Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides

MINIREVIEW

Multifunctional host defense peptides:intracellular-targeting antimicrobial peptidesPierre Nicolas

Biogenese 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, Biogenese des Signaux

Peptidiques (BIOSIPE), ER3-UPMC,

Universite Pierre et Marie Curie, Batiment A

– 5eme etage, Case courrier 29, 7 Quai

Saint-Bernard, 75005 Paris, France

Fax: +1 44 27 59 94

Tel: +1 44 27 95 36

E-mail: [email protected]

(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 ⁄disruptionis 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 permeabilizationversus 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 varyingantimicrobial potencies exhibitdisparate extents of membranepermeabilization 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 exhibittemporal dissociation betweenmicrobial membrane permeabilizationand 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 caninfluence the outcome of an in vitrostudy 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 ofantimicrobial 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 entermicrobial 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


peptide-induced membrane permeabilization ⁄disruptionof 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 antimicrobialpeptides

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-



Tab

le1.

Am

ino

acid

sequence,

mem

bra

ne-b

ound

str

uctu

reand

suggeste

din

tern

aliz

ation

mechanis

mand

eff

ect

on

mic

robia

lfu

nctions

of

intr

acellu

lar-

targ

eting

antim

icro

bia

lpeptides.

Hydro

phobic

resid

ues

are

inbold

.a,

carb

oxam

itaded.

Nam

eS

equence

Mem

bra

ne-b

ound

Str

uctu

reU

pta

ke

mechanis

mIn

tracellu

lar

targ

ets

Pyrr

hocoricin

VD

KG

SY

LP

RP

TP

PR

PIY

NR

NR

evers

etu

rns

at

the

term

ini

bridged

by

an

exte

nded

segm

ent

Recepto

r⁄d

ockin

gcom

ponent

on

inner

mem

bra

ne

Dnak;

DN

Aand

pro

tein

synth

esis

Apid

aecin

GN

NR

PV

YIP

QP

RP

PH

PR

IR

ecepto

r⁄d

ockin

gcom

ponent

Dnak;

DN

Aand

pro

tein

synth

esis

Dro

socin

GK

PR

PY

SP

RP

TS

HP

RP

IRV

Recepto

r⁄d

ockin

gcom

ponent

Dnak;

DN

Aand

pro

tein

synth

esis

Bacte

necin

-7R

RIR

PR

PP

RLP

RP

RP

RP

LP

FP

R

PG

PR

PIP

RP

LP

FP

RP

GP

RP

IP

RP

LP

FP

RP

GP

RP

IPR

P

Poly

-pro

line-I

Ihelix

?M

IC:

recepto

r⁄d

ockin

gcom

ponent

>M

IC:

mem

bra

ne

perm

eabili

zation

⁄dis

ruption

DN

A?

His

tatin-5

DS

HA

KR

HH

GY

KR

KFH

EK

HH

SH

RG

YA

mphip

ath

ica-h

elix

<M

IC:

recepto

r-m

edia

ted

endocyto

sis

(heat

shock

pro

tein

70,

perm

ease);

MIC

:tr

ansie

nt

mem

bra

ne

leakage

(mem

bra

ne

pote

ntial-dependent)

Vacuole

(nonle

thal)

MitochondrialF

1F

0-A

TP

ase

Bufo

rin-2

TR

SS

RA

GLQ

FP

VG

RV

HR

LLR

KA

mphip

ath

ica-h

elix

Tra

nsie

nt

toro

idalpore

sD

NA

?

Indolic

idin

[K6,8

,9]-

Indolic

in

ILP

WK

WP

WW

PW

RR

a

ILP

WK

KP

KK

PW

RR

a

Exte

nded

boat-

shaped

am

phip

ath

ic

str

uctu

re

No

upta

ke

Unknow

n

DN

Asynth

esis

?

Magain

in-2

GIG

KFLH

SA

KK

WG

KA

FV

GQ

IMN

SA

mphip

ath

ica-h

elix

Tra

nsie

nt

toro

idalpore

s?

Poly

phem

usin

IR

RW

CFR

VC

YR

GFC

YR

KC

Ra

Am

phip

ath

icb-h

airpin

with

two

dis

ulfi

de

bonds

Tra

nsie

nt

pore

s?

?

Tachyple

sin

IK

WC

FR

VC

YR

GIC

YR

RC

Rb-H

airpin

with

two

dis

ulfi

de

bonds

Tra

nsie

nt

pore

sD

NA

?

Ple

uro

cid

in(P

-Der)

ALW

KTM

LK

KA

AH

VG

KH

V

GK

AA

LTH

YLa

Am

phip

ath

ica-h

elix

MIC

:dis

ord

ere

dtr

ansie

nt

pore

s

>M

IC:

mem

bra

ne

perm

eabili

zation

Macro

mole

cula

rsynth

esis

Cry

ptd

in-4

GLLC

YC

RK

GH

CK

RG

ER

VR

GTC

GIR

FLY

CC

PR

R

Triple

-str

anded

b-s

heet

with

thre

e

dis

ulfi

de

bonds

Tra

nsie

nt

pore

sor

defe

cts

?

Tritr

pticin

TW

F

TP

A

VR

RFP

WW

WP

FLR

R

VR

RFP

FFFP

FLR

R

VR

RFA

FFFA

FLR

R

Am

phip

ath

ictu

rnstr

uctu

reU

pta

ke

not

show

n

Upta

ke

not

show

n

? ?



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 F1F0-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



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



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 ⁄Lysresidues (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



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 workingas antimicrobial peptides, andantimicrobial peptides workingas 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 G1 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



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-



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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Multifunctional host defense peptides: intracellular-targeting antimicrobial peptides

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