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Ann. N.Y. Acad. Sci.

ISSN 0077-8923
ANNALS OF THE NEW YORK ACADEMY OF SCI ENCES
Issue: Antimicrobial Therapeutics Reviews
Peptide antimicrobials: cell wall as a bacterial target
Nannette Y. Yount
1,2
and Michael R. Yeaman
1,2,3,4
1
Division of Infectious Diseases, Los Angeles County,
2
Los Angeles Biomedical Research Institute,
3
Division of Molecular
Medicine, Los Angeles County, Harbor-UCLA Medical Center, Torrance, California.
4
David Geffen School of Medicine, UCLA,
Los Angeles, California
Address for correspondence: Michael R. Yeaman, Divisions of Molecular Medicine and Infectious Diseases, Harbor-UCLA
Medical Center, Los Angeles Biomedical Research Institute, 1124 West Carson Street, Torrance, CA 90502.
MRYeaman@ucla.edu
Endogenous host defense peptides (HDPs) are among the most ancient immune mediators, constituting a rst line
of defense against invading pathogens across the evolutionary continuum. Generally, HDPs are small (<10 kDa),
cationic, andamphipathic polypeptides, oftenbroadly classiedbasedonstructure. Ineukaryotes, major HDPclasses
include disulde-stabilized(e.g., defensins), and-helical or extended(e.g., cathelicidins) peptides. Prokaryote HDPs
are generally referred to as bacteriocins, colicins, or lantibiotics, many of which undergo extensive posttranslational
modications. One target for prokaryotic and eukaryotic HDPs is the bacterial cell wall, an essential structural
feature conserved among broad classes of bacteria. A primary building block of the cell wall is peptidoglycan, a
macromolecular complex that arises through a series of reactions including membrane translocation, extracellular
anchoring, and side chain cross-linking. Each of these steps represents a potential target for HDP inhibition, leading
to bacteriostatic or bactericidal outcomes. Thus, understanding the relationships between HDPs and cell wall targets
may shed light on new peptide antimicrobial agents and strategies to meet the daunting challenge of antibiotic
resistance.
Keywords: host defense peptides; peptides; defensins; antibiotic resistance
Introduction
Historically, host defense peptides (HDPs) have
beendemonstrated to inactivate prokaryotic cells by
targeting a number of essential physical or metabolic
processes at extracellular, plasma membrane, and/or
intracellular sites. Overall, HDPs may be orga-
nized into three basic categories regarding target:
(1) plasma membraneactive peptides are thought
to act in a multistage process where they electro-
statically bind to a membrane surface, aggregate
to form superstructures, and disrupt membrane
integrity; (2) a second group of peptides act on
intracellular targets to inhibit transcriptional, trans-
lational or other processes; and (3) cell wallactive
peptides target precursors, mechanisms, and/or es-
sential intermediates in peptidoglycan, lipopolysac-
charide (LPS) or other biosynthetic pathways
interfering with functional cell wall synthesis and
ensuing bacterial replication. Interestingly, most cell
wallactive HDPs characterized to date have been
isolated from prokaryotic sources, although certain
prokaryotic HDPs are knowntotarget plasma mem-
brane and/or intracellular sites. In contrast, the ma-
jority of HDPs from eukaryotic sources have tradi-
tionally been thought to act via mechanisms that
target bacterial membrane and energetics systems,
and cognate mitochondrial processes in eukaryotic
pathogens. In this review, prototypic peptides tar-
geting the bacterial cell wall are considered with an
emphasis ondata fromrecent andcomparative stud-
ies illustrating targets and mechanisms reported to
date.
Basic structure and function of microbial
cell walls
Microbial cell walls are essential structural com-
ponents that serve to shape the cell itself and
mediate functional imperatives, such as reproduc-
tive division, extracellular localization of adhesins,
invasions, or other virulence factors, and prevent
doi: 10.1111/nyas.12005
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Peptide antimicrobials Yount & Yeaman
cell lysis due to high internal osmotic pressure. The
cell wall further serves as a structural scaffold to
anchor many membrane components. In bacteria,
the cell wall differentiates Gram-positive and Gram-
negative organisms. In Gram-positive bacteria, the
cell wall comprises multiple layers of peptiogylcan
interspersedwithteichoic acidandlipoteichoic acid.
In Gramnegatives, an outer plasma membrane con-
taining lipopolysaccharide encases a thinner layer
of peptidoglycan tethered to the outer leaet of the
cytoplasmic membrane. In fungi, the cell wall com-
prises a complex network of glucans, chitin, and
other biopolymers.
The prokaryotic cell wall and its synthetic
machinery comprise targets for specic classes
of antimicrobial agents, including the clinically
important -lactams (e.g., penicillins, carbapen-
ems, monobactams) and glycopeptides (e.g.,
vancomycin, daptomycin, and teichoplanins). In
particular, one of the most commonly targeted
components of the cell wall is lipid II, which is
an essential building block of peptiodoglycan
that is highly conserved among broad classes of
bacteria. Lipid II comprises a membrane-bound
bactoprenol carrier (C55-P) linked via pyrophos-
phate to the disaccharide N-acetyl-muramyl-
pentapeptide-N-acetyl-glucosamine (MurNAc-
ppGlcNAc) precursor (Fig. 1). The synthesis of lipid
II is initiated on the cytoplasmic side of the plasma
membrane wherein translocases MraY and MurG
catalyze the addition of UDP-activated sugars
(UDP-N-acetyl muramic acid and UDP-N-acetyl
glucosamine) to the bactoprenol carrier to produce
the lipidI andlipidII intermediates, respectively. Af-
ter the addition of UDP-N-acetyl glucosamine, the
lipid II membrane-bound complex is translocated
to the extracytosolic side of the membrane where it
is exposed to the extracellular milieu. Involvement
of lipid III (undecaprenolpyrophosphate-N-
acetyl-glucosamine) and lipid IV (undecaprenol-
pyrophosphate-N-acetyl-glucosamine-N-acetyl-
mannosamine) precursors are integral to the
biosynthesis of wall teichoic acid and lipoteichoic
acid.
1
The nal steps in peptidoglycan synthe-
sis involve polymerization of the pentapeptide
bridge cross-linkage by glycosyl-transferases and
transpeptidases (also known as penicillin-binding
proteins) on the extracellular facet of the mem-
brane. Excellent reviews of this process can be
found elsewhere.
2,3
Within the eld of HDP mechanisms of action
there are several criteria by which peptides are typ-
ically classied in relation to putative targets. Evi-
dence tosupport a membrane-permeabilizing mode
of action would include membrane depolarization,
loss of cytoplasmic components (e.g., ions [K
+
],
adenosine nucleotide triphosphate (ATP), or other
metabolites), and rapid lysis. By comparison, cell
walltargeting compounds typically do not cause
extensive depolarizationor loss of cytoplasmic com-
ponents and often take up to one cell cycle to
cause microbial cell death (for example, by disrupt-
ing macromolecular synthesis necessary for repli-
cation). One particularly diagnostic measure of cell
wallactive compounds is a measured accumulation
of peptidoglycan precursors. Evidence from various
studies frequently supports one or the other of these
mechanisms, except in cases where the peptide in-
activates cells using both of these processes.
Prokaryotic peptides targeting prokaryotic cell
walls
One of the earliest documented evolutionary de-
fense mechanisms developed by unicellular organ-
isms was the generation of antimicrobial metabo-
lites or macromolecules as a means of survival
or gaining a competitive advantage in the face of
other microbes. The spectrum of these compounds
is vast, and includes both chemically synthesized
and gene-encoded antimicrobial substances. Of
the ribosomally synthesized HDPs, many undergo
signicant posttranslational processing to yield
chemically complex compounds often containing
modied amino acids. Although Gram-negative
bacteria frequently produce relatively large HDPs
(often >50 residues; described in detail else-
where
4,5
), Gram-positive organisms generally pro-
duce a broad range of smaller peptides (2040
amino acids) that share many chemical features
(e.g., amphipathicity, cationicity) with HDPs from
eukaryotes. Further, many of these HDPs are syn-
thesized as precursors, with anionic pro-peptide se-
quences that may serve to attenuate these micro-
bicidal agents until they are compartmentalized or
deployed.
6
One of the most well characterized groups of
Gram-positive and ribosomally synthesized antimi-
crobial peptides are the class I bacteriocins known
as lantibiotics. After transcription, these peptides
undergo extensive posttranslational processing to
128 Ann. N.Y. Acad. Sci. 1277 (2013) 127138
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Yount & Yeaman Peptide antimicrobials
Figure 1. Basic lipid II/peptidoglycan biosynthesis pathway. Lipid II is an essential building block of peptiodoglycan that is
highly conserved among broad classes of bacteria. Lipid II comprises a membrane-bound bactoprenol carrier (C55-P) linked
via pyrophosphate to the disaccharide MurNAc-ppGlcNAc (NAM, NAG) precursor. The synthesis of lipid II is initiated on the
cytoplasmic side of the plasma membrane, wherein translocases MraY and MurG catalyze the addition of UDP-activated sugars
(UDP-NAMand UDP-NAG) to a bactoprenol carrier to produce the lipid I and lipid II intermediates, respectively. After the addition
of UDP-NAG, the lipid II membrane-bound complex is translocated (yellow arrow) to the extracytosolic leaet of the membrane,
where it is exposed to the extracellular milieu. The nal steps in peptidoglycan synthesis involve polymerization of the pentapeptide
bridge cross-linkage by glycosyl-transferases and transpeptidases (PBPs) on the extracellular facet of the membrane. Peptidoglycan
is integrated with the plasma membrane and decorated with teichoic acid (TA), lipoteichoic acid (LTA), and/or a variety of virulence
factors such as adhesins or invasins. After the transfer of a single peptidoglycan subunit to the growing peptidoglycan chain, the
bactoprenol carrier is translocated to the cytosolic side of the membrane such that the process can begin anew.
generate a diverse array of molecules with modi-
ed residues. Among these residues are dehydrated
serine (dehydroalanine) andthreonine (dehydrobu-
tyrine), which undergo Michael-addition reactions
using cystine sulfhydral (-SH) groups as substrates
to catalyze reactions yielding lanthionine and -
methyllantionine rings.
7,8
Lantibiotics are broadly classied as type A, type
B, or heteromeric two-peptide agents. Type A lan-
tibiotics are typically cationic and amphipathic, and
are structurally elongated and exible molecules
(and 35 amino acids). A prototypic type A lan-
tibiotic is nisin, with other members of the fam-
ily, including subtilin (Bacillus subtilis), epidermin
(Staphylococcus epidermis), Pep 5 (S. epidermis), lac-
ticin 481 (class AII; Lactococcus lactis), and nukacin
ISK-1 (class AII; Staphylococcus warneri). Type B
lantibiotics are typically shorter (20 amino acids),
although they still contain many intermolecular
rings, making them more rigid and globular than
type A lantibiotics. Representative type B lantibi-
otics include mersacidin and actagardine. A third
group, the two-peptide lantibiotics, comprises two
independently transcribed peptides that are
Ann. N.Y. Acad. Sci. 1277 (2013) 127138
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2013 New York Academy of Sciences. 129
Peptide antimicrobials Yount & Yeaman
elaborated concomitantly and act synergistically to
effect microbial killing.
Nisin is a 34-amino acid peptide with two well-
denedamphipathic domains (Table 1).
9
It is widely
used in the food industry and has potent activity in
the nanomolar (nM) range against a broad range
of Gram-positive,
32
and to a lesser extent Gram-
negative, organisms.
33
A number of studies have
demonstrated that nisin acts by a two-step mecha-
nisminwhichthe peptide rst binds to lipidII
34
and
then inserts into the bacterial membrane to create
a pore. NMR studies have shown that in the dock-
ing step nisin binds to the pyrophosphate moiety
of lipid II via direct hydrogen (H-) bondmediated
interactions with several backbone residues.
11
The
architecture of this peptide structural motif has been
termed a pyrophosphate cage, as the orientation of
the nisin binding pocket surrounds the pyrophos-
phate domain of lipid II. It has been postulated
that this interaction with lipid II may contribute
to the very low resistance levels seen for nisin, as
it would likely be very difcult for an organism to
alter this highly conserved component of the lipid
II synthesis pathway. When resistance to nisin is ob-
served, it is typically not due to alterations in lipid II
structure, but tomore generalizedmechanisms such
as electrostatic shielding and cell wallthickness
alterations.
3
In addition to interactions with lipid II, a re-
cent study has demonstrated that nisin also binds to
lipid III and lipid IV to interfere with biosynthesis
of cell wall teichoic and/or lipoteichoic acids;
1
this
phenomenonmay also apply to other type Alantibi-
otics, as gallidermin also binds to lipid III and lipid
IV.
1
Thus, beyond interfering with cell wall biosyn-
thesis, nisin may also inhibit the formation of other
essential structural components of the greater cell
envelope.
Nisin is one of the most potent HDPs, with activ-
ity in the nMrange. It has been postulated that tight
docking with lipid II may facilitate its membrane
pore formation process. Nisin can be demonstrated
to form pores, as evidenced by a collapse of ion gra-
dients and loss of amino acids, K
+
, and ATP. As for
many other HDPs, the membrane active component
of nisin is likely mediated by its cationic C-terminal
domain, and studies using anionic model mem-
branes demonstrate increasing vesicle lysis with in-
creasing anionic lipid composition.
12
Further, bind-
ing lipid II may signicantly enhance membrane
lysis, as the pyrophosphate domain is localized im-
mediately adjacent to the plasma membrane. There-
fore, once the N-terminal domain of nisin has been
docked to pyrophosphate, the cationic C-terminal
would ostensibly be free to electrostatically interact
with the anionic phospholipid head groups of the
plasma membrane.
Although the molecular details of the binding
interaction between nisin and lipid II have been
mapped with ne detail, other type A lantibiotics
may bind lipid II at alternate sites. The related type
Alantibiotic clausin, fromBacillus clausii, has a very
similar ring structure to nisinandalso interacts with
lipid II (Table 1). However, binding studies with
various lipid II substrates suggest that clausin binds
the rst few amino acids of the pentapeptide rather
than the pyrophosphate domain.
13
Type B lantibiotics, such as mersacidin and ac-
tagardine, have also been demonstrated to effect
microbicidal efcacy through targeted interactions
with cell wall components. This class of lantibiotics
contains peptides of shorter length than type A
compounds (e.g., 20 vs. 35 residues), and yet
are still highly cyclized with four intramolecular
thio-ether rings. Sucha structural congurationim-
poses signicant conformation restrictions on type
Blantibiotics, making themmore rigid and globular
than type Apeptides. Howthis structural difference
translates to mechanistic specicity is not fully un-
derstood, but may relate to calciuminteractions (see
below). Type B lantibiotics are also typically neutral
or negative in charge, and for the most part do not
have a membrane-active component.
The most well characterized type B lantibi-
otic is mersacidin, produced by Bacillus species
(Table 1). Mersacidin, the rst lantibiotic shown
to interact with lipid II, interferes with a subsequent
membrane-associated transglycosylation step.
14
In
murine infection models, mersacidin exhibited in
vivo efcacy against methicillin-resistant Staphylo-
coccus aureus (MRSA) intraperitoneal challenge
35
and rhinitis.
36
The mechanismby which mersacidin
and other members of this class interact with lipid
II differs fromthat of nisin and other type Alantibi-
otics. Mersacidinandother type Blantibiotics have a
conserved lipid II binding domain that, NMR data
suggest, engages Ca
2+
.
37
A putative Ca
2+
-binding
pocket has also been proposed for the related lan-
tibiotic actagardine, and it has been postulated
that association with this ion may enhance lipid II
130 Ann. N.Y. Acad. Sci. 1277 (2013) 127138
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Yount & Yeaman Peptide antimicrobials
Table 1. Structureactivity correlates of selected cell walltargeting HDPs
Primary Bind Accum. Bind
Biochemical target lipid lipid Glc Bind Lyse
classication Source Peptide spectrum II II Pre Cer LPS CM Refs.
Prokaryotes
Class I bacteriocins
Type A
lantibiotic
Lactococcus lactis Nisin G+ x x x 1012
Bacillus clausii Clausin G+ x 13
Type B
lantibiotic
Bacillus sp. HIL
Y-84,54728
Mersacidin G+ x 14
Actinoplane s
garbadinensis
Actagardine G+ x 14,15
Two-peptide
lantibiotics
Lactococcus lactis Lacticin 3147 G+ x x 16
Haloduracin G+ x x 17
Class II bacteriocins Lactococcus lactis Lactococcin 972 Lacto
cocci
x 18
Cyclic lipopeptides
and related
Empedobacter
haloabium
Empedopeptin G+ x 19
Actinoplanes spp. Ramoplanin A2 G+ x 20,21
Actinoplanes
teichomyceticus
Teicoplanin G+ x 22
Streptomyces
roseosporus
Daptomycin G+ x 23
Eukaryotesinvertebrate
Fungi
Defensin CS- Pseudoplectania
nigrella
Plectasin G+ x x 24
Eurotium
amstelodami
Eurocin G+ x 24
Aspergillus oryzea Oryzeacin G+ x 24
Plant
Defensin CS- Raphanus sativus RsAFP2 Fungi x 25
Dahlia merckii DmAMP-1 Fungi x 26,27
Mollusk
Defensin CS- Mytilus
galloprovinciali
Gallicin G+ x 24
Oysters Crassostrea gigas Cg-Defh-1, -2
and -m
G+ x x 28
Arthropod
Defensin CS- Lucilia sericata Lucifensin G+ x 24
Heliothis virescens Heliomicin Fungi x 25
Eukaryotesvertebrate
-Defensin Homo sapiens hBD-3 Broad x x x ? 29
-Defensin Homo sapiens HNP-1 Broad x x x 30
Cathelicidin Homo sapiens LL-37 Broad x 31
Broad = agents with activity against Gram-positive bacteria, Gram-negative bacteria, fungi, and some viruses; G+ =
Gram-positive bacteria; Fungi =members of the phylogenetic Fungi kingdom, including molds and yeasts.
Ann. N.Y. Acad. Sci. 1277 (2013) 127138
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Peptide antimicrobials Yount & Yeaman
binding by shielding the deprotonated Glu17
residue at the active site.
15
A third class of cell wallactive lantibiotics is the
two-peptide lantibiotic family, members of which
have been shown to synergistically act on Gram-
positive bacteria. These peptides are ribosoma-
lly translated from two separate genes and, like
other lantibiotics, undergo extensive posttransla-
tional processing to generate a number of unusual
and thioether-containing lanthionine rings. Stud-
ies suggest that these peptides may act by combin-
ing two mechanisms of action in which one pep-
tide binds lipid II and the other forms a membrane
pore.
16
Representatives from the two-peptide lantibiotic
family include lacticin 3147 and the recently charac-
terizedhaloduracins and. Lacticin3147, isolated
from L. lactis,
38
consists of synergistically acting
peptides A1 and A2, demonstrating that mutations
disrupting the ring structure in either peptide are
inactive.
39
Lacticin 3147 has activity against MRSA
and is being considered for a veterinary applica-
tion to treat mastitis.
40,41
For haloduracin, it has
been shown that Hal can directly inhibit bacterial
growth, whereas Halcannot; together, the peptides
synergize to produce a high level of activity.
42
In
particular, Halinhibits the penicillin-binding pro-
tein 1b (PBP1b)catalyzed transglycosylation step
of peptidoglycan synthesis by binding lipid II with
2:1 stoichiometry.
17
Together, the two peptides in-
teract with lipid II with a 1:2:2 stoichiometry (lipid
II, Hal, Hal). The Hal and Hal peptides struc-
turally and chemically resemble globular type B and
elongated type A lantibiotics, respectively. Overall,
haloduracin has efcacy similar to nisin and has
been shown to cause depolarization of transmem-
brane potential () and liberation of K
+
ions,
42
in addition to binding lipid II.
Distinct from the lanthionine containing class I
bacteriocins are the class II bacteriocins that are
often characterized as heat stable and lacking post-
translational modication. Although many class II
bacteriocins are thought to act via a membrane per-
meabilizing step, several are also known to have
a cell walltargeting mechanism. One representa-
tive class II bacteriocin with a putative cell wall
mediated mechanism of action, Lcn972, is a rel-
atively large bacteriocin (66 amino acid residues)
and differs from other class II bacteriocins in that
it is heat labile. Lcn972 is cationic and hydrophilic,
and exerts its microbicidal activity against lacto-
cocci via binding lipid II. The fact that lactococci
are often commensual or benecial symbionts with
mammalian hosts emphasizes the potential for such
peptides to serve as modulators of normal ora.
Mutants with resistance to Lcn972 notably contain
higher levels of muropeptides containing tripep-
tide side chains, indicating that for this bacteri-
ocin peptidoglycan remodeling may contribute to
resistance.
18
Although nonribosomally synthesized peptides
are not a focus of this review, it is useful to con-
sider the family of cyclic or semicyclic lipopeptide
and lipoglycopeptide antiinfectives for comparison.
Members of this group include vancomycin, dap-
tomycin, telavancin, and dalbavancin. These agents
may also interfere with the lipid II pathway, and
some share biophysical (e.g., Ca
2+
binding) and
structural features with certain type B mersacidin-
like lantibiotics. Cyclic lipopeptides are produced
via a complex enzymatic process, generating a
macrocyclic ring structure with an attached lipid
adduct. Representative cyclic lipopeptides in pre-
clinical development and clinical use include ramo-
planin, empedopeptin, and daptomycin.
Ramoplanins are a family of glycolipodepsipep-
tides produced by Actinoplanes spp. fungi. The most
well-known members of this family are teicoplanin
and ramoplanin A2. Synthesis of ramoplanins is
exceptionally complex, involving many enzymatic
steps that produce a 49-member ring structure from
an initial 17 amino acid template.
20
A number of
studies have suggested that ramoplanins likely act
through the binding and sequestration of lipid II,
inhibiting subsequent biosynthesis of peptidogly-
can. For example, an initial NMR report indicated
that ramoplanin formed a complex with the mu-
ramyl carbohydrate, intact pyrophosphate, and rst
two amino acids of the pentapeptide in lipid I and
lipid II.
21
These studies also suggested that ramo-
planin adopts a backbone fold similar to that of
the ribosomally synthesized type B lantibiotics mer-
sacidin and actagardine, which also target lipid II.
The potential for a convergent evolutionary process
in this respect is intriguing. Subsequent NMR stud-
ies have demonstrated that ramoplanin A2 forms
an amphipathic U-shaped dimer in lipid micelles.
20
This dimer appears to partially embed in the target
membrane bilayer anchored by the N-acyl chains
of each monomer, facilitating interaction with
132 Ann. N.Y. Acad. Sci. 1277 (2013) 127138
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2013 New York Academy of Sciences.
Yount & Yeaman Peptide antimicrobials
membrane-bound lipid II. Ramoplanin A2 has ac-
tivity against vancomycin- and -lactam-resistant
MRSA in vitro, suggesting that it interacts with a
molecular epitope on lipid II that is distinct from
those recognized by vancomycin or lactam an-
tibiotics. Teicoplanin is approved for certain types
of infections (e.g., vancomycin-resistant Clostrid-
ium difcile), while ramoplanin A2 is currently in
late phase clinical development.
20
Yet another member of the cyclic lipopeptide
family is daptomycin, an antibiotic with potent ef-
cacy against clinically important, multidrug resis-
tant Gram-positive organisms. Daptomycin is pro-
duced by enzymatic processes during Streptomyces
roseosporus fermentation to generate a 13-member
amino acid cyclic lipopeptide with a decanoyl lipid
side chain. In contrast to the preclinical stage of
development for many peptides considered here,
daptomycin is currently in clinical use, with ap-
proval for treatment of complicated skin and soft
tissue infections, as well as S. aureus bacteremia and
endocarditis.
43
Although initial reports suggested
that daptomycin may function through inhibition
of lipoteichoic acid synthesis, subsequent studies
have demonstrated that this is not the case,
43
and
that daptomycin likely inactivates microorganisms
via calcium-dependent binding of the lipophilic tail
to the bacterial plasma membrane.
23
This mem-
brane interaction leads to potassium efux, mem-
brane depolarization, inhibitionof macromolecular
compound synthesis, and cell death.
Other bacterial phyla are also known to elaborate
similar molecules. For example, empedopeptin is
an amphoteric, cyclic lipo-depsipeptide produced
by the Gram-negative bacterium Empedobacter
haloabium. Although it contains a 14-carbon myris-
tic tail, empedopeptin has a net negative charge
andremains water soluble. Empedopeptinmay have
eventual clinical considerations, as preclinical stud-
ies suggest signicant in vitro and in vivo activity
against antibiotic-resistant pathogens, low overall
toxicity, and a favorable pharmacologic prole.
19
Using membrane preparations and lipid II precur-
sor components, empedopeptin was shown to bind
to lipid II with a 2:1 stoichiometry. Reminiscent to
some lantibiotics, the binding site of empedopeptin
on lipid II includes the pyrophosphate group, the
rst sugar residue of MurNAc, and a portion of
the stem peptide and undecaprenyl chain. Notably,
Ca
2+
ions are essential for the interaction between
this net negatively charged peptide and lipid II.
Empedopeptin does not appear to cause membrane
depolarization.
19
Given their structural similarities,
it is possible that the tripropeptins and plusbacins
may also target lipid II in a similar fashion.
19
In
these respects, there appears to be a mechanis-
tic structureactivity relationship among certain
HDPs and recently developed antibiotics such as
daptomycin.
44
Eukaryotic peptides targeting microbial cell
walls
HDPs have been characterized from all eukary-
otes that have been evaluated for them, from fungi
to humans. Eukaryotic HDPs are typically small
and cationic, and are structurally classied into
ve major groups: (1) cysteine-stabilized (e.g., de-
fensins); (2) cysteine-stabilized loop (e.g., prote-
grins, tachyplesins); (3) linear -helical (e.g., LL-
37, kinocidin helices); (4) enriched in one or more
specic amino acidresidues (e.g., Bac5); or (5) com-
binations of the above. Although certain families of
HDPs, particularly those of higher eukaryotes, show
evidence of extensive gene duplication, and in some
cases positive selection, their structural (cysteine-
stabilization, -helicity) and biophysical (amphi-
pathicity, cationicity) features are often highly con-
served. Moreover, many such molecules may form
multimeric superstructures, further extending their
functional repertoire. For example, conformation
mediated by the presence or absence of lipid en-
vironments suggests that amphipathic HDPs may
exist as soluble monomers in solution, or as multi-
mers or in alternate congurations once they reach
their microbial targets.
When the rst eukaryotic defensin peptides were
characterized nearly 30 years ago mechanism of ac-
tion studies indicated these molecules were able to
rapidly and efciently permeabilize articial mem-
brane bilayers.
45
Subsequently, most research has
upheldthese ndings andfurther delineatedthe bio-
chemical details of this process. Hence, to date, the
conventional view for the mechanism of action for
defensin class HDPs is via inactivation of suscepti-
ble microbes through a membrane-permeabilizing
activity. Historically, few if any specic receptor
ligand targets have been validated for this family of
HDPs. However, within the last fewyears, a number
of groundbreaking studies have demonstrated that
certain members of the defensin family, from both
Ann. N.Y. Acad. Sci. 1277 (2013) 127138
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2013 New York Academy of Sciences. 133
Peptide antimicrobials Yount & Yeaman
lower and higher eukaryotes, bind lipid II and kill
microorganisms ina way that suggests a targetedcell
wallactive mechanism of action.
24,29
In addition,
these studies demonstrate (1) accumulation lipid II
and its precursors; (2) a lack of membrane depolar-
ization; (3) norapidloss of intracellular metabolites;
and (4) a relatively slow, versus immediate, process
of cell death. Although the generalizability of a cell
wallactive mechanism for defensin class peptides
remains to be determined, these observations do
suggest that certain members of this family may act
via a cell walltargeted mechanism.
Invertebrate defensins
Plectasin is a novel member of the invertebrate
class of defensins, the cysteine-stabilized (CS-
) defensins, isolated fromthe saprophytic fungus
Pseudoplectania nigrella. In this family, the overall
conformation consists of a double or occasionally
triple-stranded antiparallel -sheet domain teth-
ered, via disulde linkages, to an -helical domain.
This structural motif is highly conserved in inver-
tebrates, with representative lower (plant/fungal)
and higher (insect/mollusk) proteins being nearly
superimposable.
46,47
Initial evidence suggesting that plectasin might
act via a cell walltargeting mechanism of ac-
tion came from transcriptional proling data.
In these studies, exposure to plectasin led to a
gene expression prole in Gram-positive bacte-
rial strains that was similar to that of other cell
wallactive agents, such as vancomycin and peni-
cillin.
48,49
Subsequently Schneider et al.
24
carried
out a number of experiments to support a cell
wallactive mechanism. Through these studies it
was evident that plectasin targeted lipid II, and
that this was likely the mode of bacterial killing
due to the fact that (1) the peptide did not
cause depolarization; (2) the peptide did not incur
leakage of K
+
, tetraphenylphosphonium (TPP
+
),
bis-(1,3-dibutylbarbituric acid)trimethine oxonol
(DiBAC
4
), ATP, metabolites, or liposomal carboxy
uorescein; (3) the peptide did not inhibit the in-
corporation of radiolabeled precursors into DNA
or protein; (4) the kinetics of killing occurred over
the course of one bacterial generation; and (5) the
antibacterial effect led to a buildup of the ultimate
precursor of lipid II (UDP-MurNAc-pentapeptide)
in plectasin-treated cells, similar to vancomycin.
Moreover, NMR studies revealed that plectasin in-
teracts specically with lipid II. In particular, bind-
ing of plectasin to lipid II occurred at a 1:1 stoi-
chiometry and was dependent on hydrogen bond-
ing interactions between a cluster of approximately
eight residues in plectasin and the solvent-exposed
pyrophosphate domain of lipid II.
24
Validation of
the relevance of this interaction was provided by
saturation mutagenesis studies indicating that even
a conservative substitution for two of these residues
(e.g., A31G and K38R) abolished the antimicrobial
efcacy of plectasin.
24
Xiong et al.
50
recently investigated the
in vivo efcacy and pharmacology of NZ2114, a
plectasin-engineered antiinfective peptide in pre-
clinical evaluation. In these studies, the phar-
macokinetic/pharmacodynamic characteristics and
in vivo efcacycompared to vancomycin or
daptomycinwere evaluated in an experimental
rabbit infective endocarditis (IE) model due to
MRSA. At 5, 10, or 20 mg/kg intravenous injec-
tion twice daily, NZ2114 signicantly decreased
MRSA densities in cardiac vegetations and kidneys.
NZ2114 efcacy was dose dependent and at the
20 mg/kg regimen had signicantly greater efcacy
than vancomycin (P < 0.001) and equivalent ef-
cacy to daptomycin. Remarkably, only NZ2114 (10
or 20-mg/kg doses) prevented posttherapy relapse
in tissues, while bacterial counts in vancomycin-
or daptomycin-treated animals increased unabated.
The in vivo efcacy of NZ2114 correlated with the
maximum concentration of free, unbound fraction
of drug in serum as a function of MIC, area un-
der the concentration curve as a function of MIC,
and the cumulative percentage of a 24-hour period
that the drug concentration exceeds the MIC un-
der steady-state pharmacokinetic conditions. The
superior efcacy of NZ2114 in this MRSA IE model
suggests the potential for further development of
this compound for treating serious MRSA infec-
tions. Thus, plectasin efcacy was equivalent, or su-
perior, to that of vancomycin or daptomycin against
MRSA, and revealed favorable pharmacologic fea-
tures of NZ2114 in this rigorous infection model.
50
However, the commercial entity pursuing plectasin
as a novel antiinfective agent has thus far chosen to
suspend its development.
To further probe whether cell walltargeted
mechanisms of killing by CS- defensins were
limited to plectasin, interactions with lipid II were
also assessed for other members of this peptide
134 Ann. N.Y. Acad. Sci. 1277 (2013) 127138
c
2013 New York Academy of Sciences.
Yount & Yeaman Peptide antimicrobials
family.
24
These studies reavealed that fungal (eu-
rocin [Eurotium amstelodami]; oryzeacin [As-
pergillus oryzea]), arthropod (lucifensin [Lucilia
sericata]) and mollusk (gallicin [Mytilus gallo-
provincialis]) defensins also had specic binding
interactions with lipid II in a 1:1 stoichiometry
and inhibited the peptidoglycan synthesis pathway.
These observations have subsequently been con-
rmed in a second report in which the interaction
of three CS oyster defensins, Cg-Defh-1, -2,
and -m, and lipid II was measured.
28
In this study,
the oyster defensins did not compromise mem-
brane integrity in S. aureus but did inhibit cell
wall synthesis and led to accrual of the UDP-N-
acetylmuramyl-pentapetide (lipid I) cell wall pre-
cursor. Further, surface plasmon resonance (SPR)
measurements of the avidity of the binding interac-
tion between Defh-1, -2, or -m and lipid II demon-
strated a rapid, strong, and essentially irreversible
interaction. This study also demonstrated afnity
of oyster defensins for lipid II by complementary
antagonization assays and thin-layer chromatogra-
phy measurements. Taken together, the above ob-
servations provide strong evidence that plectasin, as
well as CS- defensins from other invertebrates,
likely have specic interactions with lipid II, and
hence may inactivate microorganisms via a cell wall
targeting mechanism of action.
Mammalian defensins
Before the observations described earlier, inverte-
brate defensin inhibition of cell wall structure or
function was not expected. However, these observa-
tions have now opened the door to previously un-
foreseen mechanisms of other defensin-class HDPs.
Specically, several recent studies now suggest that
human - and -defensin peptides also bind lipid II
and use a cell wallmediated mode of action against
target organisms. As these mammalian defensins
have previously been reported to have plasma mem-
brane targeting activity, the new data afford an
opportunity to revisit the established paradigms
regarding defensin-class peptide mechanisms of
action.
Human -defensin (hD-3) is a prototypic HDP
elaborated in skin and mucosal surfaces. In 2008,
a study by Sass et al.
51
measured the effect of ex-
posure to hD-3 on gene expression in S. aureus.
In this investigation, hD-3 induced alterations in
the S. aureus core cell wall stress regulon in a man-
ner similar to that of the cell wallactive antibiotic
vancomycin. This observation is consistent with the
concept that hD-3 perturbs specic component(s)
of the cell wall, triggering upregulation of cell wall
stress response pathways. To investigate this effect
directly, an additional study using hD-3 with pu-
ried cell wall synthesis components showed that
hBD-3 blocked the transglycosylation step in S. au-
reus normally carried out by PBP2, leading to a
buildup of UDP-MurNAc-pentapeptide, the ulti-
mate soluble precursor of lipid II.
29
Such an ac-
cumulation is typically associated with agents that
interfere with peptidoglycan synthesis and is not
normally correlated with depolarization or loss
of cellular metabolites essential for the synthesis
of lipid II. Further, hBD-3 inhibited puried en-
zymes required for cell wall synthesis that use the
bactoprenol-bound lipid II as a substrate. Finally,
transmission electron microscopy revealed that ex-
posure to hD-3 caused apparent ssures in the
cell wall that allowed for protrusions of membrane-
bound cytoplasmic contents of S. aureus. Similar
observations have been noted for cell wall active
compounds such as penicillins.
52
Taken together,
this body of evidence suggests that hD-3 acts via a
mechanism that includes a cell wallactive compo-
nent in its activity against S. aureus.
Human neutrophil defensin-1 (HNP-1) is the
hallmark HDP expressed in granular context of the
neutrophil. Like hD-3, evidence for a cell wall
targeting mechanism of HNP-1 action has been re-
cently demonstrated.
30
In this study, SPR was used
to characterize the binding afnity between HNP-1
and lipid II, showing high afnity and stereospecifc-
ity. For example, the afnities of stereoisomeric
forms of HNP-1 for lipid II were measured and
demonstrated that peptides containing d-amino
acids hadanapproximately vefoldreducedbinding
to lipid II, and that a linear l-amino acid form had
no apparent binding. These data implicate a specic
chiral interaction between a mammalian defensin
and a precursor for bacterial cell wall synthesis. If
correct, these observations are among the rst ex-
amples of specic targets for defensin antimicrobial
mechanisms.
Mammalian cathelicidins
The majority of cell wallactive peptides identi-
ed to date have been shown to interact with pep-
tidoglycan, which is abundant in Gram-positive
Ann. N.Y. Acad. Sci. 1277 (2013) 127138
c
2013 New York Academy of Sciences. 135
Peptide antimicrobials Yount & Yeaman
bacteria. Yet, there are also examples of peptides
that appear to interact with Gram negativespecic
cell wall elements.
5356
However, given the complex-
ity of the Gram-negative cell surface, the molecular
details of these interactionsand ultimate mecha-
nisms by which they contribute to cell deathare
less clearly understood.
One peptide for which there is a documented
interaction with a Gram-negative cell wall compo-
nent, LPS, is the human cathelicidin LL-37. LL-37
is an -helical peptide expressed by many epithelial
and myeloid tissues. Although not a direct mea-
sure of microbicidal activity, numerous studies have
demonstrated that LL-37 binds LPS with high afn-
ity,
53,54,56
and hence may limit its endotoxic prop-
erties as well as downstream immunological signal-
ing events. In one recent study, the specic inter-
action between three LL-37 homologues (human,
orangutan, and dusky leaf monkey sequences) and
bacterial exopolysaccharides (Pseudomonas aerug-
inosa, Inquilinus limosus, or Burkholderia cepa-
cia) was demonstrated using spectroscopic, uo-
rescence, and atomic force microscopy.
31
Overall,
bindingafnity of the LL-37homologues was shown
to increase with an increasingly negative charge of
the respective exopolysaccharide target. A unify-
ing model in which cationic peptides interact with
anionic bacterial targets has been previously pro-
posed.
57
The mechanism by which HDPs such as
LL-37 may lead to death or inhibition of bacteria
has recently been adapted to Gram-negative organ-
isms, as described by Dathe and colleagues.
54
In
their model, the cationic peptide is initially elec-
trostatically attracted to the LPS-rich anionic sur-
face of the Gram-negative organism. After this ini-
tial binding step, peptides accumulate and a breach
is created in the outer surface, ultimately expos-
ing the target plasma membrane to peptide ac-
tion. Support for the biological signicance of the
documented HDPLPS binding interactions is pro-
vided by well-characterized HDP-resistance mecha-
nisms in Gram-negative Salmonella and related or-
ganisms. In these organisms, resistance to cationic
antimicrobial peptides is conferred via activation
of PhoP/PhoQ and PmrA/PmrB two component
regulatory systems, which lead to increased incor-
poration of positively charged l-4-aminoarabinose
subunits into LPS and other adaptive responses.
57
This molecular modication results in a more neu-
tral or even cationic surface charge, and hence re-
duced electrostatic attraction for cationic antimi-
crobial peptides, affording increased survival of or-
ganisms. Although the above studies are not a direct
measure of LL-37s mechanism of action, they do
suggest that members of this classical -helical HDP
family may have specic interactions with certain
Gram-negative cell wall component structures.
Fungal cell wall targets of HDPs
The discussion above has considered HDPs of fun-
gal origin that appear to target the bacterial cell
wall. Beyond the immediate scope of that report,
a precedent has also been demonstrated for fungal
defensinclass peptides, and by corollary plant and
invertebrate orthologues, as having fungal cell wall
directed mechanisms of action. For these cases, a
signicant body of evidence exists to suggest that
fungal, plant, or invertebrate defensins having anti-
fungal efcacy exert direct microbicidal effects via
specic binding interactions with fungal cell wall
sphingolipid or ceramide components. This process
has been characterized in detail for the radish anti-
fungal protein-2 (RsAFP2), Dahlia merckii antimi-
crobial protein-1 (DmAMP-1), and tobacco bud-
worm heliomicin defensins, among others. RsAFP2
and heliomicin have been shown to bind fungal
glucosylceramides (GlcCer) at different structural
motifs.
25
Of potential clinical relevance is the ob-
servation that these peptides do not recognize hu-
man GlcCer, which is structurally distinct fromfun-
gal GlcCer. This observation may explain the high
level of selective toxicity toward fungal cells.
25
The
specic interaction of RsAFP2 and fungal cell wall
targets has been demonstrated by the loss of re-
active oxygen species generation (ROS), and sub-
sequent cell death, only in C. albicans strains de-
cient in GlcCer synthesis.
58
Similarly, DmAMP-1
appears to specically target mannosyl-diinositol-
phosphorylceramide (M[IP]
2
C), which may con-
tribute to cell death.
26,27
These and other important
new advances in the molecular and cellular mecha-
nisms of plant defensins that appear to involve the
fungal cell wall have been reviewed recently.
59
Prospectus
Emerging evidence now supports the concept that
HDPs have evolved to exploit specic, highly con-
served targets and functions involved in biosyn-
thesis of the bacterial cell wall. It should be
noted that such a mechanism does not necessarily
136 Ann. N.Y. Acad. Sci. 1277 (2013) 127138
c
2013 New York Academy of Sciences.
Yount & Yeaman Peptide antimicrobials
preclude other complementary or membrane-
directed modes of action. Given the evolutionary
success of HDPclasses discussed earlier, it is likely or
even probable that such peptides have many mech-
anisms of action so as to minimize emergence of
resistance. In any case, the cell wall is among the
most accessible microbial targets, and peptides may
initially interact with this component of bacterial
cells. As the cell wall is integral to prokaryotic cell in-
tegrity, reproduction, virulence expression, or other
functions, targeting and disrupting peptidoglycan
synthesis may lead to irrecoverable dysfunctions in
the target cell. Recent studies have now provided
specic examples by which HDPs of several classes
target lipid II or other cell wall biosynthetic pro-
cesses to interrupt peptidoglycan synthesis, translo-
cation, or extracellular maturation. These discover-
ies may signicantly inuence at least two funda-
mental aspects of biomedical science: (1) mechanis-
tic and evolutionary understanding of perhaps the
most ancient arm of molecular host defenses; and
(2) potential insights into development of novel an-
tiinfective peptides that exploit the highly conserved
process of cell wall generation in prokaryotes.
Acknowledgment
This manuscript was supported in part by research
Grant R01 AI39001 from the National Institutes of
Health to MRY.
Conicts of interest
The authors declare no conicts of interest.
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