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Vol. 55, Issue 1, 27-55, March 2003
Division of Infectious Diseases, Harbor-University of California Los Angeles (UCLA) Medical Center; St. John's Cardiovascular Research Center, Harbor-UCLA Research and Education Institute, Torrance, California; and UCLA School of Medicine, Los Angeles, California
Abstract
I. Introduction
II. Mechanisms of Antimicrobial Peptide Target Specificity and Selective Toxicity
A. Comparative Membrane Architecture and Energy
1. Membrane Composition, Hydrophobicity, and Charge.
2. Membrane Asymmetry.
3. Microbial Ligands for Antimicrobial Peptides.
4. Transmembrane Potential.
B. Antimicrobial Peptide Structure-Based Selective Toxicity
C. In Vivo Preferential Affinity for Microorganisms versus Mammalian Cells
D. Antimicrobial Peptide Localization to Restrict Exposure of Vulnerable Host Tissues
E. Themes in Target Affinity and Selective Toxicity of Antimicrobial Peptides
III. Mechanisms of Antimicrobial Peptide Action
A. Structural Determinants of Antimicrobial Peptide Activity
1. Conformation ().
2. Charge (Q).
3. Amphipathicity (A) and Hydrophobic Moment (MH).
4. Hydrophobicity (H).
5. Polar Angle ().
B. Common Themes in Structural Determinants of Antimicrobial Peptides
C. Initial Peptide Interactions with Membrane Targets
1. Electrostatic Interactions.
2. Receptor-Mediated Membrane Interactions.
D. Events Subsequent to Initial Membrane Binding
1. Threshold Concentration.
2. Conformational Phase Transition.
3. Self-Association and Multimerization.
4. The Barrel-Stave Mechanism.
5. The Toroid Pore or Wormhole Mechanism.
6. The Carpet Mechanism.
E. Mechanisms of Cell Death
1. Membrane Dysfunction.
2. Inhibition of Extracellular Biopolymer Synthesis.
3. Inhibition of Intracellular Functions.
F. Synergy among Antimicrobial Peptides
G. Themes in Mechanisms of Action of Antimicrobial Peptides
IV. Mechanisms of Antimicrobial Peptide Resistance
A. Constitutive and Inducible Resistance
B. Constitutive (Passive) Resistance
1. Inherent Mechanisms of Resistance to Antimicrobial Peptides.
2. Altered Membrane Energetics.
3. Electrostatic Shielding.
4. Niche-Specific Resistance.
C. Inducible (Adaptive) Resistance
1. Coordinate Microbial Responses to Antimicrobial Peptide Stress.
2. Adaptive Mechanisms of Resistance to Antimicrobial Peptides.
3. Proteases and Peptidases.
4. Extracellular Structural Modifications.
5. Resistance Modifications of the Cytoplasmic Membrane.
6. Efflux-Dependent Resistance Mechanisms.
7. Modification of Intracellular Targets.
V. Prospectus: Therapeutic Targets of Antimicrobial Peptides
A. Reconstitution or Potentiation of Conventional Antibiotic Efficacy
B. Unique and Specific Microbial Targets
C. Targeting Strategic Microbial Response Pathways
D. Engineering New Anti-Infectives Based on Peptide Structure and Function
VI. Summary
Acknowledgments
References
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Abstract |
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Antimicrobial peptides have been isolated and characterized from tissues and organisms representing virtually every kingdom and phylum, ranging from prokaryotes to humans. Yet, recurrent structural and functional themes in mechanisms of action and resistance are observed among peptides of widely diverse source and composition. Biochemical distinctions among the peptides themselves, target versus host cells, and the microenvironments in which these counterparts convene, likely provide for varying degrees of selective toxicity among diverse antimicrobial peptide types. Moreover, many antimicrobial peptides employ sophisticated and dynamic mechanisms of action to effect rapid and potent activities consistent with their likely roles in antimicrobial host defense. In balance, successful microbial pathogens have evolved multifaceted and effective countermeasures to avoid exposure to and subvert mechanisms of antimicrobial peptides. A clearer recognition of these opposing themes will significantly advance our understanding of how antimicrobial peptides function in defense against infection. Furthermore, this understanding may provide new models and strategies for developing novel antimicrobial agents, that may also augment immunity, restore potency or amplify the mechanisms of conventional antibiotics, and minimize antimicrobial resistance mechanisms among pathogens. From these perspectives, the intention of this review is to illustrate the contemporary structural and functional themes among mechanisms of antimicrobial peptide action and resistance.
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I. Introduction |
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Human subtlety will never devise
an invention more beautiful, more simple, or more direct than does
Nature
because in her inventions, nothing is
lacking
and nothing is superfluous...
Leonardo
da Vinci
Antimicrobial peptides represent ancient host defense effector molecules present in organisms across the evolutionary spectrum. Fundamental differences exist between microbial and mammalian cells that may represent targets for antimicrobial peptides. Among these, significant distinctions include membrane composition and architecture, energetics such as transmembrane potential and polarization, and structural features including sterols, lipopolysaccharide and peptidoglycan. Disparities such as these appear to translate to varying degrees of selective toxicity among distinct antimicrobial peptides, relating to peptide and target cell properties, as well as the biological settings in which the two interact.
Although hundreds of antimicrobial peptides have now been characterized as having widely diverse sequences, these peptides have been classified into relatively few conformational paradigms. Therefore, it may be argued that a high degree of degeneracy exists within the conformation code governing structure-activity relationships among antimicrobial peptides. Many of these molecules, within and beyond conformational classes, exhibit mechanisms of action that are highly complex and non-identical. Moreover, new evidence points to targets that lie interior to the cytoplasmic membrane as being important in antimicrobial mechanisms of these peptides. Thus, the assumption that antimicrobial peptides are uniform and indiscriminant membrane detergents is obsolete. Recognition of the sophisticated and thematic structure-activity relationships underlying distinct mechanisms of action among antimicrobial peptides will facilitate a more complete appreciation of their likely multiple roles in antimicrobial host defense.
Antimicrobial peptides have evolved as integral components of strategic and carefully regulated mechanisms of immunity to infection. However, microbial pathogens have not been passive to this evolutionary procession. Rather, prokaryotic and eukaryotic pathogens devote a considerable portion of their genomes to expressing complex and coordinately regulated countermeasures designed to subvert antimicrobial peptide targeting and mechanisms of action.
A clearer understanding of these parallel systems will advance two important, yet elusive goals. First, an awareness of the mechanisms employed by antimicrobial peptides will significantly improve our understanding of how these molecules act to defend against infection. Second, insights into these strategies will facilitate new opportunities and approaches to discover and develop pharmacologic agents that enhance or optimize immune mechanisms and suppress the ability of pathogens to subvert these mechanisms.
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II. Mechanisms of Antimicrobial Peptide Target Specificity and Selective Toxicity |
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Polypeptides that exert antimicrobial activity have been isolated
from essentially every tissue in which they have been sought. This
intriguing observation has contributed to divergent interpretations regarding the potential functions of many of these peptides in antimicrobial host defense: peptides that may have little or no relevance in antimicrobial host defense can be demonstrated to inhibit
or kill microorganisms in defined or austere conditions in
vitro
versus
complementary peptides of varying structures, tissue
sources, antimicrobial mechanisms, potencies, and/or spectra function
in consort to provide optimal host defense against infection.
A pivotal consideration in this regard is the degree to which an antimicrobial peptide distinguishes between microbial and host cells in settings of potential toxicity. Evidence continues to mount in support of the concept that inherent structures or functions of microbial versus host cells contribute to selective antimicrobial discretion of some peptides. Alternatively, antimicrobial peptide access to potentially vulnerable host tissues may be limited by localization and/or or highly regulated expression. The following discussion highlights these themes as supported by recent studies.
A. Comparative Membrane Architecture and Energy
All biological membranes are in effect composed of a fluid mosaic of proteins and phospholipids. In some organisms, sterols and glycerides also contribute to the surface topology and biochemical architecture of biomembranes. Yet, fundamental differences exist between microbial and host membranes that represent potentially selective targets for antimicrobial peptides. Moreover, central to the potential pharmacologic application of antimicrobial peptides is the degree to which they differentiate, or may be engineered to differentiate, between microbial targets and normal host cells.
1. Membrane Composition, Hydrophobicity, and
Charge.
The elementary component of essentially all
biomembranes is the phospholipid bilayer. By definition, such bilayers
are amphipathic, having both hydrophobic and hydrophilic domains.
However, based on composition and influenced by cell energetics,
biomembranes of prokaryotic versus eukaryotic cells differ
significantly. For example, phosphatidylcholine
(PC1) and
phosphatidylethanolamine (PE) normally have no net charge. Moreover,
sphingomyelin (SM), a close analog of PC containing a palmitoyl
residue, is also neutrally charged. In many membrane systems, the
amounts of PC and SM are inversely related. Sterols such as cholesterol
and ergesterol, found in eukaryotic but rarely in prokaryotic
membranes, are also generally neutral. In contrast, hydroxylated
phospholipids phosphatidylglycerol (PG), cardiolipin (CL; effectively a
dimer of PG), and phosphatidylserine (PS), sustain a net negative
charge. From these perspectives, it follows that the net charge of a
biomembrane is based largely upon its phospholipid stoichiometry and
architecture (Fig. 1). Cell membranes composed predominantly of PG, CL, or PS tend to be highly
electronegative; such compositions are found in many bacterial
pathogens. On the contrary, bilayers enriched in the zwitterionic
phospholipids PE, PC, or SP
commonly found in mammalian cytoplasmic
membranes
are generally neutral in net charge. These characteristic
membrane charge properties may also be compounded by differences in
electrochemical gradients of prokaryotic versus eukaryotic cells (see
below). Sterols within membranes may further differentiate mammalian
and fungal cells from prokaryotes (Tytler et al., 1995
; see below) as
potential targets for antimicrobial peptides. Moreover, it is
intriguing to note that peptides with primarily antifungal activity,
such as many of those isolated from plants, tend to be relatively rich
in polar neutral amino acids, suggesting a unique structure-activity
relationship (Hancock and Chapple, 1999
).
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2. Membrane Asymmetry.
The compositional and architectural
characteristics of prokaryotic and mammalian membranes are neither
static nor symmetric. Distinctions between microbial and mammalian
cells as targets for antimicrobial peptides include the configuration
of phospholipid bilayer components. Recent evidence indicates that the
distribution of phospholipids within cytoplasmic membranes is highly
asymmetric. For example, only 2% of the total PE content in bovine
erythrocytes is oriented toward the outer membrane leaflet
(Florin-Christensen et al., 2001
). Differences among asymmetric
distribution, compositional stoichiometry, and saturation of
phospholipid bilayers also significantly influence membrane phase
transition and fluidity (Bayer et al., 2000
; Verkleij and Post, 2000
;
McIntosh et al., 2001
). These differences may extend to the inner and
outer cytoplasmic membrane leaflets, or those of the outer membrane of
Gram-negative bacteria or enveloped viral pathogens (which generally
exhibit properties of their corresponding host cells). Accordingly, the
charge and amphipathicity of the inner and outer membrane leaflets also
vary considerably (Fig. 1). For example, in human erythrocytes, most
glycosylated lipids (glycolipids), PC, PS, and SM are positioned on the
exoplasmic membrane leaflet. Alternatively, when present, neutral or
anionic phospholipids are typically localized on the cytoplasmic
leaflet. Thus, differences in electronegativity resulting from leaflet asymmetry likely provide a further dimension influencing the relative affinity of antimicrobial peptides for biomembranes.
3. Microbial Ligands for Antimicrobial Peptides.
The fact
that D-and L-amino acid versions of
antimicrobial peptides generally show little selectivity in binding
suggests that stereospecific receptors are not present on target
microbial cells (see Section III.). However, certain
structures may be crucial for selective affinity of peptides for
microbial pathogens. For example, Teuber and Bader (1976)
demonstrated
that radioactive mono-N-acetyl-14C or
native polymyxin B absorbed to isolated cytoplasmic and outer membranes
of Salmonella typhimurium within 60 s of exposure.
Moreover, polymyxin B exhibited sigmoidal binding kinetics, suggesting
saturation of cytoplasmic and outer membranes, with approximately 30 and 60 nmol of peptide bound per milligram of membrane, respectively. Importantly, based on the stoichiometry of LPS, PG, CL, and PE in the
membranes, these investigators calculated that the theoretical binding
capacities of polymyxin B were almost identical to the binding
properties if LPS, PG, and CL were modeled to function as specific
receptors for this peptide. This robust concordance between theoretical
and experimental approximations of polymyxin B binding capacities,
along with parallel binding and killing kinetics, argues that membrane
anionic constituents themselves function as pseudoreceptors for this
cationic peptide. Thus, electronegative ligands (e.g., PG, CL, LPS)
likely provide impetus for the initial interaction between cationic
peptides and certain pathogens (also see Sections III. and
IV.).
4. Transmembrane Potential.
Another fundamental difference
between microbial and mammalian cells can be found in the charge
separation between the extracellular and intracellular aspects of the
cytoplasmic membrane. This electrochemical gradient, resulting from
differing extents and rates of proton flux across the membrane, is
termed the transmembrane potential (
). The difference in 
between certain microorganisms and host cells may provide a means of
selective targeting of microorganisms by cationic antimicrobial
peptides. For example, normal mammalian cells exhibit a 
ranging
from
90 to
110 mV. However, bacterial pathogens in logarithmic
phase growth commonly exhibit 
of
130 to
150 mV. Such
significant differences in membrane electrochemistry have been
hypothesized as additional parameters guiding selective toxicity of
antimicrobial peptides, through a mechanism that has been termed
self-promoted uptake (Hancock, 1997
[also see Section III.]).
B. Antimicrobial Peptide Structure-Based Selective Toxicity
Many antimicrobial peptides are believed to exist in relatively
unstructured or extended conformations prior to interaction with target
cells. Others are held in specific conformations by intramolecular
bonds. Upon binding to pathogen membranes, peptides may undergo
significant conformational dynamics to helical or other structures that
effect antimicrobial activity (see below). There is mounting evidence
supportive of the concept that inherent and/or dynamic conformations
among antimicrobial peptides impact their selective toxicity.
Furthermore, peptides may have distinct antimicrobial versus host
cytotoxic conformers and/or undergo conformational phase transition,
self-association, or oligomerization within target pathogen
but not
host cell
membranes, as a means for selective toxicity (also see
Section III.).
Tam et al. (2000)
recently examined the influence of conformation on
membranolytic selectivity of antimicrobial peptides. In these studies,
antimicrobial activity and human cell cytotoxicity were assessed in
conformationally restricted cyclic and noncyclic analogs of
protegrin-1, an 18-amino acid cationic peptide exhibiting broad-spectrum antimicrobial activity. Antimicrobial assays in relatively low- and high-salt conditions revealed cyclic protegrins exert differential antimicrobial profiles against Gram-positive and
Gram-negative bacteria, fungi, and human immunodeficiency virus-1. As
compared to protegrin-1, the most constrained analog (a
cyclic-tricystine protegrin termed ccPG-3) displayed a 10-fold decrease
in hemolytic propensity to human cells and up to a 30-fold increased
membranolytic selectivity against specific target pathogens. However,
an analogous cyclic protegrin lacking a disulfide bond, or a cyclic
mimic of protegrin-1 with one disulfide bond, exhibited antimicrobial
and cytotoxic profiles equivalent to protegrin-1. Interestingly,
circular dichroism showed that even cyclized protegrins stabilized by
disulfide bonds display
-strand structure in water/trifluoroethanol or phosphate-buffered environments. These findings suggest that conformational dynamics subsequent to initial binding contribute to
antimicrobial peptide activity in selected membrane environments.
Related studies by Unger et al. (2001)
provide additional insights into
the structural basis for selective toxicity of antimicrobial peptides.
These investigators examined the interaction of linear versus cyclic
counterparts of melittin and magainin analogs (peptides displaying
non-identical selective toxicity toward mammalian cells) with membrane
models in vitro. As compared with linear versions, the cyclized
peptides were less efficient in initial binding to phospholipid
membranes. However, at normalized bound concentrations, linear and
cyclic analogs retained equivalent potencies to induce membrane
permeabilization. When bound to phospholipid membranes, these cyclized
peptides reverted to ~75% of the helical structure of their linear
analogs. Even more importantly, the cyclic melittin analog exhibited
increased antibacterial activity, with reduced hemolytic propensity,
whereas the cyclic magainin exhibited opposite biological functions.
These observations were interpreted to suggest that conformation
influences initial interactions of peptides with membranes, as well as
ensuing disruptive actions on target membranes. Collectively, these
findings emphasize the potential role for conformational dynamics
subsequent to initial binding interactions in selective toxicity of
antimicrobial peptides. In addition, the above studies lend insights
into the potential for engineered conformational constraints to further
dissociate antimicrobial activity from host cytotoxicity.
Recent studies by Oren and colleagues (1999)
also shed light on the
relationship between quaternary structure and selective toxicity among
antimicrobial peptides. Human cathelicidin LL-37 is an antimicrobial
peptide cytotoxic to both bacterial and mammalian cells. This peptide
exists in equilibrium as monomers and oligomers in solution at low
concentration but appears to undergo self-association within
zwitterionic (mammalian-like) and electronegative (bacterial-like) artificial phospholipid membranes in vitro. Interestingly, in these
models, LL-37 effected a detergent-like or carpet mechanism (see
Section III.) in disrupting both membrane types, suggesting a structure-induced membrane perturbation in either setting. Supportive of this interpretation was the finding that the peptide conformed to a
predominantly
-helical configuration oriented parallel with the
surface of zwitterionic membranes. Thus, a propensity to assume an
invariable helical conformation and multimerize within lipid membranes
of differing compositions may reduce the ability of antimicrobial
peptides to exert selective toxicity against microorganisms versus host cells.
Experiments focusing on cationic antimicrobial peptides of varying
structures and origins extend this theme of peptide interaction with
model membranes of distinct phospholipid compositions (Zhang et al.,
2001
). In these studies, test peptides were uniformly cationic but
varied in conformation, including
-helical,
-sheet, extended, and
cyclic motifs. Regardless of conformation, all test peptides interacted
with and penetrated into lipid monolayers composed of anionic PG, as
measured by the release of preloaded calcein dye. In comparison, only
-helical and extended peptides interacted with monolayers composed
of more zwitterionic PC, albeit to a lesser extent than with the
anionic lipids. Interestingly, a
-sheet peptide induced rapid
phospholipid translocation (movement of lipid from the inner facet to
the outer facet of the membrane) at concentrations less than required
for membrane permeabilization. Similarly, Kol et al. (2001)
demonstrated that the ability of peptides of comparable conformation to
induce phospholipid translocation was greater for those containing
proportionately more lysine or histidine residues, compared with
tryptophan. From these examples, it appears that antimicrobial peptides
not only interact with biomembranes of specific composition and
asymmetry but may also promote remodeling of these membrane properties
within target cells.
C. In Vivo Preferential Affinity for Microorganisms versus Mammalian Cells
Recently, Welling et al. (2001)
tested the hypothesis that
cationic antimicrobial peptides may discriminate between microbial cells and host tissues in vivo. Studies evaluated whether such peptides
specifically accumulate in sites of infection, compared with sterile
inflammatory lesions, due to preferential avidity for microorganisms.
Peptide affinity and specificity for pathogens in vivo was assessed by
intravenous injection of 99mTc-labeled synthetic
derivatives of human ubiquicidin or lactoferrin into animals
experimentally infected with Staphylococcus aureus, Klebsiella
pneumoniae, or C. albicans. As controls, sterile
inflammatory sites were induced by the introduction of heat-killed
microorganisms or purified LPS into thigh muscle. Labeled human
defensin, human polyclonal IgG, and ciprofloxacin were examined as
comparative agents. The 99mTc-labeled peptides
and defensin accumulated at a significantly higher rate and to a
greater extent in bacteria- and C. albicans-infected lesions
in mice and rabbits, compared with non-infected but inflamed tissues.
These data were interpreted to indicate that the peptides distinguish
between microorganisms and host tissues, and in doing so, accumulate at
sites of infection in vivo.
In related studies, this same group examined the potential
pharmacologic utility of antimicrobial peptides to localize to sites of
infection (Welling et al., 2000
). Biodistribution scintigraphy suggested that the 99mTc-labeled peptides were
rapidly removed from the circulation by renal excretion. However,
despite this rapid clearance, the radiolabeled peptides efficiently
discriminated between infected and non-infected tissue, with up to
5-fold increased binding to target versus nontarget tissues within
1 h in rabbits. Collectively, these results indicate that
antimicrobial peptides rapidly localize and accumulate at sites of
infection, likely due to preferential affinity for peptides to
associate with target microorganism surfaces rather than non-infected tissues.
D. Antimicrobial Peptide Localization to Restrict Exposure of Vulnerable Host Tissues
Selective toxicity among antimicrobial peptides
or the lack
thereof
involves complex interactions between peptide and target cells
as indicated above (also see Section III.). However, it is
also likely that these peptides may be rendered less harmful to the
host simply through strategic localization or expression that minimizes
their interaction with potentially vulnerable host tissues. For
example, many antimicrobial peptides known in vertebrates are secreted
onto relatively inert epithelial surfaces, such as the tracheal,
lingual, or intestinal mucosa of mammals, or the skin of amphibians. In
addition, this localization
along with rapidly inducible
expression
places antimicrobial peptides in key positions to intervene
at perhaps the earliest of opportunities to prevent microbial
colonization or infection.
A similar, albeit more complex mechanism likely contributes to
selective toxicity of antimicrobial peptides found in granules of
phagocytic leukocytes. The fundamental antimicrobial functions of
professional phagocytes include internalization of pathogens (phagocytosis), subjecting them to the harsh microenvironment of the
phagolysosome. Neutrophils, monocytes, and macrophages of various
mammalian species contain among the most potent antimicrobial peptides
known
defensins (see below). However, defensins may also exhibit among
the least selective toxicity of any host defense peptides, often
exerting membrane permeabilizing and other harmful effects on
microorganisms and mammalian cells alike. Phagocytes normally
interiorize and expose pathogens to lethal concentrations of these
peptides within the maturing phagolysosome, rather than degranulating
these potentially injurious components into the extracellular milieu.
Within the restricted confines of the phagolysosome, defensins and
other antimicrobial peptides are present in very high relative
concentrations, where they may act harshly and synergistically with one
another, along with oxidative killing mechanisms. In this way,
defensins may be constrained to granules of mammalian phagocytes to
minimize their potential for host cytotoxicity. Moreover, Shafer et al.
(1986)
and Yeaman (1997)
have suggested that antimicrobial activities
of defensins and platelet microbicidal proteins are potentiated in
mildly acidic conditions, such as those found in the maturing phagolysosome.
Beyond the scope of this review, some antimicrobial peptides may also
perform other important functions contributing significantly to
antimicrobial host defense, including interfering with host cell
receptor access to pathogens, recruitment of leukocytes to sites of
infection, as well as potentiate their antimicrobial activities
(Yeaman, 1997
; Yeaman and Bayer, 1999
; Cole et al., 2001
; Tang et al.,
2002
). For example, Zhang et al. (2002)
have recently found that CD8+
cytotoxic T lymphocytes elaborate
-defensins 1, 2, and 3 in
contributing to host defense against human immunodeficiency virus-1.
Conceivably, these peptides act directly to alter or damage the human
immunodeficiency virus virion, or indirectly by interfering with
receptor targeting, eventual uncoating or replication, and/or enhanced
intracellular destruction. Thus, the extracellular secretion of
antimicrobial peptides at concentrations or in settings that do not
result in host toxicity may play important roles in immunity. Through
such strategies, the antimicrobial functions of peptides and phagocytes
may be mutually amplified, while minimizing the potential for
concomitant host cell toxicity.
E. Themes in Target Affinity and Selective Toxicity of Antimicrobial Peptides
Antimicrobial peptides display highly variable abilities to discriminate between microbial targets versus normal host cells. The governing rules for differences in selective toxicity among such peptides remain to be fully elucidated. However, several themes relating to the structural and functional properties of peptides as they relate to their potential targets include: 1) compositional divergence conveying differential electrostatic affinities for microbial versus host cells; 2) conformational dynamics that promote peptide activation or self-association in microbial membranes, but not others; 3) target cell energetics that accelerate or retard peptide interactions with target versus host membranes, respectively; and 4) limitations in the access of antimicrobial peptides with poor selective toxicity to potentially vulnerable host tissues.
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III. Mechanisms of Antimicrobial Peptide Action |
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A striking feature among antimicrobial peptides as a group is their overall conservation of structure and charge themes across diverse phyla. Whether synthesized non-ribosomally with D- and L-amino acids, or from genetically encoded messenger RNA, antimicrobial peptides form amphipathic structures and are often cationic at physiological pH. As outlined above, amphipathicity and net charge are characteristics understandably conserved among many antimicrobial peptides. Furthermore, charge affinity is likely an important means conferring selectivity to antimicrobial peptides. In the context of these paradigms, the following discussion highlights current concepts relating to the molecular basis of antimicrobial peptide mechanisms of action.
A. Structural Determinants of Antimicrobial Peptide Activity
An essential requirement for any antimicrobial host defense or
therapeutic agent is that it has a selective toxicity for the microbial
target relative to the host. Ideally, such compounds have affinity for
one or more microbial determinants that are easily accessible, common
to a broad spectrum of microbes, and relatively immutable. Nature has
apparently yielded a class of molecules that meets these constraints in
the evolution of antimicrobial peptides. Antimicrobial peptides
initially target microbial cells, and thus fulfill criteria outlined
above for identifying molecular determinants of pathogens that are
accessible and broadly conserved. As a group, antimicrobial peptides
have amphipathic features that mirror phospholipids, thus allowing them
to interact with and exploit vulnerabilities inherent in essential
microbial structures such as cell membranes. In the following section,
several aspects of antimicrobial peptide structure relevant to
antimicrobial activity and selective toxicity are considered
thematically. Specifically, structural parameters such as conformation
(
), charge (Q), hydrophobicity (H),
hydrophobic moment (MH), amphipathicity
(A), and polar angle (
), are examined in some detail. It
is important to note that these molecular determinants are
interdependent, and therefore, modification of one parameter often
leads to compensatory alterations in others. This holistic view of
peptide structure-activity relationship relates to each of these key
properties influencing mechanisms of action of antimicrobial peptides
(Fig. 2). The following discussion is
considered in this context.
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1. Conformation (
).
Although antimicrobial peptides differ
widely in sequence and source, several themes in their
three-dimensional topology appear predominant, and peptides have been
categorized accordingly. The two largest groups are the
-helical and
-sheet peptides, whereas the majority of remaining peptides can be
classified as those that are enriched in one or more amino acid
residues [e.g., proline-arginine or tryptophan-rich (Hancock, 1997
)].
Other classification schemes are based upon peptide source (e.g.,
neutrophils or other leukocytes), precursor (e.g., cathelicidin,
derivatives of cathelin), extent of intramolecular bonds (e.g.,
cysteine array or cyclization in peptides), or other parameters.
-helical antimicrobial peptides are abundant in the
extracellular fluids of insects and frogs and frequently exist as extended or unstructured conformers in solution. Many of these peptides
only become helical upon interaction with amphipathic phospholipid
membranes. The
-sheet peptides represent a highly diverse group of
molecules at the level of primary structure. Despite such differences,
these peptides share common features, including amphipathic
composition, with distinct hydrophilic and hydrophobic surfaces. Less
is known about the structures adopted by the proline-arginine-rich and
tryptophan-rich peptides. However, examples of conformations distinct
from prototypic
or
structures have also been identified. For
example, certain proline-arginine-rich peptides, and tryptophan-rich
indolicidin, conform to polyproline helical type II structures (Boman
et al., 19932. Charge (Q).
Many of the antimicrobial peptides
characterized to date display a net positive charge, ranging from +2 to
+9, and may contain highly defined cationic domain(s). Cationicity is
undoubtedly important for the initial electrostatic attraction of
antimicrobial peptides to negatively charged phospholipid membranes of
bacteria and other microorganisms (see Fig. 2), and mutual
electroaffinity likely confers selective antimicrobial targeting
relative to host tissues. The fact that bacterial membranes are rich in
the acidic phospholipids PG, PS, and CL confers their overall negative
charge. Moreover, LPS and teichoic or teichuronic acids of
Gram-negative and Gram-positive bacteria, impart additional negative
charge to the surfaces of these respective organisms. Target cell

is typically up to 50% greater in prokaryotes than in most
mammalian cells. Thus, it has been proposed that such a chemiosmotic
potential may act in an electrophoretic manner to concentrate
positively charged peptides on microbial surfaces (also see
Section II.).
3. Amphipathicity (A) and Hydrophobic Moment
(MH).
Nearly all antimicrobial peptides form
amphipathic structures upon interaction with target membranes.
Amphipathicity can be achieved via a multitude of protein
conformations; however, one of the simplest and perhaps most elegant is
the amphipathic helix. The amphipathic
-helix has a periodicity of
three to four residues and is optimal for interaction with amphipathic
biomembranes. While the extent of amphipathic helicity influences
peptide activity against negatively charged membranes, it may have an
even more pronounced effect in rendering peptides hemolytic against
zwitterionic or neutral membranes. Thus, a high degree of helicity
and/or amphipathicity yielding a segregated hydrophobic domain, is
correlated with increased toxicity toward cells composed of neutral
phospholipids (Dathe and Wieprecht, 1999
).
-helical content in governing antimicrobial
peptide activity. In a similar study with magainin analogs, the
relative role of hydrophobic moment on membrane binding and
permeabilization was examined (Wieprecht et al., 1997
-sheet antimicrobial peptides are also amphipathic. This
amphipathicity is characterized by a variable number of
-strands, with relatively few or no helical domains, organized to create both
polar and non-polar surfaces. These
-strands are frequently anti-parallel, and are stabilized by a series of disulfide bonds, with
as many as eight cysteines in some peptides [e.g., plant defensins
(Sitaram and Nagaraj, 1999
-defensins). The conformational rigidity observed in
many
-sheet antimicrobial peptides in aqueous solution may also
promote multimerization, limiting exposure of hydrophobic facets to
hydrophilic environments. This configuration contrasts with that of
higher degrees of freedom among the
-helical peptides in similar
solutions. A number of
-sheet peptides have been shown to exist as
dimers in aqueous solution, including the human defensin HNP-3, as
determined by X-ray crystallography. The proposed mechanisms by which
HNP-3 and other defensins or antimicrobial peptides perturb target
membranes involve amphipathicity and hydrophobic moment. For example,
insertion of the hydrophobic peptide face into the lipid bilayer, and
association of the charged arginine side chains with polar lipid head
groups, relies upon three-dimensional separation of hydrophobic and
charge. Once associated with the membrane, the amphipathic nature of
-sheet peptides likely enables their formation of transmembrane
channels. Several models have been proposed to explain the exact
mechanism by which these peptides may form and traverse the channel
(see below); however, the precise conformation adopted by such peptides
in the hydrophobic membrane environment remains to be determined.
However, as in
-helical peptides, it is now apparent that highly
segregated amphipathicity strongly influences
-sheet peptide
disruption of neutral membranes. These findings have led to studies
demonstrating that residue-specific modifications in hydrophobicity
enhance selectivity among cationic peptides. For example, studies using
synthetic derivatives of gramicidin S have revealed that reductions in
hydrophobicity significantly increase selective toxicity against
microorganisms, with approximately 10,000-fold increase in the
estimated therapeutic index of such peptides (Kondejewski et al.,
19994. Hydrophobicity (H). Peptide hydrophobicity, defined as the percentage of hydrophobic residues within a peptide, is approximately 50% for most antimicrobial peptides. Hydrophobicity is an essential feature for antimicrobial peptide membrane interactions, as it governs the extent to which a peptide can partition into the lipid bilayer. Although hydrophobicity is required for effective membrane permeabilization, increasing levels of hydrophobicity are strongly correlated with mammalian cell toxicity and loss of antimicrobial specificity. Therefore, many antimicrobial peptides are moderately hydrophobic, such that they optimize activity against microbial cell membranes.
The relationship between peptide hydrophobicity and membrane permeabilization was examined in an interesting study by Wieprect and coworkers (1997)
1, compared with a
Kapp of 7,400 M
1 for the least hydrophobic peptide. This
difference in binding affinity exemplifies the extent to which
hydrophobicity influences membrane binding and permeabilization.
Furthermore, differences in membrane perturbation were achieved with
relatively minor changes in net peptide hydrophobicity, indicating the
relative significance of hydrophobic features on these interactions.
5. Polar Angle (
).
Polar angle is a measurement of the
relative proportion of polar versus nonpolar facets of a peptide
conformed to an amphipathic helix. For example, in a hypothetical
-helical peptide, in which one facet is exclusively composed of
hydrophobic residues and the other solely composed of charged residues,
the polar angle would be 180o. A reduced
segregation between these domains or an increased hydrophobic
proportion of the helix would proportionately reduce the polar angle.
In numerous studies of native and synthetic peptides, a smaller polar
angle (and therefore a greater hydrophobic surface) is associated with
increased capacity to permeabilize membranes (Dathe et al., 1997
;
Wieprecht et al., 1997
; Uematsu and Matsuzaki, 2000
). The polar angle
has also been shown to correlate with the overall stability and
half-life of peptide-induced membrane pores. In a recent study by
Uematsu Matsuzaki (2000)
, the effects of polar angle on membrane
permeabilization and pore formation were compared. Two model peptides
with polar angles of 100o and
180o showed functional similarities with native
-helical antimicrobial peptides in forming amphipathic helices,
selective targeting of negatively charged membranes, and creating
toroid or lipid-containing pores (see below). Results from these
studies indicated that peptides with smaller polar angles induced
greater membrane permeabilization, translocation, and pore formation
rates (Uematsu and Matsuzaki, 2000
). However, although the rate of pore
formation was greater for peptides with smaller polar angles, the rate
of pore collapse was higher. These results suggest that peptides with
smaller polar angles achieve less stable pore structures compared with
peptides having larger polar angles. Greater stability of pores formed by the latter peptides could result from larger charged surfaces, and/or more peptide molecules per channel. These concepts are consistent with those observed in native peptides, showing that peptide
PGLa (
= 100o) is more easily
translocated than magainin 2 (
=
180o; Matsuzaki, 1998
). These results indicate
that hydrophobic and hydrophilic stereogeometries in antimicrobial
peptides play significant roles influencing the process and
consequences of membrane interaction and disruption.
B. Common Themes in Structural Determinants of Antimicrobial Peptides
The existence of a broad diversity in antimicrobial peptide
sequences and structures underscores the reality that no single antimicrobial peptide sequence has emerged as singularly effective against all pathogens in all settings. Moreover, Nature may have sustained such diversity as a strategy to prevent or delay evolution of
microbial resistance to antimicrobial peptides. Nonetheless, a
circumspect analysis of structural parameters associated with differential antimicrobial activity versus host cell toxicity among
peptides reveals several themes. Conservation in secondary structure
may be key to three-dimensional configurations facilitating antimicrobial activity of distinct peptides. Generally, extremes of
certain features, such as charge, amphipathicity, hydrophobic moment,
or polar angle may disfavor peptide antimicrobial activity and
selective toxicity. A minimum threshold of charge, perhaps as low as
+2, appears necessary for antimicrobial peptide selectivity toward
microorganisms. This property is likely important for a number of
reasons: 1) initial electrostatic attraction to negatively charged
microbial membranes; 2) potential to displace membrane-associated cations; and 3) a strong trans-negative 
of many microorganisms may facilitate cationic peptide transitions in orientation on the
membrane, entry into the polar membrane core, and/or translocate peptides from exoplasmic to cytoplasmic membrane facets. A moderate level of amphipathicity, independent of or in context of polarization of charge, appears to be more favorable in these respects. Segregation of charge and hydrophobicity paralleling the inherent amphipathicity of
the target lipid bilayer may also promote peptide integration into and
disruption of the microbial membrane. A third theme is that selectivity
among membrane-lytic peptides may rely on moderate degrees of
hydrophobicity, as excessive hydrophobicity may increase selectivity
for zwitterionic membranes, increasing mammalian cytotoxicity. Thus,
selective antimicrobial activity results from a delicate balance among
three-dimensional hydrophobic and electrostatic interactions between an
antimicrobial peptide and its target (Dathe et al., 1996
, 1997
).
C. Initial Peptide Interactions with Membrane Targets
As outlined above, antimicrobial peptides are inherently structured to target and interact with biomembranes. More importantly, the initial interaction with the target surface significantly influences subsequent peptide dynamics and membrane-disrupting effects. As discussed below, the basis of this initial interaction integrates biochemical as well as biophysical aspects of the peptide and the target membrane.
1. Electrostatic Interactions.
There is widespread acceptance
that the initial mechanism by which antimicrobial peptides target
microbes occurs via an electrostatic interaction. For example, cationic
antimicrobial peptides and negatively charged lipid membranes of
bacteria provide for a mutual and vigorous attraction. This supposition
has been borne out by numerous studies in which a strong correlation
between peptide charge and membrane binding activity has been
demonstrated (Bessalle et al., 1992
; Vaz Gomes et al., 1993
; Matsuzaki
et al., 1997
; Dathe et al., 2001
). This view is also supported by the
conservation of positive charge within many antimicrobial peptides
isolated from organisms across the evolutionary spectrum. The facts
that electrostatic forces are active over relatively long molecular distances and that lysine and arginine interactions with phosphate groups in lipid bilayers are particularly strong (Mavri and Vogel, 1996
) likely contributes to the initial attraction and
membrane-targeting step of many antimicrobial peptides.

) of most bacterial
membranes likely compounds the biophysical forces driving the
interaction between cationic peptides and target pathogens. Studies
supporting this theory have shown that a membrane potential as low as
20 mV increases the binding constant of the cationic peptide
tachyplesin 200-fold (Matsuzaki, 1997
is required
for nisin activity (Breukink and Kruijff, 1999
relative to that of mammalian cells may be a significant factor contributing to charge-mediated peptide selectivity.
2. Receptor-Mediated Membrane Interactions.
Early studies
using all D-enantiomers of native and model peptides
demonstrated equivalent antimicrobial activities of D- and
L-isoforms. Thus, the prevailing dogma supported a
non-receptor type interaction for antimicrobial peptides with most
pathogen membranes (Bessalle et al., 1990
; Wade et al., 1990
). Since
then, several studies suggest there may be important exceptions to this generalization. Perhaps the most well characterized example is that of
nisin, a small, cyclic, non-ribosomally produced peptide that has been
used in the food industry for several decades. Nisin exhibits
antimicrobial activity in the nanomolar range and specifically binds to
bacterial lipid II, a membrane bound component involved in
peptidoglycan synthesis. When exposed to nisin, vesicles containing lipid II exhibit an ~1000-fold increase in fluorescein leakage compared with vesicles lacking lipid II (Breukink and Kruijff, 1999
).
It has been proposed that this specificity in nisin activity relates to
a specific receptor-like interaction with lipid II and the proximity it
confers to this peptide relative to the microorganism. Notably, lipid
II is believed integral to peptidoglycan synthesis, and nisin is
considerably more active against peptidoglycan-rich Gram-positive
organisms than Gram-negative organisms (Breukink and Kruijff, 1999
).
Likewise, Brotz et al. (1998)
have recently demonstrated that the
lantibiotic mersacidin interferes with transglycosylation and
peptidoglycan synthesis in Gram-positive bacteria by direct targeting
of lipid II. In addition, tachyplesin has been demonstrated to have a
specific affinity for LPS (Hirakura et al., 2002
). Moreover, a number
of studies have now shown non-equivalent activities for native
all-L peptides, versus their all-D enantiomers
(Fehlbaum et al., 1996
; Vunnam et al., 1997
). For example, in
intriguing studies using PR-39, a proline- and arginine-rich peptide of
porcine origin, the all-D enantiomer showed 1000-fold
differences in species-specific activity against bacterial organisms
(Vunnam et al., 1997
). These studies suggest receptor-type interactions
may be important for some peptides in targeting specific epitopes on
the microbial surface.
D. Events Subsequent to Initial Membrane Binding
Perhaps one of the more controversial issues within the field
revolves around the fate of antimicrobial peptides following their
initial interaction with biological membranes. The mechanism(s) by
which peptides may permeabilize and traverse microbial membranes are
not entirely clear and likely vary for different peptides. Uncertainty
stems in part from technical difficulties associated with
first-principle determinations or molecular modeling of peptide-lipid interactions. Attempts to crystallize antimicrobial peptides within a
native lipid environment have been largely unsuccessful, and other
methods of structure determination have various limitations. Conventional CD is an excellent tool for determining peptide secondary structures, such as
-helices. However it necessitates the use of
optically clear solutions and provides little information as to the
relative size of conformer regions or their location (Blondelle et al.,
1999
; Sitaram and Nagaraj, 1999
). Similarly, infrared spectroscopy
(e.g., FTIR) is an important tool particularly well suited to study
-sheet peptide conformations, but also has technical limitations.
Fluorescence spectroscopy is convenient, and its high level of
sensitivity allows for a small sample size. However, data are often
highly dependent on the solvent or membrane mimetic system used.
Nuclear magnetic resonance (NMR) studies also offer a powerful means to
obtain structural information at the single residue or domain levels
but can be limited by relatively slow rates of molecular reorientation.
Recently developed methods include reverse phase-high pressure liquid
chromatography-based and surface plasmon resonance; however,
these techniques are limited to the extent they represent protein-whole
microorganism interactions. Therefore, at the present time, the most
comprehensive assessments of peptide-lipid structure often come from a
combinatorial approach wherein a variety of methodologies are employed,
and the results are considered collectively.
The following discussion considers events subsequent to peptide-target
binding that may significantly influence peptide mechanisms of action
and/or selective toxicity. Functional themes are reviewed using
examples of prevailing models for these processes, which are proposed
to occur via specific and nonspecific mechanisms. These data should be
interpreted in the context of the specific biophysical methods
employed; the particular conditions and assays utilized for assessing
peptide antimicrobial activities are beyond the scope of this review.
However, it should be understood that the potencies, spectra, and/or
mechanisms of antimicrobial peptide action could be highly dependent
upon conditions of testing. For example, media pH, osmotic and ionic
strength, temperature, and viscosity (e.g., in peptide diffusion
assays)
individually and in combination
may significantly influence
peptide antimicrobial activities.
1. Threshold Concentration.
At some point following initial
membrane binding, peptides enter a second stage of membrane
interaction, frequently referred to as the threshold concentration. In
this phase, peptides begin to enter and traverse the lipid bilayer via
a number of possible mechanisms, ultimately extending their
antimicrobial action to targets interior to the cell membrane.
Conceptually, the threshold concentration necessary to drive such
events results from accumulation of peptides on the target surface.
Parameters that likely influence this threshold include peptide
concentration, propensity to self-assemble or multimerize, as well as
phospholipid membrane composition, fluidity, and head group size (Yang
et al., 2000
). Additionally, it is important to note that individual
peptide-membrane interactions can vary such that one type of peptide
may act via multiple mechanisms dependent on conformation dynamics of
the peptide or target membrane remodeling.

of many bacterial membranes. It is postulated
that membrane potential oriented in this way electrophoretically draws
cationic peptides into the nonpolar membrane environment, effectively
reducing the energy barrier for pore formation. For example, nisin,
which requires a considerable 
for activity, has been shown to
lose its voltage dependence when an N-terminal lysine is replaced with
leucine (Breukink and Kruijff, 19992. Conformational Phase Transition.
A key event occurring
after membrane binding is the process of peptide structural or
conformational phase transition, most well documented for
-helical
antimicrobial peptides. Numerous studies using various biophysical
methodologies show that many antimicrobial peptides are disordered in
aqueous environments, exhibiting extended or random coil conformations
in this setting (Bello et al., 1982
; Dathe and Wieprecht, 1999
).
However, many such peptides rapidly assume highly structured
amphipathic
-helical conformation upon interaction with phospholipid
bilayers or in membrane mimetic solvents. Interestingly, a number of
peptides require a negatively charged bilayer to undergo this
transition. For example, the frog skin peptide PGLa, disordered when
exposed to membranes composed of the zwitterionic PC and SM membranes, adopts a helical structure in the presence of membranes composed of PG
and PE (Latal et al., 1997
). Similarly, magainins only undergo a
helical transition when interacting with anionic membranes as demonstated by CD, (Matsuzaki et al., 1989
, 1991
),
vibrational/Raman-FTIR (Williams et al., 1990
; Hirsh et al., 1996
) and
NMR (Bechinger et al., 1993
; Hirsh et al., 1996
). Examination of
cecropin analogs revealed that the extent of
-helical conformation
is proportionately dependent on the amount of negatively charged
phospholipid within the model membrane (Wang et al., 1998
). One
mechanism by which such a relationship may promote peptide order relies
on the inherent phospholipid packing within the bilayer. For example,
interactions of the peptide with the phospholipid head groups may
promote an optimal periodicity within the charged residues of the
peptide, promoting folding of the
-helix. As discussed above, this
change in conformation would also likely alter peptide hydrophobic
moment and polar angle. Another potentially important aspect of the
conformational phase transition is that it may prevent indiscriminant
membranolytic activity until the peptide identifies an appropriate
target surface. Thus, a lack of bioactive structure at nontarget sites
may be an important means by which antimicrobial peptides minimize
host-cell toxicity.
-sheet antimicrobial peptides are typically much more
ordered in aqueous solution and membrane environments, due to
constraints imposed by disulfide bonds or cyclization of the peptide
backbone. For example, the secondary structure of tachyplesin, a cyclic
-sheet peptide that contains a type-II
-turn, is largely
unchanged as the peptide moves from an aqueous environment to that of a
membrane-mimetic (Oishi et al., 1997
-sheet peptides are likely relatively stable upon
interaction with target cell membranes. However, it is possible that
the quaternary peptide structures proposed for some
-sheet peptides
in aqueous solution are dissociated upon interaction with the membrane
surface. In contrast to
-helical peptides, the potential
monomerization of such peptides could also facilitate antimicrobial
mechanisms or selective toxicity.
3. Self-Association and Multimerization. Considerable evidence suggests that antimicrobial peptides may self-associate or multimerize following initial interactions with target membranes. These peptide-peptide and peptide-lipid interactions within membranes likely create complex structures associated with specific antimicrobial peptide mechanisms of action. However, the potential for a peptide to form quaternary structures is fundamentally related to the inherent composition and conformation of the peptide in its monomeric form. For example, peptides with well defined hydrophobic and hydrophilic domains may efficiently orient these facets toward respective membrane constituents, or corresponding domains in adjacent peptides. Such orientations may facilitate amphipathic peptides partitioning more deeply into the hydrophobic membrane core than would likely occur otherwise. Assembly of peptide complexes in this way may create the existence of transmembrane pores or channels, which meay be selective or non-selective. For example, peptide structures may assume configurations in which hydrophobic surfaces are aligned toward the membrane such that a hydrophilic channel is lined only by polar and charged facets of individual peptides.
A number of models for antimicrobial peptide membrane permeabilization have been proposed. Given the variability in microbial membrane ultrastructure, a given peptide may act via different mechanisms in distinct membrane environments. The models described below here have been largely derived from results examining activities of individual peptides or analogs against artificial membrane systems. It should be pointed out that there is no universal consensus among investigators in this regard. Therefore, the following models are compared with illustrate advances in proposed mechanisms of antimicrobial peptide action.4. The Barrel-Stave Mechanism.
The term barrel-stave
describes the overall topology of a membrane channel formed in this
mechanism of membrane permeabilization. In this model, a variable
number of channel-forming peptides are positioned in a
"barrel-like" ring around an aqueous pore. The "stave" term
refers to individual transmembrane spokes within this barrel, which may
be composed of individual peptides or peptide complexes. In this
mechanism, the hydrophobic surfaces of
-helical or
-sheet
peptides face outward, toward the acyl chains of the membrane, whereas
the hydrophilic surfaces form the pore lining (Ehrenstein and Lecar,
1977
; Breukink and Kruijff, 1999
). The initial step in barrel-stave
pore formation involves peptide binding at the membrane surface, most
likely as monomers. Upon 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 hydrophobicity of the membrane outer
leaflet. Positioning of the positively charged amino acids near the
phospholipid head groups facilitates this process. When bound peptide
reaches a threshold concentration, peptide monomers self-aggregate and
insert deeper into the hydrophobic membrane core. Aggregation allows
for a minimal exposure of the peptide hydrophilic residues to the
hydrophobic membrane interior, as the peptides adopt a transmembrane
configuration. Continued accretion of peptide monomers results in
further expansion of the membrane pore. Upon phospholipid translocation
or relaxation of the pore, peptides are transported to the inner
membrane leaflet aspect due to the concentration gradient of
surface-bound peptide, as well as trans-negative 
. An example of
such a mechanism of action has been proposed for alamethicin (Sansom,
1991
; Beven et al., 1999
; Yang et al., 2001
). Alamethicin-induced
membrane conductance has been measured to proceed as a pattern of
multistate conductance levels. This finding suggests the existence of
pores with openings of various diameters, corresponding to channels composed of four or more transmembrane-spanning peptides. However, there remain relatively few peptides for which there is compelling evidence of a barrel-stave mechanism, despite this model having been
proposed more than a decade ago. More recent studies often support the
toroid pore model (see below; Yang et al., 2000
, 2001
). These newer
data may reflect refinements in methodology and offer a clearer
understanding of biophysical properties of transmembrane pores or
channels that may incorporate lipid and peptide moieties.
5. The Toroid Pore or Wormhole Mechanism.
One of the most
well characterized peptide-membrane interactions is that of the toroid
pore. A primary difference between the toroid pore and barrel-stave
models is that in the former, lipids are intercalated with peptide in
the transmembrane channel. Therefore, this structure has been referred
to as a supramolecular complex and represents a membrane-spanning pore
lined with polar peptide surfaces as well as phospholipid head groups.
The toroid pore model has been deduced principally from experiments
using
-helical peptides, including magainins and PGLa. In this
model, peptides in the extracellular environment take on an
-helical structure as they interact with the charged and hydrophobic bacterial membrane. Helices are initially oriented parallel to the membrane surface as confirmed by NMR, fluorescence quenching, and CD (Hara et
al., 2001
). The hydrophobic residues of the bound peptides displace the
polar head groups, creating a breach in the hydrophobic region and
inducing positive curvature strain in the membrane (Hara et al., 2001
).
The introduction of strain and thinning further destabilizes the
membrane surface integrity, making it more vulnerable to ensuing
peptide interactions. At a threshold peptide-to-lipid ratio (e.g.,
estimated to be 1:30 for magainin), peptides orient perpendicular to
the membrane. At this point,