bilayer
by allowing the chains to dislocate more and forcing the head
groups together, making the peptide–lipid assembly thermodynamically more stable.
Fig. 7. Illustration of the lamellar (La) and the hexagonal phases (HI, hexagonal phase and HII, inverted hexagonal phase) of the lipid membrane. A schematic lipid molecule is
depicted in the figure, with the hydrophilic headgroup represented in red and the hydrophobic alkyl chain in grey. (Adapted from reference [258]. Copyright 2009 PMC
Biophysics).
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
165
Fig. 8. Schematic structures of lipid cubic phases. The inverse bicontinuous cubic phases Ia3d, Pn3m and Im3m are shown, together with the micellar cubic phases Fd3m and
Pm3n. The two types of inverse micelles in Fd3m (open and grey spheres) are indicated on each site of the cubic lattice, along with their polyhedral shapes. The current models
proposed for the cubic phase with space group Pm3n by Tardieu and Luzzati (A) [259], Fontell et al. (B) [260], and Charvolin and Sadoc (C) [261] are also shown. Adapted from
references [209] and [258].
The major importance of this lipid polymorphism in some biological processes has been demonstrated. However, its importance
was underestimated for many years. Some examples of the importance of the formation of non-lamellar phases, in particular, cubic
phases, have been reported in recent years and its biological relevance assessed (Fig. 8) [182,199,200].
Luzzati et al. [201] have mentioned that phase Q227 (Fd3m) may
have important biological implications. Indeed, phase Q227 is observed with some of the most common lipid components of biological membranes (PC and PE) in conjugation with some
intermediates derived from enzymatic degradation [fatty acid
(FA) and diacylglycerol (DAG)]. Evidence has shown that enzymatic
degradation induces the formation of lipid patches and the local
structures appear to adopt phases Q224 (Pn3m) and Q227 (Fd3m).
These metabolites (FA and DAG) destabilize the membrane and alter its permeability, turning it leakier and more susceptible to a
lipolytic agent. However, when FA and DAG are directly incorporated into the membrane, this leakage stops due to alteration in
the lipid topology [201].
Another example arises from studies performed on Archaebacteria, a particular group of organisms with challenging physiology
that thrive in extreme conditions of pH, temperature, and ionic
strength. A prominent difference between archaebacteria and
other organisms resides in the chemical structure of the lipid molecules that have considerable implications on the colonization of
such adverse environments. This aspect is paramount if one considers the molecular adaptations that these bacteria had to undergo in order to survive in such an extreme habitat (in excess salinity
and at temperature above 80 °C). One important example of the
biological relevance of such phases arises from a study by Deatherage et al. [202]. The plasma membrane of Sulfolobus solfataricus is
supported by a protein layer, the S-layer, that remarkably exhibits
a precise epitaxial relationship with the maximally hydrated Q224
phase of the polar lipid extract, suggesting that the plasma membrane is tightly folded in vivo according to the symmetry of phase
Q224. The result is a system of two mutually intertwined and
unconnected 3D water channels that resembles the cubic membranes. The biological implications of such a complex interconnected channel network were assessed by Luzzati et al. [201].
One end of the channels of one of the two networks communicates
with the cytoplasm; the other end is closed by the S-layer. The
channels of the other network communicate with the extracellular
medium via the channels of the S-layer and must end on the cytoplasmic side. In this structure, the hydrocarbon layer is topologically equivalent to a lipid bilayer constituting a 2D septum
separating the cytoplasm from the extracellular medium. The total
area of this septum, however, is highly variable according to the
thickness of the cubic crystallite. According to Luzzati et al. this
model may have important biological implications: by a proper
poising of passive ionic permeability across the bilayer and ion diffusion along the water channels, a steady state may be established,
leading to a smooth pH gradient along the two channel systems. As
a result, the local pH difference across the hydrocarbon layer,
against which the protons must be actively pumped, may be much
smaller than the difference between the cytoplasmic and the external pH (6.5 and 2, respectively). Furthermore, the unusual metastability of the cubic phase may also be biologically relevant, and it
appears to be associated with the bipolar lipid molecular nature
(archaeal tetraether bipolar lipids). These molecular nature bear
two headgroups, each anchored at opposite polar/apolar interfaces: any phase transition involving a migration of lipid molecules
is obstructed by the presence of two independent diffusion processes, therefore turning the transition more difficult to occur.
The presence of bipolar lipids, likely to preserve the high-temperature native organization of the membrane at low temperature, is
believed to be the evolutionary response taken by these bacteria,
and other thermophilic archaebacteria, to this challenge.
Many authors have established that the physical properties of
the membrane are modulated by proteins and peptides and they
may induce non-lamellar phases by altering the curvature strain,
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the topology of the membrane and the spatial arrangement of lipids and water molecules. On the other hand, they may also act by
changing the physical properties of the lamellar phase since it is
known that the spatial disposition and packing of lipids in the
membrane is closely controlled by the cell, in order to maintain
the flat, mostly planar morphology of the membrane
[149,182,199,203]. Therefore, how can antimicrobial peptides
modulate the membrane dynamics (shape, phase, membrane-pressure profile and curvature strain) and which are the implications
for their biological activity?
Epand had previously suggested that the ability of membrane
active peptides to alter lipid polymorphism mainly resides on five
factors: hydrophobicity, charge conformation and self-association,
steric effects and mode of insertion [203,204]. Some examples of
the formation of non-lamellar phases by antimicrobial peptides
have been reported by some authors. Sevcsik et al. [205] have
shown that LL-37 induces a peptide-associated quasi-interdigitated phase in negatively charged phosphatidylglycerol (PG) model
membranes. Polyphemusin-1 and the variant PV5 were shown to
decrease the lamellar to inverted hexagonal phase transition temperature of PE, indicating the induction of negative curvature strain
and promotion of the HII phase [206]. The formation of this hexagonal phase could account for a lipid–peptide intermediate in the
mechanism of action of these peptides. The authors have proposed
that this peptide acts by a translocation mechanism involving five
steps, specifically the initial electrostatic interaction with the anionic phospholipid membrane, the partition and insertion of the peptide into the hydrophobic core of the membrane, the peptide
aggregation and formation of the non-bilayer intermediate with a
random distribution of peptide between the core and the inner
and outer leaflets of the membrane, collapse of this intermediate
and redistribution of the peptide along both sides. The accumulation of the peptide in the cytoplasm, as observed by Powers et al.
[132], would be explained on the basis of lipid composition asymmetry between the two leaflets of the membrane, which would account for the release of the peptide from the inner leaflet and
accumulation in the cytoplasm [182].
Regarding the formation of cubic phases in particular, a wellknown antimicrobial peptide, gramicidin S (GS), has the ability
to form such phases in relevant biological mimicking membrane
systems [207,208]. Prenner et al. have initially studied the interactions of the cyclic peptide GS with single-component lipid bilayers and membrane polar lipid extracts of Acholeplasma (A.)
laidlawii B and E. coli [208]. 31P nuclear magnetic resonance
(NMR) spectroscopy and X-ray diffraction have revealed that the
binding of GS to membranes solely composed of PC, PS, CL or
SM presented an axially symmetric 31P NMR powder patterns
throughout the entire temperature range studied, suggesting the
absence or little effect on the membrane structure with respect
to the formation of non-lamellar phases. However, in the presence
of PE, PG or a nonlamellar phase-forming PC and even on the heterogeneous lipid mixtures of A. laidlawii B and E. coli membranes,
an isotropic component is observed in the 31P NMR spectra at high
temperatures, which is consistent with the formation of a cubic
phase [209]. It was observed that the relative intensity of this
component was increased with increasing temperature and GS
concentration. Recently, Staudegger et al. have demonstrated that
lipid extracts of these two bacteria induce the formation of inverted cubic phases of the space groups Pn3m and Im3m and that
its formation was observed in physiologically-relevant conditions
[210]. Therefore, the possibility of natural occurrence of ‘‘cubic
phase domains’’ in the membrane and the rearrangement of the
bilayer structure and dynamics by the peptide, leading to monolayer curvature stress and to formation of the cubic phase, could
be assigned as a mechanism of action and concur to their biological activity.
Zweytick et al. [83] have recently studied the interaction of human lactoferrin derivatives, VS1–13 and VS1–24, with E. coli total
lipid extracts. The formation of non-lamellar phases was detected
by SAXS (small-angle X-ray scattering), where two cubic phases
of the space group Pn3m and Im3m could be assigned, coexisting
with the lamellar phase. The ability to induce cubic phase formation was also correlated with the antimicrobial activity as the most
potent antimicrobial peptide VS1–24 was a stronger promoter of
cubic phases than its less active counterpart VS1–13. Therefore,
it is tempting to assume that the formation of cubic phases and,
more generally non-lamellar phases, may indeed be related to
the mechanism of action of antimicrobial peptides and their potency towards pathogens.
Interestingly, some authors have shown that some AMPs, such
as protegrin-1, PGLa and gramicidin S, were also able to form cubic
phases on pure POPE membranes, which is one of the main components of bacterial membranes. It was shown that GS had the strongest effect whereas melittin fully stabilized the lamellar phase
[211]. Nisin was also shown to form cubic phases with POPE membranes and it was suggested that the insertion of the peptide in the
bilayer shifts the amphiphilic balance by increasing the hydrophobic contribution, which would be the basis of the changes in the
polymorphic state of PE. This was supported by the fact that the
presence of cholesterol in the PE bilayer inhibits the membrane
perturbation by nisin, most likely due to the presence of the sterol
and its effect on membrane structure and fluidity [212]. Another
study using X-ray diffraction and NMR has shown that the presence of 1% alamethicin introduces a large region of cubic phase into
the phase diagram of bulk PE membranes [213].
It is highly conceivable that peptide-mediated non-lamellar
phase formation in bacterial membranes may provide a mechanism of action and have biological consequences. Haney et al. have
highlighted some important aspects on this concept [182]. The local destabilization of the membrane structure by altering lipid
packing and sorting of different lipids seems plausible, particularly
on PE-enriched domains with a natural propensity to form nonlamellar phases or regions with high negative curvature strain. In
this context, Haney et al. propose that the different PE content
on bacterial membranes and the extent of non-lamellar phase formation may be the basis of different bacterial susceptibilities to
AMPs [182].
Epand et al. have recently reviewed the importance of membrane domains and the loss of their stability and functional properties upon binding of antimicrobial peptides. We suggest that
this can be viewed in the light of non-lamellar phase induced formation, as it may induce an unstable membrane where such natural domains may lose their biological function due to the formation
of a cubic phase and disorganization of ’functional’ lipid domains
[137], in similar fashion as suggested for the peptide-induced lipid
lateral and phase segregation mechanism.
Moreover, bicontinuous cubic phases are characterized by
water channels that weave their way through a single continuous
bilayer equally divided into two inter-linked but separate aqueous
sub-volumes. Therefore, the formation of this complex network of
water channels across the structure may contribute to the leakage
of ions and other metabolites and consequent loss of membrane
function as a permeability barrier.
Recently, a new concept has emerged that establishes a close
relationship between alterations in the lipid structure and the
modification of cell signaling events. Amphitropic enzymes comprise a class of proteins whose activities are modulated by the
reversible translocation to membrane surfaces in response to local
fluctuations on membrane dynamics and organization. This translocation may be regulated by the membrane lipid composition and
by the membrane physical properties [199]. Two well-recognized
examples of amphitropic enzymes that respond to lipid polymor-
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
phism are protein kinase C and CTP:phosphocholine cytidylyltransferase [199].
Protein kinase C (PKC) is a family of enzymes important in cell
growth and proliferation, differentiation and cell signaling. PKC
consists of several isoforms that require phospholipid, Ca2+ and
diacylglycerol as cofactors for activation [214]. It is known that
PKC activity is influenced by the presence of non-lamellar forming
lipids and the basis of its binding to lipids has been recently reviewed [214]. Giorgione et al. have studied the activity and membrane binding of PKC in lipid bicontinuous cubic phases and
hexagonal phases using MO/PS and DEPE/alamethicin as cubic
phase lipid systems. These phases were shown to trigger the
PKC-catalyzed phosphorylation of histone and it was further
shown that the specific activity of the enzyme bound to cubic
phase membranes is much greater than that bound to phospholipid in the lamellar phase. Since alamethicin is a well-known antimicrobial peptide produced by the fungus Trichoderma viride, the
ability to alter the activity of an enzyme involved in cell signaling
establishes a possible correlation between non-lamellar phase formation and modulation of cell signaling events. The capability to
alter cell metabolism by other compounds, such as anticancer
agents, have been recently reported in a representative study by
Martínez et al. [215]. The anticancer drug, 2-hydroxy-9-cis-octadecenoic acid (Minerval) increases the tendency of PE membranes
to organize into nonlamellar (hexagonal HII) phases, promoting the
subsequent recruitment of protein kinase C (PKC) to the cell membrane with inhibition of the growth of cancer cells and antitumor
effects in animal models of cancer without apparent histological
toxicity [215]. Therefore, we can only speculate that the cubic
phase triggered by antimicrobial peptides may possibly generate
the very same effect, i.e. it can modulate important cell signaling
pathways by altering the activity of amphitropic enzymes. This
seems to be, at least, reasonable, although to the best of our knowledge, an example of an antimicrobial peptide that induces alterations on cell signaling in vivo has not yet been reported.
From all the studies described, it is now clear that AMPs have
the ability to modulate the physical properties of the membrane
and, as a result of their interaction with the membrane, induce lipid phase and topological changes, alter the membrane curvature
167
strain and consequently the membrane morphology or modify
the spatial arrangement of lipids and water molecules. All of these
effects carry relevant in vivo implications on biological membrane
structure and ultimately on cell viability. The functional and integral knowledge of such interactions is therefore of major importance in the elucidation of AMPs mechanisms of action upon
binding to the membrane. The relationships that might be derived
between the mechanism of action and its influence on membrane
structure will lead to a more comprehensive understanding of the
structure–activity paradigm for these peptides, which may be used
in the design of novel compounds with improved properties.
6. Intracellular targets of antimicrobial peptides: an alternative
mechanism of action?
Another area of intensive focus regarding AMP biology has recently emerged, providing a new point of view to the mechanisms
by which antimicrobial peptides cause cell death by acting by a
non-permeabilizing mechanism or by targeting intracellular components [48]. For a long time, it was assumed that their antimicrobial action was mainly mediated by membrane disruption. In fact,
AMPs were mainly assumed to act through the formation of transmembrane pores, leading to leakage of ions and metabolites, depolarization of the membrane potential and impairment of cellular
energetics and viability. Currently, an increasing amount of studies
has given evidence that membrane permeabilization alone appears
insufficient to cause cell death by some AMPs and therefore other
complementary or novel mechanisms have recently been reported
(Table 2).
Skleravaj et al. have demonstrated that several Bac5 and Bac7
fragments did not permeabilize E. coli although a decrease in the
number of viable organisms could be observed [216]. One of the
most interesting studies has shown the existence of AMP-mediated
activation of proteases, such as phospholipase A2, whose activity is
markedly enhanced by magainin 2, indolicidin and temporins B
and L in the presence of calcium [217]. This synergistic activity
has been pointed out by some authors as a major connection between AMPs and immune elements in an infection context [1].
Table 2
Intracellular components targeted by AMPs. The antimicrobial peptides buforin II and pleurocidin have been shown to inhibit DNA and RNA synthesis without disrupting the
membrane. Protein synthesis is another macromolecular target for antibacterial peptides such as indolicidin and PR-39. Several antibacterial peptides have been shown to act on
other intracellular processes, such as enzymatic activity. The ATPase activity of DnaK, an enzyme involved in chaperone-assisted protein folding, is targeted by pyrrhocidin, while
inhibition of enzymes involved in the modification of aminoglycosides has also been demonstrated. Cell wall synthesis may also be compromised by the action of some peptides,
such as mersacidin.
Antimicrobial peptides
DNA and cell division
Buforin II [218], tachyplesin I [266], ABP-CM4 [267], polyphemusin [132],*
pleurocidin and magainin 2 [268]
Hexapeptide WRWYCR [221]
dermaseptin [223], HNP -1 and -2 [269], PR-39 [270], indolicidin [271] and plant thionins [272]
PR-39 [220], PR-26 [220], indolicidin [271] and microcin 25 [273]
Enzymatic activity and protein synthesis
Purothionin [274]
Pyrrhocoricin [225], Bac7 [275]
Pleurocidin [223], dermaseptin [223], HNP -1 and -2 [269], PR-39 [270], indolicidin [271]
and plant thionins [272]
*
Intracellular target/mode of action
DNA binding
DNA repair enzymes
Inhibition of nucleic-acid synthesis
Septum formation
Ribonucleotide reductase
Inhibition of DnaK chaperone
Inhibition of protein synthesis
Cell wall
Nisin Z [142], plectasin (fungal defensin) [276] and mesarcidin [277]
plant antimicrobial protein-2 (Ac-AMP2), tachycitin and penaeidins [278]
Cell wall precursor lipid II
Chitin-binding activity
Eukaryotic Organelles
Histatin-5 [20] and hLF(1–11) [87]
ABP-CM4 [267] and BMAP-28 [279]
Cationic a-helical neuropeptides [280]
Energetic metabolism impairment (mitochondria)
Mitochondria
Energetic metabolism failure (autophagic-like cell death)
Possible direct PM1-biotin/DNA binding.
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For those peptides that gain access to intracellular targets, it is
essential to cross the membrane and translocate to the cytoplasm.
Buforin II, a linear AMP derived from proteolytic processing of histone H2A with a proline hinge, is an example of peptides that do
not permeabilize membranes but still penetrate this physical barrier and accumulate in the cytoplasm [218]. The mechanism by
which this peptide is translocated was revealed by combinatorial
studies by fusing the proline-hinge region of buforin II with the
N-terminal helix of magainin 2. It was shown that these hybrid
peptides translocated bacterial cytoplasmic membranes and accumulated in the cytoplasm, yet presenting antimicrobial activity.
Once in the cytoplasm, peptides usually concentrate and unleash
many different processes, such as the alteration of septum formation in cytokinesis, inhibition of cell-wall, nucleic-acid and protein
synthesis and suppress certain protein functions with an intrinsic
metabolic cost [219].
PR-39 is a proline–arginine-rich neutrophil peptide and its Nterminal 1–26 fragment, PR-26, were shown to induce filamentation of Salmonella enterica serovar Typhimurium (Salmonella
typhimurium). The deficiency in septum formation is characterized
by an extremely elongated morphology and this may arise from
the blocking of DNA replication or the inhibition of membrane proteins involved in septum formation [220]. More recently, Su et al.
have demonstrated that a hexapeptide, WRWYCR was capable of
inhibiting S. typhimurium growth within murine macrophages
[221]. This peptide acts by inhibiting unrelated DNA repair enzymes, thus trapping and preventing resolution of the Holliday
junction intermediate. In general, WRWYCR and its D-stereoisomer, wrwycr, were shown to be bactericidal against both Gram-positive and Gram-negative bacteria by promoting DNA breaks,
chromosome segregation defects and filamentation of cells [221].
The antimicrobial peptide, microcin B17, is also believed to inhibit an intracellular target within E. coli. This peptide has been
suggested to specifically inhibit DNA replication by targeting
DNA gyrase [222]. At the MIC, pleurocidin and dermaseptin both
inhibit nucleic acid and protein synthesis without damaging the
E. coli cytoplasmic membrane [223]. A study by Gifford et al. has
demonstrated that Lfcin B localizes in the cytoplasm and inhibits
macromolecular synthesis of DNA, RNA, and proteins [224]. Kragol
et al. [225] have recently showed that the insect antibacterial peptides pyrrhocoricin, drosocin and apidaecin bind to the bacterial
heat shock protein DnaK, and this inhibition is associated with cell
death since binding DnaK prevents chaperone-assisted protein
folding and inhibits its associated ATPase activity by impairing
the peptide-binding pocket of DnaK. The peptide permanently
closes the cavity and inhibits chaperone-assisted protein folding.
An important feature that is usually disregarded is the action of
antimicrobial peptides on eukaryotic organelles, which has proven
to be important in immunological resolution of fungal infections
and even in cancer cases. One such example is the activity displayed by some lactoferrin derived-peptides. For instance, human
lactoferricin hLF (1–11) binds to a receptor on the fungal cell membrane, accumulates in the cytoplasm and induces the loss of ATP
and the dissipation of the electrochemical gradient by targeting
energized mitochondria [87,226]. Helmerhorst et al. found that
the cationic peptide histatin-5 binds via Ssa1/2p surface protein,
enters the cell and causes a depletion in mitochondrial membrane
potential in Candida albicans [20]. Another example was found by
Mader et al. who described that LfcinB is able to permeabilize
the membrane without immediate disruption and mainly targets
mitochondria, inducing apoptosis in Jurkat T-leukemia cells by loss
of transmembrane potential and cytochrome c release [227].
Although the number of examples of AMPs intracellular action
is growing in the literature, the information available is still scarce
and does not yet allow a comprehensive and a clear perspective on
the mode of action of AMPs on these molecular and cellular targets.
7. Mechanisms of antimicrobial peptide resistance
In order to survive in a specific environment, pathogens have
developed several mechanisms of resistance to counteract the host
defense. This is important in the context of the present review with
regards to the role of antimicrobial peptides in innate immunity.
The current view states that such mechanisms are difficult to develop since the action of the majority of antimicrobial peptides is
quite unspecific, targeting mostly the negatively-charged membrane of pathogens, when compared with that of most classic antibiotics, which have more specific molecular targets [14]. In the
case of AMPs, the resistance would involve an overall organization
of the whole membrane beyond the capacity for the pathogen to
rearrange the cell wall and maintain its viability. However, a variety of studies have already documented the existence of some
resistance mechanisms to a given peptide through constitutive or
inducible mechanisms in a diverse and dynamic manner. These
bacterial strategies target some key steps, such as the antimicrobial peptide attachment, peptide insertion and membrane
permeability.
7.1. Membrane electrostatics and structural modifications
Part of the unspecific action of antimicrobial peptides arises
from their electrostatic interaction with the anionic phospholipids
of the membrane, which simultaneously confers its selectivity towards pathogens [24]. Therefore, one of the evolutionary mechanisms found by microorganisms consists on a membrane with a
reduced net negative charge without leading to significant modification of membrane fluidity or enzymatic activities (Fig. 9) [228].
Early studies by Dorrer and Teuber have shown that some pathogens such as Pseudomonas fluorescens adaptively change the electronegativity of the cytoplasmic membrane by decreasing the
content of anionic phospholipids (PG and CL) and increasing cationic ornithine–amide lipid composition when incubated in a poor
phosphate limited medium and that this induces a resistance to
cationic polymyxin B [229]. On the other hand, some bacteria modified the membrane by increasing the content of cationic constituents as observed on some S. aureus strains. They present an
unusual resistance to defensins and protegrins and such feature
has been linked to lysine modification of PG in the cytoplasmic
membrane, forming lysyl-PG, which appears to be under the mprF
operon control [230].
The teichoic acid polymers found in the cell walls of Gram-positive bacteria have strong anionic properties as all the phosphate
groups in their glycerolphosphate repeating units contain negative
charges. Streptococcus agalactiae and Listeria monocytogenes partially neutralize this negative charge by modifying teichoic acid
with D-alanine residues that bear positively charged amino groups
by the products of the dltABCD operon [231,232].
The presence of a capsular polysaccharide or glycocalyx shielding the pathogens from AMPs and other compounds is undoubtedly one of the most effective means of circumventing hostdeployed agents (Fig. 9) [24]. Capsular polysaccharides, formed
from the oligomerization of anionic monomer subunits, are typically negatively charged and therefore can bind antimicrobial peptides and partially shield the cytoplasmic membrane from peptide
action. P. aeruginosa exhibits an unusual propensity to infect tissues in which dysfunctional salt transport results in abnormal tissue physiology, abnormal phagocyte function, and increased local
ionic strength, as it can be seen in some pathologies like cystic
fibrosis [27]. A study by Friedrich et al. [233] suggests that the glycocalyx of alginic acid, an anionic and the main capsular exopolysaccharide constituent produced by virulent strains of P.
aeruginosa, is necessary to sequester cationic antimicrobial pep-
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
169
Fig. 9. Antimicrobial peptide mechanisms of resistance. The most common and specific mechanisms of resistance triggered by pathogens to decrease their susceptibility to
AMPs are described.
tides present in mucosal secretions, before they can achieve
threshold concentrations and reach the membrane, thereby conferring partial resistance to the membranolytic action of the peptides.
AMPs initially target and interact with microbial structures
exterior to the cytoplasmic membrane. Thus, microbial pathogens
have developed mechanisms by which these targets may be modified to resist peptide targeting and circumvent the resulting antimicrobial mechanisms. One of the most important mechanisms is
the particular susceptibility of Gram-negative bacteria to peptides
that specifically target LPS (Fig. 9). The alteration of the structure of
LPS through mechanisms such as lipid A acylation, 4-amino-4deoxy-L-arabinose and palmitate derivation of lipid A, aminoarabinose and myristoylation in E. coli have been reported as mechanisms of resistance employed by some Gram-negative bacteria.
For instance, resistance to polymyxins has been consistently linked
to the presence of LPS with a less anionic lipid A, to acylation with
an additional fatty acid, or to the presence of an O antigen [234].
Phosphorylcholine is a component of LPS in some bacteria and
may mimic PC present in mammalian cell membranes, which are
usually less susceptible to AMPs activity. Haemophilus influenzae
was shown to exhibit resistance to cationic antimicrobial peptide
exposure correlated with the relative amount of phosphorylcholine
present on its surface. Interestingly, this species resisted killing by
antimicrobial peptides only when choline was provided, which is
necessary for phosphorylcholine modification of LPS. Such feature
was evidenced by Lysenko et al. that showed that H. influenzae ex-
presses unusually high levels of phosphorylcholine and exhibited
decreased susceptibility to the antimicrobial peptide LL-37/
hCAP18 expressed in the upper respiratory tract [235]. Such envelope modifications are important to understand their impact as a
mechanism of resistance and represent a reliable strategy in microbial resistance to antimicrobial peptide-based immune mechanisms in medical pathology. This interesting aspect provides
further evidence of the specificity of the action of AMPs and the
ability of microorganisms to take evolutionary advantage of such
discrimination to avoid or, at least, decrease their susceptibility
to AMPs. In addition, these findings emphasize the difficulty to access biological relevant processes in vitro, since resistance in vivo is
dependent on strategies and specific microenvironments relevant
to immunoavoidance.
7.2. Membrane electrical potential
The main mechanism by which cationic antimicrobial peptides
may selectively target pathogen cells is the electrostatic adsorption
due to their higher negative electrochemical potential [4,24]. Indeed, the presence of a transmembrane electric potential can lower
the free energy of the insertion state relative to the surface adsorption state for peptides possessing a dipole moment, such as the
case of helical peptides. Although this might seem to be a biophysical parameter that would necessarily remain constant, a number
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of studies now suggest that some organisms may evade host-defense mechanisms by modulating this parameter.
Early studies with the type-I (highly cationic) and type II adefensins have demonstrated that the antimicrobial activity occurs
in a different way for the two peptides since the last class exhibits
maximum activity against highly energized cells [236]. Therefore,
the magnitude of the electronegative potential of the membrane
is important for the action of some antimicrobial peptides and it
may explain the different pattern of action of related peptides or
why some peptides are inactive against some strains. Some S. aureus strains with constitutive reductions in DW display reduced
susceptibilities to some but not all platelet microbicidal peptides
tested, as reported by Yeaman et al. [237].
However, there are some peptides that are insensitive to the
transmembrane potential, as observed for protegrin-like peptides,
which present no dipole moment [4238]. This aspect is particularly
important because this ability to subvert the membrane potential
and compromise the bacterial integrity and survival can be of major importance for the design and rational use on therapeutics
against a specific set of microorganisms.
Despite the fact that the cell metabolic status affects the transmembrane potential of the membrane, there are also other molecular targets (e.g. organelles) whose membrane energetics accounts
for the mechanism of action of some peptides. This antimicrobial
peptide resistance pattern has been observed in the eukaryotic
cells like C. albicans. Gyurko et al. [239] have shown that mutants
of C. albicans deficient in respiration resulting from mutations in
mitochondrial DNA were drastically more resistant to histatin-5
when compared with their parental counterpart and that the bactericidal activity was significantly lower against the parental strain
in the presence of inhibitors of respiration. These findings suggest
that the anticandidal activities of histatin-5 require a threshold level of cellular energetics involving mitochondrial ATP synthesis,
that is, energized mitochondria are required to exert its action.
hLF(1–11) was reported to target energized mitochondria in C. albicans by interacting with the inner mitochondrial membrane, thus
affecting mitochondrial output, e.g., generation of ATP and reactive
oxygen species (ROS) [87].
7.3. Sensor-transducer response systems
One of the most ancient mechanisms of resistance used by
microorganisms to face environmental challenges such as variations on ionic strength, pressure and temperature, is the existence
of very sophisticated sensor-transducer response systems through
which they alter their genetic and metabolic pattern until favorable conditions are reinstated. The finding that this kind of systems
also exists as a mechanism to resist antimicrobial peptide stress
was reported by Hong et al. [240], who demonstrated that sublethal concentrations of cecropin A prompted a pattern of genomic
response that is distinguishable from that of lethal concentrations
and distinct from global stress response systems such as heatshock or oxidative response, stressing the importance of genomic
induction in sensing and prompting a coordinate response to
AMPs’ exposure (Fig. 9).
Studies of PhoP/PhoQ regulon in Salmonella and most recently
the finding of YdeI/OmdA and its interaction with OmpD/NmpC
in the same genera are representative of this type of mechanisms.
Indeed, it was shown that Salmonella enterica presents at least
three different sensor-kinase systems to modify gene expression
in the presence of AMPs, namely PhoP-PhoQ, PmrA-PmrB, and
RcsB-RcsC-RcsD. In the first case, the regulon comprises a sensor
kinase, PhoQ, and a transcriptional activator, PhoP, which is encoded in response to environmental signals including changes in
extracellular magnesium or calcium concentrations, pH or even
after infection (phagocytosis). Besides, it encodes inducible surface
and secretor enzymes that modify lipopolysaccharide, lipid and
protein constituents of the outer membrane as well as proteases
that likely degrade certain AMPs. PhoP–PhoQ also regulates the
PmrA–PmrB two-component regulatory system, which is involved
in the resistance of P. aeruginosa to polymyxin B and cationic antimicrobial peptides from human neutrophils in low-Mg2+ conditions. This is due to amino acid substitutions in the regulatory
protein PmrA, which confers resistance to polymyxin by decreasing the overall negative charge of the lipopolysaccharide (LPS),
which in turn lowers the affinity for AMPs and other cationic peptide antibiotics [241].
The ydeI gene is regulated by the RcsB-RcsC-RcsD pathway and
encodes a 14-kDa predicted oligosaccharide/oligonucleotide binding-fold (OB-fold) protein important for polymyxin B resistance in
virulence in mice. ydeI is additionally regulated by the PhoP-PhoQ
and PmrA-PmrB sensor-kinase systems, which confer resistance to
cationic AMPs by modifying lipopolysaccharide (LPS). Two independent biochemical methods found that YdeI co-purifies with
OmpD/NmpC, a member of the trimeric b-barrel outer membrane
general porin family. Genetic analysis indicates that OmpD contributes to polymyxin B resistance, and both YdeI and OmpD are
important for resistance to cathelicidin antimicrobial peptide. YdeI
is localized in the periplasm, where it could interact with OmpD. A
second predicted periplasmic OB-fold protein, YgiW, and OmpF,
another general porin, also contribute to polymyxin B resistance
[242].
7.4. Proteases and peptidases
A growing number of microorganisms have presented a large
set of proteolytic agents that cleave host-derived AMPs (Fig. 9),
which is pointed out to become one of the most important mechanisms of resistance to AMPs and a great challenge in the future
when these peptides will be translated into therapeutics. As described above, PhoP/PhoQ-like systems regulate many metabolic
strategies employed by bacteria to resist antimicrobial peptides
and trigger coordinate responses in order to counteract the peptide
action. Among these, the PgtE protein was recently demonstrated
by Guina et al. to be an outer membrane endopeptidase in Salmonella that confers resistance to a-helical AMPs [243]. Thus, a variety of amphipathic and cationic antimicrobial peptides are
potential substrates of PgtE protease. However, PgtE protease does
not confer Salmonella increased resistance to antimicrobial peptides exhibiting amphipathic b-sheet conformation induced by
intramolecular disulfide bonds (e.g. defensins or protegrins). This
probably creates a steric hindrance that is protective against the
activity of PgtE protease. More recently, this protease was shown
to affect the complement system activity, which further extends
its activity on other immune branches in order to subvert the host
defense system [244]. Other proteases have also been implicated in
AMPs resistance of S. aureus and E. coli [245], namely the heatshock serine protease DegP that seems to confer a considerable
resistance to E. coli in the presence of lactoferricin B in vitro.
Due to limitations on peptide proteolytic stability, a number of
structural modifications have been proposed to overcome this
problem, leading to enhanced AMP biological lifetimes and therapeutic index. For this purpose, natural and even synthetic AMPs
with specific structures were submitted to structural modifications
conferring resistance to proteolytic degradation. One of the most
prominent examples derives from indolicidin, a small cationic peptide amide composed of 13 amino acids, five of which are tryptophan residues. The group of Ösapay et al. has built a synthetic
modified form of indolicidin, named X-indolicidin, which was produced by deprotonation of two indole side chains, yielding an
intrachain ditryptophan configuration in which the Trp-6 and
Trp-9 residues are covalently linked. This variant X-indolicidin
V. Teixeira et al. / Progress in Lipid Research 51 (2012) 149–177
was shown to be more resistant to trypsin and chymotrypsin
digestion, suggesting that ditryptophan stabilizes an indolicidin
conformer resistant to certain proteases [246]. More recently,
Fernández-Reyes et al. have produced various modified lysinetrimethylated analogues of a cecropin A-mellitin hybrid [CA(1–
7)M(2–9)], which exhibits an improved proteolytic (trypsin cleavage) stability profile, although protease shielding does not seem to
be the only contributing factor to the improved performance of the
constructed trimethylated analogues [184]. Overall, these studies
have provided evidence that natural and engineering-improved
synthetic AMPs can be a very useful tool to increase peptide stability in solution for future therapeutical purposes.
7.5. Efflux-dependent resistance mechanisms
Porin-mediated efflux is widely studied as a relevant resistance
mechanism to classical antibiotics in order to avoid intracellular
accumulation and the possibility to reach a threshold concentration after which they exert their antibacterial action. Recently, it
has also emerged as a mechanism by which microbial pathogens
may oppose antimicrobial peptide action (Fig. 9). Early studies by
Shafer et al. provided evidence of the existence of this kind of
mechanism. In Neisseria gonorrhoeae, the resistance to AMPs is
mediated in part by an energy-dependent efflux system termed
mtr [247]. Evidence indicates the MtrCDE complex mediates the efflux of antibiotics, dyes and detergents, suggesting that this mechanism also protects the pathogen against AMPs within and beyond
the genitourinary tract.
A similar mechanism was also observed in Yersinia and the efflux of AMPs appears to be mediated by a temperature-regulated
system involving the efflux pump/potassium antiporter complex
formed by the RosA and RosB proteins [248]. Importantly, the
RosA/RosB gene regulon appears to be specific, since it is inducible
in the presence of AMPs and in vivo, and it has been suggested to
enhance the survival of the organism in the acidic and antimicrobial peptide-rich phagolysosome in polymorphonuclear leukocytes. The presence of efflux systems was also found in Grampositive bacteria and fungal pathogens. For example, the plasmid
encoded gene qacA mediates staphylococcal-resistance to multiple
organic cations, like AMPs and polyamines, via a proton motive
force-dependent efflux pump. A study by Kupferwasser et al. demonstrated that an S. aureus plasmid containing qacA provides resistance to antimicrobial peptide tPMP-1 [249]. Notably, the
expression of qacA did not appear to impart cross-resistance to
other structurally distinct cationic peptides, including a defensin,
protamine, or the lantibiotics pep5 or nisin. More recently, Bina
et al. have provided evidence that RND efflux systems in Vibrio
cholerae were responsible for resistance to AMPs [250].
The existence of such efflux pumps illustrates the importance of
these systems, not only to block the passage of classical antibiotics
but also to provide a means of protection by which antimicrobial
peptides are not allowed to reach lethal concentrations and to trigger their biological effects.
8. Development of antimicrobial peptides for clinical
applications: a novel advance in therapeutics
There is a very alarming increase in pathogenic microorganisms
that are multi-resistant to commercially available antibiotics, some
of them acquiring resistance in very short periods of time (decades) [6,8,10,14,49,135,140,228]. Vancomycin resistant enterococci (VRE) and methicillin resistant S. aureus (MRSA) are
occurring with increasing frequency and represent a serious threat
in hospitals worldwide. In the last 25 years, MRSA incidence has
raised more than 10-fold and VRE has witnessed similar increases
171
within the last 15 years [73]. This has been attributed to the excessive (and often inappropriate) use of antibiotics in human and animal health care for the treatment and prevention of infections, as
well as, to the increased use of immunosuppressive chemotherapeutic regimes. Indeed, the last statistics of the World Health Organization have highlighted a very important and serious global
health problem that has pressed the scientific community to develop novel antibiotics with improved properties and mechanisms of
action. Nowadays, the situation presents itself very disquieting because only few antibiotics that provide successful response to
infection are available; during the period of 1983–1987 only 16
new compounds appeared, and that number decreased to only 7
between 1998 and 2002 [166]. For some of them, studies have already documented mechanisms of resistance in a few microorganisms [166].
To face this situation, considerable progress has been made in
the field of microbial genomics which has provided a more comprehensive understanding of the mechanisms to countermeasure
the action of host immune response. The development of molecular biology tools brought forth a number of strategies for creating
antibiotics with better therapeutic index, such as metalloantibiotics, the new generation of macrolide antibiotics, or even the development of adhesin-based vaccines, which induce antibody
responses at the mucosal surface that prevent attachment and
abrogate colonization, and therefore block the primary stages of
infection [251]. Another emerging strategy is based on host antimicrobial peptides that can provide a first line of defense against
a wide range of pathogenic microorganisms. For the therapeutics
purpose, a new array of natural and synthetic antibiotics derived
from combinatorial libraries has emerged as a reliable alternative
to overcome the ultimate problem of worldwide antibiotic
resistance.
The potential therapeutic applications for AMPs or derived products may arise from the amplification of antimicrobial therapy
when conjugated with themselves or with conventional antibiotics
[25,28,252]. Such synergism has already been applied for classic
antibiotics in order to improve the effectiveness of the treatment,
particularly for multi-resistant strains. For instance, the use of sulfonamides and diaminopiridines (trimethoprim) allow the inhibition of tetrahydrofolic acid necessary for nucleic acids and protein
synthesis. An obvious application of these peptides comes into view
given their propensity to permeabilize target microbial membranes
and form transient or permanent pores, which may facilitate conventional agents in overcoming surface resistance mechanisms
such as reduced uptake or enhanced efflux through microbial
pumps. On the other hand, some synergistic interactions between
peptides themselves have proved to be efficient in overcoming bacterial resistance, e.g. in that imposed by the LPS leaflet of Gram-negative bacteria, like the combined use of closely related temporins A/
B and L [25]. A study by Giacometti et al. has demonstrated the potential of combinational therapy, such as the verified synergy of
cecropin A-melittin hybrid peptides with b-lactams [253]. Moreover, some peptides act only at the intracellular level and may also
be used in conjugation with classical antibiotics, like buforin II
(DNA) and fluoroquinolones (DNA gyrase or topoisomerase IV),
amplifying its antimicrobial action on a specific or similar target.
Current knowledge regarding the relationship between peptide
structure and function as well as the mechanism of action has been
applied in the rational design of antimicrobial peptide variants
with a broad spectrum of activity. However, in spite of its large potential as therapeutic agents in many areas [6,7,10,13,26,254], the
development of such agents has proved to be difficult due to
important aspects concerning its manufacture, pharmacokinetics,
site of action delivery and specific properties concerning the class
of microorganisms they are active against. Furthermore, their stability in the infection context still remains an issue, since most
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studies reported are mainly performed in vitro without taking into
account the very specific physiological environment of infection in
which antimicrobial peptides act on.
8.1. A promising future for antimicrobial peptides as reliable
antibiotics?
Presently, several studies have pointed out the exceptional potential of antimicrobial peptides as universal and encouraging antibiotics for the treatment of many diseases, such as inflammatory
and auto-immune diseases. However, their applicability in the
market has proceeded at a slow rate and less than a few hundred
peptides have been evaluated to date for clinical potential, a number that is quite small compared to the figures for many antibiotic
development programs, particularly classical antibiotics. Despite
the promising attributes and recent successes in demonstrating
the efficacy of antimicrobial peptides already in animal disease
models, there are considerable challenges in the clinical application of candidate peptide therapies. Some of these obstacles were
already summarized, namely the cost of peptide synthesis since
it is an expensive and time-consuming process [44]. Besides, some
difficulties have already been mentioned, e.g. the susceptibility of
peptides to proteolytic degradation in vivo and its consequences
on the pharmacokinetics and interaction with pathogen, the lack
of consistent information regarding the potential toxicity of
amphipathic peptides to host cells, etc. [44]. Nevertheless, probably the most important feature relies on lack of consistent knowledge on adequate antimicrobial activity of natural and synthetic
AMPs under specific physiological conditions.
There has been limited success with those antimicrobial peptides that have entered into clinical trials because no peptide has
yet been approved by Food and Drug Administration (FDA) for clinical applications. The most advanced AMPs are currently two indolicidin-based antimicrobial peptides, MBI-226 and MX-594AN
that have been developed by Migenix (Vancouver, British Columbia, Canada) for the treatment of catheter-related infections and
acne, respectively. MBI-226 (Omiganan) showed promising results
for secondary end points in phase IIIa trials, resulting in a 40%
reduction of catheter colonization and a 50% decrease in tunnel
infections, although its primary end point of a reduced rate of
infections was not achieved. Another known peptide with clinical
trials currently underway is IB-367 (Iseganan), a protegrin-derived
peptide targeted for oral mucositis in cancer patients (Intrabiotics
Pharmaceuticals, Inc. Mountain View, CA). Based on the pig peptide
protegrins, IB-367 possesses broad-spectrum activity in vitro
against bacteria and fungi; however, in clinical trials it failed to
prevent or reduce oral mucositis compared with a placebo. Unfortunately, increased rates of pneumonia and mortality were observed in patients receiving the peptide, which eventually halted
the clinical trials.
AM-Pharma Holding BV have announced that they have completed phase I clinical trials with the 11-mer peptide from the Nterminus of human lactoferrin, hLF (1–11). It was shown that this
peptide was effective in animal models of osteomyelitis and other
bacterial infections. AM-Pharma is also targeting the prevention of
infections in patients undergoing stem cell, especially allogeneic
transplantation, with common high post-operative rates of severe
infection and mortality. The treatment of patients with hematological malignancies by means of hematopoietic stem cell transplantation (HSCT) has been recently considered [255]. Similarly,
DermaGen AB (Pergamum) has received promising results from a
clinical Phase I/IIa study of a novel antimicrobial peptide (DPK060) treatment for atopic dermatitis. The peptide has shown a
broad spectrum of activity and exhibited both bactericidal and fungicidal activities. In the clinical trial, the company’s candidate drug
clearly reached its primary objective, demonstrating a significant
reduction of total microbes in eczemas compared to placebo. In
addition to good safety and tolerability performance, the candidate
drug also showed a trend towards improved eczema status (the research was performed in the website http://www.dermagen.se/ at
20th February 2011).
The 21 kDa recombinant N-terminal fragment of Bactericidal/
permeability-increasing protein (BPI), rBPI21, has recently completed Phase III clinical trials of parenteral use for meningococcaemia and it was shown to be also a promising agent against
septic shock [256].
Although none of the peptides in the examples described above
have obtained the approval from FDA for clinical use, the accumulation of evidence so far has enhanced the optimism on the conceptualization of antimicrobial peptides as encouraging therapeutic
agents in the prevention and management of diverse clinical
conditions.
9. Concluding remarks and future directions
The study of antimicrobial cationic host defense peptides in the
past few years has shown their importance as therapeutic weapons. Many studies have provided consistent evidence that these
peptides have such potential as they display activity towards a
wide spectrum of infectious bacterial, viral, fungal, and parasitic
pathogens and even cancer cells. In addition, many reports have
claimed diverse immunomodulatory activities of such host defense
peptides, providing an additional incentive when considering these
peptides as a new class of therapeutic agents.
The structure–activity studies have identified common traits
among their properties, namely their cationic nature and amphipathic structure, which in part justify their action and selectivity
towards pathogens. Differences in biochemical and biophysical
properties of pathogens versus host cells provide an additional basis for their selective toxicity. Moreover, many mechanisms of action have provided insight into how the peptides target the
membrane either by promoting its permeabilization, altering its
topology or by crossing it in order to gain access to intracellular
targets, such as nucleic acids, enzymes or even eukaryotic organelles. However, some microorganisms were successful in circumventing this host defense mechanism and the understanding of
the balance between the action of the peptide and the resistance
mechanisms triggered by the pathogen will provide further evidence for the evolutionary maintenance of this ancient branch of
immunity. These insights may provide novel strategies or templates from which new peptides can be designed to improve the
prevention or treatment of infections.
Overall, the comprehensive knowledge of the elusive aspects of
their mechanism of action and the improvement of their properties
will definitely contribute to consolidate their potential and the
claims of these peptides as the next-generation antibiotics to prevail over the alarming worldwide antibiotic crisis.
Acknowledgments
The authors acknowledge Fundação para a Ciência e Tecnologia
(FCT-Portugal) for financial support to CIQ(UP), Unidade de Investigacão 81 and to REQUIMTE (Rede de Química e Tecnologia). Dr. S.
Connell, University of Leeds, UK, is gratefully acknowledged for
assistance during the reviewing process of this manuscript.
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