Tài liệu Báo cáo khoa học: Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides docx

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

timent A
–5e
`
me e
´
tage, Case courrier 29, 7 Quai
Saint-Bernard, 75005 Paris, France
Fax: +1 44 27 59 94
Tel: +1 44 27 95 36
E-mail:
(Received 1 May 2009, revised 25 July
2009, accepted 29 July 2009)
doi:10.1111/j.1742-4658.2009.07359.x
There is widespread acceptance that cationic antimicrobial peptides, apart
from their membrane-permeabilizing ⁄ disrupting properties, also operate
through interactions with intracellular targets, or disruption of key cellu-
lar processes. Examples of intracellular activity include inhibition of
DNA and protein synthesis, inhibition of chaperone-assisted protein
folding and enzymatic activity, and inhibition of cytoplasmic membrane
septum formation and cell wall synthesis. The purpose of this minireview
is to question some widely held views about intracellular-targeting anti-
microbial peptides. In particular, I focus on the relative contributions of
intracellular targeting and membrane disruption to the overall killing
strategy of antimicrobial peptides, as well as on mechanisms whereby
some peptides are able to translocate spontaneously across the plasma
membrane. Currently, there are no more than three peptides that have
been convincingly demonstrated to enter microbial cells without the
involvement of stereospecific interactions with a receptor ⁄ docking mole-
cule and, once in the cell, to interfere with cellular functions. From the
limited data currently available, it seems unlikely that this property,

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

target cells has been increasingly challenged by the
realization that: (a) information on the membrane
interactions and activity of antimicrobial peptides
obtained in vitro using simple artificial membrane
bilayers, or in vivo using intact microbial cells, is not
clearly correlated with the observation of microbial
death; (b) several antimicrobial peptides may recognize
and inactivate cellular targets in vitro, such as nucleic
acids, proteins, enzymes, and organelles, their mecha-
nism of action being postulated to involve transloca-
tion across the plasma membrane in a nonlethal
manner; (c) regardless of which model of antimicrobial
peptide-induced membrane permeabilization ⁄ disruption
is correct, they all offer the peptide the possibility of
rapidly crossing the cytoplasmic membrane and reach-
ing macromolecular targets in the cell interior; and (d)
most antimicrobial peptides show strong similarities in
charge, structure and membrane interactions with cell-
penetrating peptides, which are thought to enter mam-
malian cells by passive transport [8]. The purpose of
this minireview is to describe and critically analyze
some widely held views about intracellular-targeting
antimicrobial peptides. In particular, I focus on the
proposed mechanisms by which antimicrobial peptides
might translocate across microbial membranes to
attack cellular targets.
Microbial membrane permeabilization
versus intracellular killing
There has been increasing speculation in the last dec-
ade that antimicrobial peptide-mediated permeabiliza-

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

in permeabilizing calcein-loaded phosphatidylcholine
(PC) ⁄ phosphatidylglycerol (PG) and phosphatidyletha-
nolamine (PE) ⁄ PG vesicles [10]. Whereas dermaseptin
B2 rapidly kills bacteria [11], [1–23]-dermaseptin B2 is
devoid of antimicrobial activity and is inefficient in
permeating intact bacterial cells. The bacterium-
derived antimicrobial peptides polymyxin B and poly-
mixin E1 failed to cause significant depolarization of
the Pseudomonas aeruginosa cytoplasmic membrane
but rapidly killed the test organism. In contrast, grami-
cidin S caused rapid depolarization of the bacterial
cytoplasmic membrane at concentrations at which no
killing was observed [12]. These observations support
the concept that, for some antimicrobial peptides,
membrane perturbation and cell killing may be inde-
pendent events that occur individually or complemen-
tary to other mechanisms of action [13].
Antimicrobial peptides exhibit
temporal dissociation between
microbial membrane permeabilization
and cell death
Although there is a wealth of evidence that many anti-
microbial peptides interact and increase the permeabil-
ity of microbial membranes as part of their killing
mechanism, it is not clear whether this is a lethal step.
Intracellular-targeting antimicrobial peptides P. Nicolas
6484 FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS
In addition, several antimicrobial peptides kill micro-
bial cells in the absence of significant permeabiliza-
tion ⁄ disruption of membrane structure and functions

[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

models that mimic microbial cell membranes, whether
in the form of lipid monolayers, oriented bilayers, or
vesicles (reviewed by Bhattacharjya and Ramamoorthy
[19]; this issue). Extrapolating these in vitro data to an
in vivo model is not straightforward, and the choice of
the model system may profoundly influence the out-
come of a study of lipid–peptide interaction. An ele-
gant study of the interaction of the human cathelicidin
antimicrobial peptide LL-37 with single phospholipid
monolayers, bilayers and bilayers composed of binary
mixtures of phospholipid species predominantly used
in model membrane experiments, i.e. PC, PE, PG, and
phosphatidylserine, showed the following [20]: (a) the
effects on single lipid monolayers are not comparable
to those on the corresponding bilayers; (b) there are
four different modes of interaction of LL-37 on bilay-
ers with the four different lipids used; and (c) there are
significant differences in the mode of peptide–lipid
interaction between the binary lipid mixtures PC ⁄ PG,
PE ⁄ PG, and PC ⁄ phosphatidylserine, which all carry
the same net charge. A similar disagreement was
observed for the interaction of dermaseptin B2 with
cardiolipin ⁄ PC and PG ⁄ PC vesicles.
Peptide concentration dependence of
antimicrobial action
Research on the mode of action of antimicrobial pep-
tides in vitro has usually been conducted at high multi-
ples of the minimal inhibitory concentration (MIC) of
peptides and ⁄ or high peptide ⁄ lipid ratios. Owing to
technical limitations, these high peptide concentrations

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-

location. The precise mechanisms whereby some
antimicrobial peptides are able to translocate sponta-
neously across the plasma membrane remain largely
unknown, and may vary from peptide to peptide.
However, membrane translocation seems to be a corol-
lary of transient membrane permeabilization. There are
currently several models accounting for antimicrobial
P. Nicolas Intracellular-targeting antimicrobial peptides
FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS 6485
peptide-induced membrane permeabilization ⁄ disruption
of microbial cells. The Shai–Matzusaki–Huang unifying
model proposes that a-helical antimicrobial peptides
initially bind parallel to the membrane plane and car-
pet the surface of the bilayer [23–26], with the apolar
amino acids penetrating partly into the bilayer hydro-
carbon core, and the cationic residues interacting with
the negatively charged phosphate moieties of the lipid
head groups, hence causing membrane thinning and
positive curvature strain. To release the strain, a frac-
tion of the peptides change their orientation from par-
allel to transversal, forming transient mixed
phospholipid–peptide toroidal pores. Upon disintegra-
tion of the pores, some peptides become translocated
to the inner leaflet of the membrane [27], suggesting
that stochastic pore disassembly may be a mechanism
by which antimicrobial peptides can reach the cell inte-
rior. Note that only a few pores exist after the redistri-
bution of the peptides between the two leaflets,
because the pore formation is a cooperative process.
Therefore, the integrity of the membrane is only tran-

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-

rial membranes as patches of antiparallel amphipathic
b-sheets forming rigid and thicker lipid domains
enriched in negatively charged lipids [29,30]. These
domains become ordered, mainly owing to the inser-
tion of aromatic residues into the hydrophobic bilayer
core. Zones of different rigidity and thickness bring
about phase boundary defects that lead to permeability
induction and peptides crossing through bacterial
membranes. Thus, the peptides could pass through the
membrane and interact with intracellular targets, as do
other Arg-rich peptides (see below).
The disordered toroidal pore model proposed that a
nanometer-sized, toroidal-shaped pore is formed by a
single a-helical or b-sheeted peptide that is able to
insert into the membrane, because of the difference in
mechanical stress between the two faces of the mem-
brane, and ⁄ or because of the different electric field, i.e.
the electroporation-like mechanism [31–34]. Above a
threshold number of membrane-bound peptides, one
peptide molecule becomes deeply embedded in the
membrane interface. The membrane–water interface
becomes unstable, and solvent molecules from the pep-
tide-free interface are able to interact with hydrophylic
groups of the embedded peptide, resulting in the devel-
opment of a continuous pore. In contrast to the Shai–
Matzusaki–Huang model of the toroidal pore, only
one peptide is found near the center of the pore, and
the remaining peptides lay close to the edge of the
pore, maintaining a parallel orientation with respect to
the membrane plane. The resulting pore is sufficient to

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

Receptor ⁄ docking component on inner membrane Dnak; DNA and protein synthesis
Apidaecin GNNRPVYIPQPRPPHPRI Receptor ⁄ docking component Dnak; DNA and protein synthesis
Drosocin GKPRPYSPRPTSHPRPIRV Receptor ⁄ docking component Dnak; DNA and protein synthesis
Bactenecin-7 RRIRPRPPRLPRPRPRPLPFPR
PGPRPIPRPLPFPRPGPRPIP
RPLPFPRPGPRPIPRP
Poly-proline-II helix? MIC: receptor ⁄ docking component
> MIC: membrane permeabilization ⁄ disruption
DNA?
Histatin-5 DSHAKRHHGYKRKFHEKHHSHRGY Amphipathic a-helix < MIC: receptor-mediated endocytosis
(heat shock protein 70, permease);
MIC: transient membrane leakage
(membrane potential-dependent)
Vacuole (nonlethal)
Mitochondrial F
1
F
0
-ATPase
Buforin-2 TRSSRAGLQFPVGRVHRLLRK Amphipathic a-helix Transient toroidal pores DNA?
Indolicidin
[K6,8,9]-Indolicin
ILPWKWPWWPWRRa
ILPWKKPKKPWRRa
Extended boat-shaped amphipathic
structure
No uptake
Unknown
DNA synthesis?
Magainin-2 GIGKFLHSAKKWGKAFVGQIMNS Amphipathic a-helix Transient toroidal pores ?

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.

Pro
⁄
Arg-rich antimicrobial peptides
Pro ⁄ Arg-rich antimicrobial peptides form a heterolo-
gous group of linear peptides isolated from mammals
and invertebrates that are predominantly active against
Gram-negative bacteria. Members of this group include
pyrrhocoricin, drosocin and apidaecin from insects, and
the cathelicidin-derived peptides bactenecin, PR-39 and
prophenin from mammals [36]. The mechanism of
action by which these peptides kill bacteria involve a
stereospecific interaction with a receptor ⁄ docking mole-
cule that may be a component of a permease-type
transporter system on the inner membrane, followed by
translocation of the peptide into the interior of the cell.
Once inside the cell, the peptides interact with the tar-
get, which, for pyrrhocoricin and drosocin, has been
clearly defined as the chaperone Dnak, or interfere with
DNA and protein synthesis through binding to nucleic
acids [37–39]. Interestingly, Bac-7 has recently been
shown to inactivate bacteria via two different modes of
action, depending on its concentration: (a) at near-MIC
concentrations via stereospecific-dependent uptake that
is followed by its binding to an unknown intracellular
target, which may be DNA; and (b) at concentrations
higher than the MIC via a nonstereospecific membran-
olytic mechanism [40].
Histatin
Histatin-5 is a 24 residue, His-rich and weakly aphi-
pathic a-helical antimicrobial peptide found in human

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

the internalization and accumulation, rapid expansion
of the vacuole with a parallel loss of cell volume is
observed, leading to cell death. Histatin-5 shows poor
translocation capacity in anionic liposomes. The
dependence of histatin-5 internalization on the
membrane potential may provide an explanation for a
single rupture per cell, rather than multiple breaches,
as once there is one site of leakage, the membrane
potential is lost, and this prevents a second rupture.
Buforin
Buforin II is a 21 residue truncated analog of buforin
I, the histone H2A-derived antimicrobial peptide,
which adopts a helix–hinge–helix structure in apolar
media [43]. Buforin II kills bacteria without lysing the
cell membrane, even at five-fold the MIC. It binds
selectively to negatively charged liposomes, and trans-
locates even below the MIC across artificial bilayers
efficiently via the transient formation of toroidal pores,
without inducing significant permeabilization or lipid
flip-flop. The induction of a positive curvature strain
by the peptide on the membrane is related to the trans-
location process [44,45]. Pro11 in the hinge region of
the peptide plays a key role in the cell uptake mecha-
nism by distorting the helix and concentrating basic
residues in a limited amphipathic region, thus destabi-
lizing the pore by electrostatic repulsion, enabling effi-
cient translocation [46] Confocal laser fluorescence
microscopy on living bacterial cells shows that, even
below the MIC, the peptide penetrates the cell mem-
brane and accumulates in the cytoplasm [47]. Although

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

[K6,8,9,11]-indolicidin bind strongly to DNA in vitro,
suggesting inhibition of intracellular functions via inter-
ference with DNA ⁄ RNA synthesis. Whether indolicidin
uses its membrane-binding properties to permeabilize
the cytoplasmic membrane, activate extracellular targets
or enter the cytoplasm and exert its antimicrobial activ-
ity by attacking intracellular targets is presently unclear.
Magainin
Magainin-2, an a-helical peptide isolated from the Afri-
can clawed frog Xenopus laevis, forms toroidal transient
pores in the lipid bilayer of liposomes near the MIC,
inducing lipid flip-flop and the translocation of peptides
into the inner leaflet of the bilayer coupled to mem-
brane permeabilization. Interaction of F5W-magainin-
2, an equipotent analog of magainin-2, with unfixed
Bacillus megaterium was investigated by confocal laser
microscopy [51]. At four times the MIC, magainin-2
binds to bacteria, permeabilizes the cytoplasmic mem-
brane within seconds, and internalizes simultaneously.
The influx of fluorescent markers of various size into
the cytosol revealed that magainin-2 permeabilizes the
bacterial membrane by forming toroidal pores with a
diameter of  2.8 nm. However, there is no informa-
tion available from which to evaluate whether magai-
nin-2 disrupts key intracellular processes, and, if so, to
what extent this may contribute to its killing action.
Polyphemusin
The horseshoe crab antimicrobial peptide polyphe-
musin I is a 18 amino acid peptide that is stabilized
into an amphipathic, antiparallel b-hairpin by two

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,

dimethyl sulfate protection and bleomycin-induced
DNA cleavage revealed that tachyplesin I interacts
with the minor groove of the DNA duplex in vitro
[58]. It is not known yet whether tachyplesin I is able
to enter living cells, and whether its antibiotic activity
is due to its capacity to bind DNA or to depolarize
the cytoplasmic membrane.
Pleurocidin
Pleurocidin and dermaseptins are a-helical antimicro-
bial peptides isolated from winterflounder and frog
skin, respectively. When used at its MIC, the hybrid of
pleurocidin and dermaseptin, P-der, inhibits E. coli
growth, but does not cause bacterial death within
30 min, and demonstrates a weak ability to permeabi-
lize the bacterial membrane [59]. When used at 10
times the MIC, the peptide causes rapid depolarization
of the cytoplasmic membrane and cell death, indicating
that the cell membrane is a lethal target for the peptide
applied at high concentrations. Both sublethal and
lethal concentrations of P-der inhibit macromolecular
P. Nicolas Intracellular-targeting antimicrobial peptides
FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS 6489
synthesis within 5 min. P-der is able to translocate
across the lipid bilayers of liposomes without causing
calcein leakage or flip-flop. It has been proposed that
pleurocidin translocates in vitro from one side of the
membrane to another through disordered transient
pores, allowing the peptide to reach the cell interior
[60]. As discussed above, membrane crossing remains
to be shown in living bacterial cells. In addition, the

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,

or pores, information on the internalization and the
fate of the peptide within microbial cells, as well as the
nature of the putative intracellular target, if any, needs
to be provided to decipher whether the microbicidal
activity of cryptdin-4 is due to its membrane permeabi-
lization ⁄ disruption effect or to its ability to impede
intracellular processes.
Tritrpticin
Tritrpticin consists of 13 residues and belongs to the
cathelicidin family of antimicrobial peptides from the
bone marrow of mammals. Tritrpticin has a broad
spectrum of antimicrobial activity, and exhibits a high
content of Trp (23%) and positively charged Arg ⁄ Lys
residues (31%). It adopts a well-defined amphipathic
turn–turn secondary structure in a membrane-mimetic
environment (organic solvents or dodecylphosphocho-
line micelles [62]. At high enough peptide concentra-
tions, interaction of tritrpticin with membranes was
postulated to cause positive curvature strain, which
leads to toroidal pore formation membrane permeabili-
zation and cell death in accordance with the Shai–Mat-
zusaki–Huang model. In contrast, TWF, in which Trp
is replaced with Phe, is highly potent against both
S. aureus and E. coli, but shows very little membrane-
disrupting activity and no ability to depolarize the
membrane potential of the microbial cell targets [9].
Moreover, a lag period of about 3–6 h is required for
bactericidal activity. It was thus suggested that TWF-
mediated cell death occurs as a result of a nonmem-
branolytic mechanism, but testing of this hypothesis

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

dynamic functioning of membrane-bound multienzyme
complexes, rather than killing through interaction with
intracellular targets, has been largely ignored [22]; (e)
several putative microbial cell-penetrating peptides are
Intracellular-targeting antimicrobial peptides P. Nicolas
6490 FEBS Journal 276 (2009) 6483–6496 ª 2009 The Author Journal compilation ª 2009 FEBS
synthetic analogs of naturally occurring antimicrobial
peptides that differ from the parent molecule by one
or more amino acid substitutions – as it is well known
that the microbicidal potency and selectivity of antimi-
crobial peptides, as well as their membrane-bound
structure and mode of action, are exquisitely sensitive
even to single amino acid substitutions, the penetrating
properties of the analog may not represent that of the
parent peptide; and (f) most antimicrobial peptides
that are proposed to attack internal targets exhibit an
overrepresentation of one or two amino acids, i.e. Trp
and Pro, His, or Arg, hence resembling cell-penetrating
peptides (see below).
Cell-penetrating peptides working
as antimicrobial peptides, and
antimicrobial peptides working
as cell-penetrating peptides
A substantial number of mammalian cell-penetrating
peptides, including TP-10, pVEC, Tat, Pep-1, MAP,
and penetratin, have the capacity to work as cell-pene-
trating peptides or as antimicrobial peptides, the
threshold between these two properties relying on the
composition on the membrane and the peptide concen-
tration (Table 2). Their microbicidal action is thought

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

[68,69]. It was suggested that the microbial cell mem-
brane permeabilization might not be the only mode of
peptide uptake. For instance, the import route of
pVEC by B. megaterium is consistent with two distinct
uptake mechanisms: one operating via a transporter
with high affinity and low capacity, which is sensitive
to the chirality of the peptide and reminiscent of that
of histatin-5; and another with low affinity and high
capacity that could be caused by the membrane-
permeabilizing activity of the peptide.
Tat(47–58), an Arg-rich cell-penetrating peptide
derived from the HIV-1 regulatory protein Tat, exhibits
antimicrobial activity against Gram-positive and
Gram-negative bacteria, and antifungal activity against
Malassezia furfur, Saccharomyces cerevisiae and Tricho-
sporon beigelii in the low micromolar range [71]. Tat
showed no ability to depolarize the membrane potential
of S. aureus cells and to leak calcein-entrapped nega-
tively charged lipid vesicles. Tat peptide internalizes in
the fungal cells and rapidly accumulates in the nucleus
without causing visible damage to the cell membrane.
The penetration pathway of Tat is independent of
energy, time, and temperature. After penetration, the
peptide blocks the cell cycle process of Candida albicans
through arrest at G
1
phase.
Pep-1 is a synthetic cell-penetrating peptide com-
posed of an N-terminal Trp-rich domain and a C-ter-
minal nuclear signal domain, KKKRKV [72], which

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-

ster ovary K1 cells, showed that magainin-2 permeabi-
lized the cells, forming pores in the cell membrane that
allowed the entry of a large molecule (diame-
ter, > 23 nm) into the cytosol. Pore formation and
subsequent cell entry are closely related to cell death.
The peptide is internalized within a time scale of tens
of minutes [44,51], and once it has entered the cell,
accumulates in mitochondria and nuclei. The permea-
bilization of Chinese hamster ovary cells was accompa-
nied by extensive deformation, including membrane
budding. Whether magainin-2 kills mammalian cells by
dissipating membrane potential or damaging mito-
chondria is presently unknown. Likewise, studies of
buforin suggest a similar ability to translocate into
mammalian cells, but by a temperature-independent,
less concentration-dependent passive mechanism, and
without showing any significant cytotoxicity [51].
These observations show that mammalian cell-pene-
trating ability and microbial cell-permeabilizing ability
can coexist within a single peptide, but the unifying
rules that govern these two properties remain to be
fully elucidated.
Broadly speaking, evidence exists for two main,
simultaneous mammalian cell-entering pathways,
including direct penetration of peptides in parallel with
different forms of endocytosis, the endocytosic path-
way being a preferred form of entry of cell-penetrating
peptides, at least when attached to bioactive cargo.
The direct penetration mechanism remains elusive, and
has long been thought not to involve membrane dam-

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

with the existence of a transmembrane potential and an
appropriate balance between hydrophobicity and
hydrophylic surface groups. Interestingly, bidendate
hydrogen bonding of the guanidinium groups of prote-
grin, an Arg-rich antimicrobial peptide, with the phos-
phate groups of the bilayer was demonstrated to be
crucial for insertion and pore formation of the peptide
within bacterial membranes [78]. Molecular dynamic
simulations of the Tat peptide crossing zwitterionic
membranes suggest a mechanism of translocation that
involves thinning of the membrane bilayers with
increasing concentrations of Tat, owing to strong inter-
actions between the guanidinium groups of the peptide
and the phosphate groups on both sides of the mem-
brane bilayers [35]. This is followed by the insertion of
charged side chains into the bilayer. As the charged side
chains enter the acyl core of the membrane, water also
penetrates and solvates the charged groups, favouring
the formation of a transient pore. Once the pore is
formed, the Tat peptide translocates across the mem-
brane by diffusing on the pore walls. The fast, tran-
sient nature of the pore may explain why mammalian
cell death because of membrane leakage was not
observed with Tat [35]. This mechanism is highly
reminiscent of the disordered toroidal pore-electro-
poration mechanism proposed for some antimicrobial
peptides. This suggests that general mechanisms that
involve fluctuations of the membrane surface, such as
transient pores and the insertion of charged side
chains, may be common and central to the functions

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-

bicity, amphipathicity, and membrane-bound confor-
mation. From the limited data currently available, the
specific translocating properties of some antimicrobial
peptides are likely to be specific and limited to partic-
ular peptide families.
When looking for different parameters that could
promote the cellular penetration properties of antimi-
crobial peptides, it is noticeable that microbial cell-
penetrating antimicrobial peptides and antimicrobial
cell-penetrating peptides have very distinct sequences,
but, nonetheless usually share several characteristics,
such as their high positive net charge, clustered posi-
tive charges, and an overrepresentation of one or two
amino acids, i.e. Arg, Trp, and, albeit to a lower
extent, His. Among these peptides, Arg-rich peptides
are the most represented. As cell penetration occurs not
only in microbial cells, but also in mammalian cells, it
is tempting to assume that, despite controversies about
mechanisms and artefactual ⁄ incomplete results, the
biased amino acid composition of the peptides, and
especially the contribution of Arg residues, plays a key
role in the membrane translocation process.
Acknowledgements
This work was supported by the Universite
´
Pierre et
Marie Curie and the Association Nationale pour la
Recherche (ANR-Prob DOM). We apologize to those
authors whose work could not be cited because of
space constraints.

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