MINIREVIEW
Multifunctional host defense peptides:
intracellular-targeting antimicrobial peptides
Pierre Nicolas
Biogene
`
se des Signaux Peptidiques, ER3-UPMC, Universite
´
Pierre et Marie Curie, Paris, France
Introduction
There has been increasing interest in recent years in
describing the complex, multifunctional role that anti-
microbial peptides play in directly killing microbes,
boosting specific inate immune responses, and exerting
selective immunomodulatory effects on the host [1–4].
Furthermore, many antimicrobial peptides are quite
inactive on normal eukaryotic cells. The basis for this
discrimination appears to be related to the lipid com-
Keywords
antimicrobial peptides; cell-penetrating
peptides; dermaseptin; intracellular target;
membrane translocation
Correspondence
P. Nicolas, Biogene
`
se des Signaux
Peptidiques (BIOSIPE), ER3-UPMC,
Universite
´
Pierre et Marie Curie, Ba
ˆ

which is isolated in particular peptide families, is also shared by the hun-
dreds of naturally occurring antimicrobial peptides that differ in length,
amino acid composition, sequence, hydrophobicity, amphipathicity, and
membrane-bound conformation. Microbial cell entry and ⁄ or membrane
damage associated with membrane phase ⁄ transient pore or long-lived
transitions could be a feature common to intracellular-targeting antimi-
crobial peptides and mammalian cell-penetrating peptides that have an
overrepresentation of one or two amino acids, i.e. Trp and Pro, His, or
Arg. Differences in membrane lipid composition, as well as differential
lipid recruitment by peptides, may provide a basis for microbial cell kill-
ing on one hand, and mammalian cell passage on the other.
Abbreviations
MIC, minimal inhibitory concentration; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol.
FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS 6483
position of the target membrane (i.e. fluidity, negative
charge density, and the absence ⁄ presence of choles-
terol), and the possession, by the microbial organism,
of a large, negative transmembrane electrical potential.
There is now a widespread acceptance that antimicro-
bial peptides, apart from their membrane-permeabiliz-
ing ⁄ disrupting properties, may also affect microbial
viability by interactions with intracellular targets or
disruption of key intracellular processes. Much of the
focus in this area has been on the identification of tar-
gets in the interior of the microbial cell and the mecha-
nism by which antimicrobial peptides can enter the
microbial cell in a nondisruptive way [5–7].
The prevailing dogma that the microbicidal effects
of cationic antimicrobial peptides solely involve cyto-
plasmic membrane permeabilization ⁄ disruption of

tion ⁄ disruption of the microbial cytoplasmic
membrane is not the only mechanism of cell killing,
and that antimicrobial peptide might also operate by
entering the cells and interfering with their metabolic
function.
Antimicrobial peptides with varying
antimicrobial potencies exhibit
disparate extents of membrane
permeabilization and cell killing
Even though all cationic antimicrobial peptides are
able to interact with microbial cytoplasmic mem-
branes, and some strongly perturb bilayers, the num-
ber of studies documentating a clear dissociation
between cell death and the ability of some peptides to
permeabilize the membrane, either in vitro or in vivo,
has increased significantly during the last decade. For
example, TWF, an analog of the cathelicidin-derived
antimicrobial peptide tritrpticin, in which Trp is
replaced with Phe, is much more effective than TPA,
in which the two Pro residues of tritrpticin are
replaced with with Ala, against both Staphylococ-
cus aureus and Escherichia coli [9]. However, TWF
shows very little membrane-disrupting activity and no
ability to depolarize the membrane potential of micro-
bial cell targets, whereas TPA rapidly depolarizes the
membrane and causes rapid leakage of negatively
charged phospholipid vesicles. Dermaseptin B2 –
GLWSKIKEVGKEAAKAAAKAAGKAALGAVSE-
AVa – from frog skin and its C-terminally truncated
analog [1–23]-dermaseptin B2 are both highly effective

[14]. For some antimicrobial peptides, permeabilization
of the microbial cytoplasmic membrane and cell killing
begin concomitantly as quickly as a few minutes after
exposure [15–17]. For others, there is a considerable
lag period between these two events. For instance,
although TWF and TPA are equipotent in inhibiting
the growth of S. aureus and E. coli, TWF requires a
lag period of about 3–6 h for bactericidal activity,
whereas TPA kills bacteria after only after 30 min of
exposure [9]. Experiments based on confocal micros-
copy on living cells using the fluorescence of fluores-
cein isothiocyanate, 4¢,6-diamidino-2-phenylindole and
5-cyano-2,3-ditolyl tetrazolium chloride revealed that
sublethal concentrations of temporin L permeabilize
the inner membrane of E. coli to small compounds,
but do not allow the killing of bacteria [18]. At higher
peptide concentrations, the bacterial membrane
becomes permeable to large cytoplasmic components,
and this is concomitant with death of bacteria. This
shows that membrane permeabilization of bacteria by
temporin L and TWF is not a lethal step per se in the
absence of a catastrophic collapse of the membrane
integrity, and that peptide-mediated killing required
other additional events.
The choice of a membrane model can
influence the outcome of an in vitro
study of lipid–peptide interaction
Most models accounting for antimicrobial peptide-
induced membrane permeabilization are inferred from
data obtained with very simple, artificial membrane

are necessary to determine the three-dimensional struc-
ture of membrane-bound antimicrobial peptides and to
observe perturbation of the thermodynamic parameters
of the gel-to-crystalline phase transition of lipid mem-
brane models, lipid flip-flop, calcein release on model
liposomes, etc. However, there is no evidence that such
peptide concentrations, which provide almost full bac-
terial membrane coverage by the peptides, are really
present at the surface of bacteria during bacterial kill-
ing in vivo [21]. In addition, electron transport chains
and ion and complex nutrient transport systems
require the coordination over time and space of a net-
work of interacting proteins, coenzymes, and sub-
strates. That microbial cell death may result from
nonspecific interference of cationic amphipathic
peptides with the dynamic organization of membrane-
bound pathways rather than just from membrane
permeabilization has seldom been evaluated, and it is
hardly possible to do so in vitro through the use of
lipid membrane models [22]. The above-mentioned
data collectively suggest that, at least near the MIC,
the killing actions of some antimicrobial peptides are
complex and may involve targets in the interior of the
microbial cell.
How antimicrobial peptides may enter
microbial cells
Two general mechanisms are proposed to describe the
process by which antimicrobial peptides enter the
microbial cells, spontaneous lipid-assisted translocation
and stereospecific receptor-mediated membrane trans-

siently breached, and pores are hardly detectable in the
equilibrium state by usual biophysical approaches.
Once a threshold level of membrane-bound peptide is
reached, this may lead to disruption ⁄ solubilization of
the membrane in a detergent-like manner. The thresh-
old between the toroidal pore and the detergent-like
mechanisms of action may be related to two facets of
the cell killing mechanism relying on the peptide con-
centration: the membrane composition, and the final
peptide ⁄ lipid ratio. Because the threshold peptide con-
centrations required for membrane disruption are
always close to full bacterial membrane saturation,
doubts have arisen regarding the relevance of these
thresholds and their importance in vivo [21]. However,
rigorous calculations have demonstrated that antimi-
crobial peptides with MIC values in the micromolar
range can easily reach millimolar concentrations in a
bacterial membrane, owing to high partition constants
[28]. At this concentration level, there is a strong link
between cell death and membrane disruptive events.
On the other hand, at low peptide ⁄ lipid ratios, antimi-
crobial peptides may translocate across the plasma
membrane, perturbing its structure in a transient, non-
lethal manner, and reach the cell interior.
Another mechanism for breaching membrane perme-
ability, the lipid phase boundary defects model, pro-
posed that some b -sheeted peptides, such as cateslytin,
a 15 residue Arg-rich antimicrobial peptide resulting
from the cleavage of chromogranin A, form mainly flat
aggregates at the surface of negatively charged bacte-

allow the passage of the peptide from one side of the
membrane to the other. A similar mechanism of tran-
sient pore formation was proposed for the transloca-
tion of the HIV-1 Tat cell-penetrating peptide across
mammalian cell membranes [35].
Intracellular-targeting antimicrobial
peptides
Although there is no doubt that most cationic antimi-
crobial peptides act at high concentrations by permea-
bilizing ⁄ disrupting the microbial membrane, recent
studies and reviews have reported an ever-growing list
of peptides that are presumed to affect microbial via-
bility at low to moderate concentrations through inter-
action with one or more intracellular targets (Table 1).
Examples of intracellular activity include inhibition
of DNA and protein synthesis, inhibition of chaper-
one-assisted protein folding, inhibition of enzymatic
activity, and inhibition of cytoplasmic membrane sep-
tum formation and cell wall synthesis. Very different
amounts of data, acquired with different experimental
protocols, have been presented for individual peptides
in order to support this assumption, so that, in most
cases, straightforward interpretation of these observa-
Intracellular-targeting antimicrobial peptides P. Nicolas
6486 FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS
Table 1. Amino acid sequence, membrane-bound structure and suggested internalization mechanism and effect on microbial functions of intracellular-targeting antimicrobial peptides.
Hydrophobic residues are in bold. a, carboxamitaded.
Name Sequence Membrane-bound Structure Uptake mechanism Intracellular targets
Pyrrhocoricin VDKGSYLPRPTPPRPIYNRN Reverse turns at the termini
bridged by an extended segment

Polyphemusin I RRWCFRVCYRGFCYRKCRa Amphipathic b-hairpin with two
disulfide bonds
Transient pores? ?
Tachyplesin I KWCFRVCYRGICYRRCR b-Hairpin with two disulfide bonds Transient pores DNA?
Pleurocidin (P-Der) ALWKTMLKKAAHVGKHV
GKAALTHYLa
Amphipathic a-helix MIC: disordered transient pores
> MIC: membrane permeabilization
Macromolecular synthesis
Cryptdin-4 GLLCYCRKGHCKRGERVR
GTCGIRFLYCCPRR
Triple-stranded b-sheet with three
disulfide bonds
Transient pores or defects ?
Tritrpticin
TWF
TPA
VRRFPWWWPFLRR
VRRFPFFFPFLRR
VRRFAFFFAFLRR
Amphipathic turn structure Uptake not shown
Uptake not shown
?
?
P. Nicolas Intracellular-targeting antimicrobial peptides
FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS 6487
tions is difficult or, at best, arbitrary. Representative
examples will be used to elaborate this issue, starting
with the more documented examples and moving
towards those that are less documented.

salivary secretions that displays high candidacidal and
leishmanicidal activities at micromolar concentrations.
Previous research has indicated that histatin-5 binds
heat shock protein 70 (Ssa1 ⁄ 2), located on the cell
wall, and is subsequently transferred to a membrane
permease that transports the peptide across to the
cytoplasm in a nonlytic manner [41]. Ensuing studies
demonstrated that the uptake of histatin-5 is actually a
dichotomous event [42]. Below the MIC, the peptide
translocates into the cytoplasm of the parasite through
receptor-mediated endocytosis (see above) and is inter-
nalized into the vacuole without harmful effects on the
parasite. Under physiological concentrations, histatin-5
induces a concentration-dependent perturbation at a
spatially restricted site on the cell surface of Candida,
leading to rapid translocation of the peptide into the
cytoplasm in a nonstereospecific, receptor-independent
manner, causing only a fast but temporary depolariza-
tion and limited damage to the plasma membrane, as
shown by membrane depolarization, entrance of the
vital dye SITOX green, electron microscopy, and time-
lapse confocal microscopy on live cells. Once inside the
cell, the peptide accumulates in the mitochondrion,
inducing bioenergetic collapse of the parasite, caused
by the decrease of mitochondrial ATP synthesis
through inhibition of F
1
F
0
-ATPase. Concurrent with

buforin II was shown to bind DNA in vitro, the con-
nection between nucleic acid binding and antimicrobial
activity has not been demonstrated.
Indolicidin
Indolicidin is a Trp-rich, 13 residue antimicrobial
peptide isolated from bovine neutrophils that adopts an
extended wedge-type conformation when bound to
biological membranes. Owing to the presence of Trp
residues interspersed with Pro residues throughout the
sequence, it probably assumes a structure distinct from
Intracellular-targeting antimicrobial peptides P. Nicolas
6488 FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS
the well-described helical and b-structured peptides.
Indolicidin is active against a wide range of microorgan-
isms, including bacteria, fungi, and protozoa, and lyses
erythrocytes. Close to the MIC, indolicidin causes sig-
nificant membrane depolarization of the bacterial cyto-
plasmic membrane by forming transient pores, but does
not enter the cell and does not lead to cell wall lysis,
suggesting that there is more than one mechanism of
antimicrobial action [48]. Earlier investigations have
shown that indolicidin mainly reduces the synthesis of
DNA, rather than RNA and protein, and that inhibi-
tion of DNA synthesis causes E. coli filamentation and
contributes to the antimicrobial activity of indolicidin
[49]. Unlike indolicidin, [K6,8,9]-indolicidin and
[K6,8,9,11]-indolicidin do not depolarize the membrane
and accumulate in the cytoplasm, as shown by confocal
laser microscopy on living E. coli cells [50]. Gel-retarda-
tion assays showed that [K6,8,9]-indolicidin and

disulfide bridges [52]. It has excellent antimicrobial
activity against bacteria, demonstrating rapid killing
within 5 min of treatment. At two times the MIC,
polyphemusin I is only able to depolarize the E. coli
cytoplasmic membrane by 50% [53]. At the MIC,
polyphemusin I is able to translocate through mem-
brane bilayers of negatively charged model vesicles,
inducing flip-flop between membrane leaflets. Biotin-
labeled polyphemusin I accumulates in the cytoplasm
of E. coli within 30 min after addition, with only
modest cytoplasmic membrane disruption, and causes
disorganization of cytoplasmic structures [54]. In these
studies, permeabilization of E. coli with Triton X-100
was performed after fixation with glutaraldehyde, so as
to allow streptavidin fluorescent conjugate to access
intracellular biotin-labeled polyphemusin I. Moreover,
the mechanism of translocation and the nature of the
intracellular targets are as yet undefined.
Tachyplesin
Tachyplesin I is a cyclic b-sheet antimicrobial peptide
of 17 amino acids isolated from the hemocytes of the
horseshoe crab [55]. The peptide forms transient pores
in membranes containing acidic phospholipids, and
induces lipid flip-flop coupled to calcein leakage, the
latter being coupled to the translocation of the peptide
across lipid bilayers upon pore disintegration. The pep-
tide induced rapid inner membrane permeabilization of
E. coli at MIC, concomitant with a rapid decrease of
cell viability [56,57]. Gel-retardation assays and foot-
printing-like techniques using DNase I protection,

relative contributions of intracellular targeting and
membrane disruption to the overall killing strategy of
pleurocidin, as well as the precise mechanism by which
the peptide inhibits macromolecular synthesis in vivo,
remain to be defined.
Cryptdin
Cryptdin-4 is a 32 amino acid amphipathic antimicro-
bial peptide that adopts a triple-stranded antiparallel
b-sheet structure constrained by three disulfide bridges.
Near the MIC, cryptdin-4 induces E. coli cell permea-
bilization coupled to rapid potassium efflux, a sensitive
index of cell death. The lipid ⁄ polydiacrylate colorimet-
ric assay and fluorescence resonance energy transfer
from the Trp of the peptide to the dansyl chromo-
phore in the membrane vesicles of various lipid com-
positions suggested that cryptdin-4 inserts deep into
the membrane of highly negatively charged PG-con-
taining or cardiolipin-containing vesicles and then
translocates via transient membrane defects to the
inner membrane leaflet as a consequence of closure
and disintegration of these short-lived formations [61].
Cardiolipin seems to be the key lipid constituent con-
ferring sensitivity to cryptdin-4-induced vesicle permea-
bilization. Because this lipid is able to form domains
in E. coli cells, it was suggested that cardiolipin
domains might serve as highly charged ‘gates’ to facili-
tate movement of cryptdin-4 into and through lipid
membranes. Although these studies provide evidence
that the membrane disruptive action of cryptdin-4 is
linked to peptide translocation through lipid defects,

awaits further investigation.
A closer look shows that only a small number of the
above-mentioned antimicrobial peptides have been
convincingly demonstrated to fulfill the criteria to be
considered as microbial cell-penetrating peptides that
attack internal targets in vivo, and, of these, few spon-
taneously cross the cytoplasmic membrane. For
instance, in most cases: (a) the connection between
intracellular target binding in vitro and antimicrobial
activity has not been demonstrated, and ⁄ or the state of
integrity of the membrane has not been checked –
thus, it is not known whether the microbicidal activity
of the peptides is due to their membrane permeability
effect, their effects on intracellular targets, or a combi-
nation of these effects; (b) although a substantial num-
ber of these antimicrobial peptides have been shown to
translocate through model membrane vesicles in vitro,
detailed information on the internalization obtained
with living cells, and quantification of peptide uptake
and degradation, is still lacking – most of the confocal
and electron microscopic studies reporting internaliza-
tion of antimicrobial peptides have been conducted on
fixed cells, and the possibility that the fixation changed
the distribution of peptides cannot be ignored [63]; (c)
if intracellular targeting exists, one would expect the
peptide to evoke some degree of alteration of back-
ground transcript profiles, even if the peptide is present
at sublethal concentrations – this has seldom been
evaluated [22,64,65]; (d) the possibility that antimicro-
bial peptides interfere with the coordinated and highly

to be due to their ability to inhibit key intracellular
functions by crossing the microbial membrane, rather
than to create pores in the cell surface. Although this
picture is accepted by most authors, because observa-
tions of translocation in model membrane systems and
in living bacteria for some cell-penetrating peptides
might support the existence of uptake mechanisms
governed by lipid-assisted pore formation, quantitative
comparison of the uptake and antimicrobial effects of
these peptides in bacteria and yeasts have demon-
strated that their uptake route, intracellular concentra-
tion, fate and microbicidal effects vary widely among
peptides and microbial organisms. In several cases, the
experimental protocols that have been used suffer from
the same limitations as those mentioned above for
antimicrobial peptides, preventing a clear conclusion
to be drawn about the mechanism(s) by which these
peptides exert their antimicrobial action.
TP-10, a 21 amino acid deletion analog of the chi-
meric cell-penetrating peptide transportan, causes rapid
permeabilization of S. aureus cell membranes, followed
by cell entry, dispersion throughout the cytoplasm,
and subsequent death of the bacteria. pVEC, an 18
amino acid peptide derived from murine vascular
endothelial-cadherin protein, MAP, and penetratin,
has weak ability to depolarize the membrane potential
of S. aureus cells and the calcein-entrapped negatively
charged bacterial membrane-mimicking vesicles [66–
70]. The peptides internalize within these cell lines, but
all were degraded to various extents inside the cells

kills E. coli and Bacillus subtilis in the low micromolar
range, but has low activity against Salmonella, Pseudo-
monas, and Staphylococcus. The peptide strongly inter-
acts with negatively charged lipid bilayers, causing
local perturbation and depolarization of the membrane
potential, and crosses the membrane by a mechanism
promoted by the transmembrane potential [73]. The
mechanism of translocation is controversial. Deshayes
et al. [74] proposed a transient transmembrane-pore-
Table 2. Amino acid sequences of designed mammalian cell-pene-
trating peptides with antimicrobial activity. Hydrophobic residues
are in bold. a, carboxamitaded.
Name Sequence
Tat-[48–60] GRKKRRQRRRPQa
pVEC LLILRRRIRKQAHAHSKa
MAP KLALKLALKALKAALKLAa
TP 10 AGYLLGKINLKALAALA
Pep-1 KETWWETWWTEWSCPKKKFKVa
Penetratin RQIKIWFQNRRMKWKKa
P. Nicolas Intracellular-targeting antimicrobial peptides
FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS 6491
like structure promoted by the a-helical conformation
of the hydrophobic domain when it interacts with
membranes. This was disputed by other groups,
because no membrane leakage was observed.
Conversely, the capacity to translocate across the
mammalian cell membrane has been clearly demon-
strated for some antimicrobial peptides. Confocal laser
microscopy on fixed human cervical carcinoma HeLa
and fibroblastic TM12 cells, and on live Chinese ham-

age, because no indication of membrane disruption has
been seen at relevant concentrations of peptide. How-
ever, mammalian membrane disorganization associated
with penetration is very difficult to observe, because
the membrane repair response masks membrane distur-
bance by mobilizing vesicles within seconds to patch
any broken membranes [75].
Cell entry and ⁄ or membrane damage may be a
common feature of some antimicrobial peptides and
cell-penetrating peptides through very similar mecha-
nisms. Cell entry may involve membrane phase ⁄
transient pores or long-lived transitions that can be
dependent on peptide and membrane composition.
Differences in membrane lipid composition, as well as
differential lipid recruitment by peptides, may provide a
basis for microbial cell killing on the one hand and
mammalian cell passage on the other. For instance, the
translocation properties of Arg-rich cell-penetrating
peptides have been shown to be directly associated with
the presence of Arg residues. Transmembrane crossing
of these peptides is affected by their flexibility and am-
phipathicity, and is critically dependent on the number
and spacing of guanidinium groups [76]. In the case of
Tat peptides, replacement of Arg with Lys, or with His
or ornithine, strongly reduced the translocation ability
[77]. Charge neutralization of the guanidinium groups
through bidendate hydrogen bonding with the phos-
phate groups of the bilayer is thought to be necessary
for effective internalization into mammalian cells, and
the efficiency of the peptide uptake is directly associated

of both cell-penetrating peptides and antimicrobial
peptides.
Final comments
There is a widespread acceptance that antimicrobial
peptides, apart from their membrane-permeabiliz-
Intracellular-targeting antimicrobial peptides P. Nicolas
6492 FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS
ing ⁄ disrupting properties, may also affect microbial
viability by mechanisms that extend beyond the
plasma membrane, involving interactions with intra-
cellular targets or disruption of key intracellular pro-
cesses. So far, more than 1200 antimicrobial peptides
with different origins have been isolated or predicted.
Currently, there are only a handful of antimicrobial
peptides in the literature that have convincingly been
demonstrated to spontaneously enter microbial cells
and, once inside the cell, to interfere with cellular
functions. Without any doubt, a case-by-case system-
atic analysis of the uptake, fate and integrity of anti-
microbial peptides in living microbial cells with the
help of state-of-the-art cell biological methods,
together with the implementation of in vitro and
in vivo biochemical assays to characterize their intra-
cellular targets, should increase the panel of the
so-called intracellular-targeting antimicrobial peptides.
However, it is unlikely that the specific abilities of
some antimicrobial peptides to enter microbial cells
and impede cellular functions are also shared by the
hundreds of antimicrobial peptides that differ in
length, amino acid composition, sequence, hydropho-

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