Jack2 1 Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan;2Institut fu¨r Organische Chemie, Universita¨t Tu¨bingen, Germany Different pathways of bilayer disruption b
Trang 1Distinguishing between different pathways of bilayer disruption
by the related antimicrobial peptides cecropin B, B1 and B3
Hueih Min Chen1, King Wong Leung1, Nagendra N Thakur1, Anmin Tan1,* and Ralph W Jack2
1
Institute of BioAgricultural Sciences, Academia Sinica, Taipei, Taiwan;2Institut fu¨r Organische Chemie,
Universita¨t Tu¨bingen, Germany
Different pathways of bilayer disruption by the structurally
related antimicrobial peptides cecropin B, B1 and B3,
revealed by surface plasma resonance analysis of
immobi-lized liposomes, differential scanning calorimetry of peptide–
large unilamellar vesicle interactions, and light microscopic
analysis of peptide-treated giant unilamellar vesicles, have
been identified in this study Natural cecropin B (CB) has one
amphipathic and one hydrophobic a-helix, whereas
cecro-pins B1 (CB1) and B3 (CB3), which are custom-designed,
chimaeric analogues of CB, possess either two amphipathic
or two hydrophobic a-helices, respectively Surface plasma
resonance analysis of unilamellar vesicles immobilized
through a biotin–avidin interaction showed that both CB
and CB1 bind to the lipid bilayers at high concentration
(>10 lM); in contrast, CB3 induces disintegration of the
vesicles at all concentrations tested Differential scanning
calorimetry showed the concentration-dependent effect of
bilayer disruption, based on the different thermotrophic phase behaviours and the shapes of the thermal phase-transition curves obtained The kinetics of the lysis of giant unilamellar vesicles observed by microscopy demonstrated that both CB and CB1 effect a continuous process involving loss of integrity followed by coalescence and resolution into smaller vesicles, whereas CB3 induces rapid formation of irregular-shaped, nonlamellar structures which rapidly dis-integrate into twisted, microtubule-containing debris before being completely destroyed On the basis of these observa-tions, models by which CB, CB1 and CB3 induce lysis of lipid bilayers are discussed
Keywords: differential scanning calorimetry; lysis mechan-ism; lytic peptides; microscopic analysis; surface plasma resonance
Cationic antimicrobial peptides have now been isolated
from a wide variety of sources including bacteria,
inverte-brates, vertebrates and plants [1–7] Although their structure
and chemical nature differ markedly, their function appears
to involve protection of the producing organism from
competing or pathogenic micro-organisms, and their
acti-vity may be directed towards a variety of bacteria, protozoa,
fungi and/or viruses In bacteria, the production of cationic
antimicrobial peptides is probably a survival strategy to
obtain an ecological advantage over competitors [8–10] In
invertebrates and plants, which lack the adaptive immune
system of higher animals, these peptides represent a major
component of innate immune defence [11–13] Even
vertebrates rely heavily on the protection against infection
offered by the innate immune system, in which the defence peptides play a pivotal role [14,15]
The general mechanism by which cationic antimicrobial peptides bring about cell death appears to involve perme-abilization of the phospholipid bilayer membranes border-ing their targets However, closer inspection of this activity reveals significant differences between various peptides Several cationic antimicrobial peptides from bacteria, including the lantibiotics nisin and epidermin and the bacteriocin pediocin PA-1, are most effective against specific target organisms and utilize docking molecules present in the cell membrane in a mechanism that stabilizes the formation of ion-permeable pores [16–19] As a result, these peptides are active at nanomolar concentrations and show limited target range with little or no observable activity against membranes of eukaryotic organisms; they may be active in the absence of the docking molecules (e.g artificial bilayers), but at significantly higher peptide concentrations Conversely, many of the cationic defence peptides of eukaryotic origin are active at micromolar concentrations, but show little target specificity and may act on many membrane types including those of erythrocytes (haemolytic activity) For example, the synthesis of a variety of analogues of the 13-amino acid, tryptophan-rich bovine antimicrobial peptide indolicidin has suggested that chiral and/or sequence-specific determinants are not required for either antibacterial or haemolytic activity, although target specificity is strongly influenced by the overall physico-chemical nature of the analogue [20], and similar results
Correspondence to H M Chen, Institute of BioAgricultural
Sciences, Academia Sinica, Taipei, Taiwan 115.
Fax: + 886 2 2788 8401, Tel.: + 886 2 2785 5696 ext 8030,
E-mail: robell@gate.sinica.edu.tw
Abbreviations: CB, cecropin B; CB1, cecropin B1; CB3, cecropin B3;
ESR, electron spin resonance; DSC, differential scanning calorimetry;
GUV, giant unilamellar vesicle; LUV, large unilamellar vesicle;
PA, phosphatidic acid; PtdEtn, phosphatidylethanolamine;
RU, resonance units; SPR, surface plasma resonance.
*Present address: Department of Biophysical Chemistry, Biocenter,
University of Basel, Basel, Switzerland.
(Received 30 July 2002, revised 28 October 2002,
accepted 7 January 2003)
Trang 2have been obtained with analogues of the insect defence
peptide cecropin B [21] Thus, the cationic defence peptides,
which are active against a broad range of targets, seem to
offer the best opportunity for the study of the molecular
mechanisms involved in generalized pore formation in the
absence of specific stabilizing structures
Because of their relatively broad spectrum of activity, it
has been suggested that defence peptides may offer an
alternative source of antimicrobial chemotherapeutics
[22,23] Moreover, it has also been shown that certain
magainins (isolated from frog skin) and cecropins
(insect-derived peptides) also possess antitumour cell activities,
making these peptides of special interest as molecular
models for the study of pore formation in biological
membranes [24–27] Structure–function analysis of the
various subclasses of defence peptides, combined with
dissection of the molecular events involved in pore
forma-tion, offers the possibility to develop customized peptides
with defined activities for anti-infective and antitumour
applications
In general, two models for disruption of membrane
integrity have generally been adopted: pore formation by
the barrel-stave model, for which alamethicin and melittin
are considered prime examples, and the flip-flop action
(so-called carpet-like action) of peptides such as cecropin
P1 [28–32] However, it is not currently possible to assess the
likely mechanism of membrane disruption of a given
peptide simply on the basis of its structure Thus, we have
used specific peptides with high homology, but which
contain specific structural variations, to investigate the
relationship between these two cell-killing models [33,34]
We have previously reported the synthesis and biological
activities of three model peptides: native cecropin B (CB),
which contains both a hydrophobic and an amphipathic
helix, and the custom-designed, synthetic analogues
cecro-pin B1 (CB1) and cecrocecro-pin B3 (CB3), which contain two
amphiphilic and two hydrophobic helices, respectively
[27,35–42] Recently, we explored the kinetics of liposome
lysis using fluorescent quenching and used surface plasma
resonance (BIACore) and oriented circular dichroism [43] to
investigate the two different modes of membrane disruption
and to detect the orientation of the peptides with respect to
the membrane surface [42] Kinetic analysis suggests that
bilayer disruption occurs in two distinct steps, whereas
oriented circular dichroism provides evidence that the
peptides exist in at least two different membrane-associated
states, depending on the orientation of the helical segments
with respect to the bilayer surface Similar transitions of
peptide orientation in membrane multilayers have also been
shown for melittin, alamethicin and magainin [44,45]
In this study, we examined and compared the disruptive
mechanisms of the natural peptide (CB) and the custom
peptide analogues (CB1) and (CB3) against liposomes, large
unilamellar vesicles (LUVs) and giant unilamellar vesicles
(GUVs) using surface plasma resonance (SPR), differential
scanning calorimetry (DSC) and real-time light microscopy,
respectively The results indicate that the different
mecha-nisms of disruption of lipid bilayers observed are dependent
on the particular physicochemical characteristics of the
different peptides Investigation of such differences will
provide useful information for the future custom design of
peptidyl and mimetic drugs
Experimental procedures
Materials Phosphatidylethanolamine (PtdEtn, an egg derivative con-taining fatty acids of C16, C18 and C20) 1,2-Diacyl-sn-glycero-3-phosphatidic acid (PA) is a monosodium salt synthetic lipid (98% purity) The symmetric fatty acid of PA contains 18-carbon double chains Both PtdEtn and PA were obtained from Sigma-Aldrich (St Louis, MO, USA) and were used without further purification CM5 Sensor Chips, amine coupling kits, and BIAevaluation software were all purchased from BIAcore AB (Uppsala, Sweden) Analytical grades of Triton X-100 and SDS were obtained from Sigma-Aldrich, and Biotin-X-DHPE (N-{[6-(bioti- noyl)amino]hexanoyl)}-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine) was purchased from Molecular Probes (Eugene, OR, USA) Avidin obtained from Sigma-Aldrich is made from egg white and is chromatographically purified and a lyophilized powder One unit of avidin [12.3 UÆ(mg solid))1] will bind 1.0 lg biotin All water used
in these experiments was deionized and distilled
Peptide sequences, preparation and solutions The preparation of the 35-amino-acid peptide amides
AKAL)CONH2), CB1 (H)KWKVFKKIEKMGRNIRNGI VKAGPKWKVFKKIEK)CONH2) and CB3 (H)AIAVLGE
used throughout this study has been previously described [35]; CB contains a 10-amino-acid amphiphilic (italicized) and a 10-amino-acid hydrophobic (underlined) helix, and CB1 contains two 10-amino-acid amphiphilic helices (itali-cized) and CB3 contains two 10-amino-acid hydrophobic helices (underlined), each derived from the parent, CB The peptides were all > 95% pure, as determined by RP-HPLC, and were stored lyophilized at )20 °C until use The concentration of peptide stock solutions was determined from the net weight of peptides (the weight of the associated counter ions was not taken into consideration) and their molecular masses Concentrations measured by the above method were confirmed with a bicinchoninic acid assay (Micro BCA protein assay; Pierce Chemical Co.) A negligible deviation between these two methods was observed
Analysis of liposome–peptide interactions by SPR Peptides were dissolved in HBS-EP buffer [10 mMHepes, 0.15MNaCl, 3 mMEDTA and 0.005% surfactant P20 (or Tween 20), pH 7.4] to give a stock solution with a concentration of 500 lM, and these were further diluted as necessary in the same buffer Surfactant P20 is a 10% aqueous solution of the nonionic surfactant Polysorbate 20 The purpose of the surfactant is to reduce sample loss caused by the adsorption of hydrophobic molecules to the surfaces of the flow system of the BIAcore instrument Peptides dissolved in HBS-EP buffer are not lysed by the liposomes Avidin was prepared as a stock at 50 lgÆmL)1 dissolved in 10 mM sodium acetate (pH 4.8) To prepare biotin-containing liposomes, PtdEtn (7 mg) and PA (3 mg)
Trang 3were dissolved in 1 mL chloroform, and 50 lg
biotin-X-DHPE was added before removal of the chloroform in an
argon stream Instead of our previous use of
phosphatidyl-choline/PA (7 : 3) [27,33–42], PtdEtn used in these
experi-ments is for the comparison with a microbial lipid
The lipid film was rehydrated in 2 mL HBS-EP buffer,
sonicated for 30 s, and liposomes were produced after 30
passes through a LipoFast extruder fitted with two 100-nm
polycarbonate filter stacks
SPR experiments were conducted using a BIAcore 2000
biosensor (Biacore) Avidin was immobilized on the sensor
surface first using EDC
[1-ethyl-3-(3-dimethylaminopro-pyl)carbodiimide] and NHS (succinimide), and the activated
esters that had not bound ligand were capped with
ethanolamine The maximum avidin loading was achieved
by optimizing the flow rate used (1 lLÆmin)1) Anionic
liposomes (1 mgÆmL)1) were then immobilized on the
surface of the chip using various flow rates Peptides
dissolved in HBS-EP buffer were applied at the
concentra-tions and flow rates indicated in order to assess their effect
on the immobilized liposomes (1 lLÆmin)1is the optimum)
HBS-EP was used a running buffer An avidin-free chip
surface, liposomes without biotin, or applications of BSA
served as controls to assess the specificity of the responses
observed Peptide-induced responses were measured by
SPR, and results were collected and analysed using the
manufacturer’s software and protocols A diagram of the
construction of the biosensor surface is shown in Fig 1
Binding of peptides to the immobilized liposomes causes an
increase in resonance units (RU), whereas the disruption of
liposomes results in a decrease in RU The sensor chip
surface was regenerated by using 10 mMHCl
DSC
LUVs for use in DSC experiments were prepared from
PtdEtn and PA by dissolving 3.5 mg PtdEtn and 1.5 mg PA
(PtdEtn/PA¼ 7 : 3, w/w) in 1 mL chloroform After
removal of the solvent at room temperature under an argon
gas stream, the lipids were rehydrated in 1 mL NaCl/Piand
then sonicated for 30 s LUVs were subsequently formed
by extrusion through a LiposoFast extruder fitted with
two 100-nm polycarbonate filters for 25 repetitions The
thermotrophic phase behaviour of the LUVs was measured
in a nano differential scanning calorimeter (Calorimetry Sciences Corp., Provo, UT, USA), with temperature scans performed at a rate of 1°CÆmin)1 The DSC cells were pressurized to 0.31 kgÆcm)2 (0.30 MPa) throughout the experiment, and liposomes were used undiluted (i.e at
5 mgÆmL)1) Peptides, dissolved in NaCl/Piwere added to give final concentrations in the range 1–100 lM, and controls consisted of DSC analysis of each peptide in the absence of lipid and the liposomes without peptide Preparation of GUVs
GUVs were used for light microscopic observation of the membrane disruptive effects of the peptides (see section below) To obtain GUVs, we used the method of Akashi
et al [46] with some modification Briefly, PtdEtn and PA (7 : 3) were dissolved together in chloroform/methanol (2 : 1, v/v) to a final concentration of 10 mgÆmL)1 and stored under argon at)20 °C Subsequently, 10-lL aliquots were placed in the bottom of a 1.5-cm diameter, 10-mL glass tube, and the solvent was dried at room temperature in a stream of argon gas, to provide a thin lipid film After removal of residual solvent under high vacuum for at least
10 h, the film was prehydrated with a stream of water-saturated argon gas for 30 min at 43–45°C and then fully rehydrated by addition of 2 mL argon-saturated 1 mM MgCl2 solution for 48 h at 37°C MgCl2 concentration dependence experiments on GUV lysis induced by peptides were conducted and it was found that the electrostatic interactions between peptides and lipids are little influenced
by the addition of MgCl2 The liposomes, which appear as
an almost transparent, milky-white cloud in the middle of the solution, were collected and stored at 4°C in plastic tubes
Microscopic observation of GUVs The stored GUVs were typically diluted 10-fold to prevent liposome fusion, and 40-lL aliquots were observed on an inverted microscope (Diaphot TMD; Nikon) under phase contrast, and images were captured with a charge-coupled-device camera (microflex UFX-DX; Nikon) The unilamel-lar nature of liposomes was assessed optically; those with an appropriately thin contour were judged unilamellar and were used for further study Peptide solutions (20 lL of a 100-lM stock, final concentration 33.3 lM) were added after selection of a single GUV (diameter generally 40–120 lm)
Results
SPR analysis of peptide–liposome interactions:
effect of peptides on immobilized liposomes Analysis of chips carrying immobilized PtdEtn/PA lipo-somes containing biotin-X-DHPE revealed that the SPR response varies greatly with both the type and concentration
of the peptide applied At higher concentrations (> 10 lM)
of either CB or CB1 (Fig 2), an increase in relative response was observed, indicating a time-dependent increase in mass and suggesting that the peptides were interacting intimately
Fig 1 Diagram detailing the liposome immobilization procedure.
Liposomes were immobilized by binding of embedded biotin with
avidin-EDC/NHS, which in turn was first immobilized on the surface
of the sensor chip Peptides passing over the immobilized liposome
either bind to the liposome or disrupt it.
Trang 4with the liposome membranes Moreover, this effect was
slightly higher for CB1, as indicated by the higher relative
response (Fig 2A,C) However, at lower peptide
concen-trations (< 2.5 lM), the relative response was negative
These negative observations may be due to the lower flow
rates used in this experiment Further experiments will be
performed to clarify this Figure 2B,D shows the
non-specific binding of CB and CB1 (respectively) to the chip
surface in the absence of liposomes; the level of nonspecific
binding between peptides and sensor chip surface should,
however, be significantly lower when immobilized liposomes
mostly occupy the chip surface By contrast, SPR analysis of
the disruptive effects of CB3 generates negative RU values
for all concentrations tested (Fig 2E) About 80% of the
liposome RU is lost at 100 lMCB3 Moreover, the amount
of nonspecific binding of CB3 to the sensor chip surface in
the absence of immobilized liposome was not significant
(Fig 2F)
The negative RU values observed with CB3 at all
concentrations when passing through the immobilized
liposomes were consistent compared with the positive RU
values of peptides with nonspecific bindings Plots of
peptide concentration against the relative reduction in
mass estimated from the SPR response (data not shown)
suggested that CB3 caused a time-dependent disruption of
membrane integrity (Fig 3C); the effect was more
pronounced when higher concentrations of CB3 were
applied
DSC analysis of the effect of peptides
on PtdEtn/PA LUV thermotrophic phase behaviour The effect of CB, CB1 and CB3 on the thermotrophic phase behaviour of LUVs is shown in Fig 4 The phase-transition temperature (Tm) for the PtdEtn/PA (7 : 3) liposome varied considerably as a function of peptide concentration (data not shown) Typical examples were shown at two peptide concentrations (lower concentration at 1 lMand 20 lMfor CB/CB1 and CB3, respectively; higher concentration at
20 lMand 50 lMfor CB/CB1 and CB3, respectively) In the case of the parent peptide (CB) with liposomes, low concentrations of peptide (1 lM) lowered Tmby 1.7 °C and broadened the DSC endotherm as compared with liposome alone Higher concentrations of peptide (20 lM) resulted in DSC endotherms that split to yield multiple peaks, one of which was considerably lower than the Tmof the liposomes alone, and one of which yielded a Tmeven higher than that for LUVs in the absence of peptide These two distinguishable outcomes of concentration-dependence are comparable to those observed by SPR (Fig 2A) Similar results were obtained for the analogue CB1, although the peak splitting at higher peptide concentrations was less pronounced than for the parent peptide However, a slightly different situation could be discerned for the analogue CB3; considerably higher concentrations (20 lM) were required
to obtain a decrease in the DSC endotherm similar to that observed with 1 l CB or CB1 At even higher peptide
Fig 2 Effect of peptides CB, CB1 and CB3on biotin-loaded PtdEtn/PA (7 : 3, w/w) liposomes bound to the surface of a sensor chip and comparison with nonspecific binding to surfaces devoid of liposomes (A) and (B) Application of various concentrations (0.25, 0.5, 0.75, 2.5, 10, 50 and 100 l M ) of peptide CB to the surface of a sensor with and without (respectively) biotin-loaded PtdEtn/PA liposomes (C) and (D) Application of various concentrations (0.25, 0.5, 0.75, 2.5, 10, 50 and 100 l M ) of peptide CB1 to the surface of a sensor with and without (respectively) biotin-loaded PtdEtn/PA liposomes (E) and (F) Application of various concentrations (0.25, 0.5, 0.75, 2.5, 10, 50 and 100 l M ) of peptide CB3 to the surface of a sensor with and without (respectively) biotin-loaded PtdEtn/PA liposomes.
Trang 5concentrations (50 lM), more peak splitting was observed,
although it was not as pronounced as that measured with
the other two peptides
Control experiments (flat lines shown in each figure)
using samples of each peptide at concentrations of 20 lM
(CB and CB1) and 50 lM(CB3) in the absence of liposomes
suggested that endothermic variations observed in the
presence of liposomes arise exclusively from phase
transi-tions in the liposomes, which are effected by antimicrobial
peptides
Microscopic investigation of peptide-induced disruption of GUVs
Our initial optimization studies showed that the most stable GUVs were formed in low salt concentrations, in particular MgCl2(1 mM), and that liposome stability increased with increasing incubation time, although the relative size decreased with increasing stability (data not shown) With the optimized procedure given in Materials and Methods,
we routinely produced stable GUVs with diameters ranging between 40 and 120 lm To study the step-by-step disrup-tion of a GUV, we added the peptide soludisrup-tion and allowed it
to reach the target by diffusion When CB was added in this way (Fig 5A), no observable alteration to the GUV occurred within the first 1–2 min, but, after this initial lag phase, the liposome began to decrease in size without evidence of significant micelle or small liposome formation (Fig 5B–D) Finally, during the terminal stages of liposome disintegration induced by CB, the GUV transformed rapidly into a series of very small vesicles which eventually disappeared (Fig 5D)
Similar analysis of the effect of the analogues CB1 and CB3 revealed several differences When a GUV was exposed
to CB1 (Fig 5E), the lag phase was markedly shorter, at
30–45 s Moreover, disintegration of the GUV was hastened in comparison with that observed with CB, with small vesicles rapidly becoming visible Subsequently, 4–5 min after addition of the peptide, the liposome crum-pled to leave multivesicular and debris-like structures (Fig 5F–H) The presence of CB1 leads to a propensity to form mutlilamellar vesicles and other fused structures, not observed with the parent peptide (data not shown) By contrast, a markedly different disintegration pattern was observed when the GUV was exposed to CB3 (Fig 5I–L) After an initial lag phase of 2–3 min, the vesicles began to rapidly fluctuate in size and shape, deviating strongly from their normally spherical appearance Moreover, hair-like microtubules appeared with increasing frequency, and liposomes that had deviated significantly from their spheri-cal form either burst to leave only the microtubules or rearranged into substantially smaller, microtubule-covered spherical structures
Discussion
Cationic antimicrobial defence peptides such as cecropins [1] disrupt cell membranes, and thereby kill their targets, by associating with the membrane lipids [21] The mechanism
of bilayer disruption by cecropin A was recently shown to occur via an ion channel (pore-forming model), and this system has been extensively analysed and characterized using solid-state NMR [47–49] In addition, experiments using synthetic enantio analogues of a variety of defence peptides, including cecropins, magainins and melittin or melittin-cecropin chimaeras, have shown that these peptides
do not function via enzymatic processing or a chiral-specific receptor, but by formation of ion-conducting pores com-posed of self-aggregates [21,50] However, an alternative model to the barrel-stave pore formation, involving a
parallel arrangement of peptides on the membrane surface (carpet-like model) has also been suggested for the antimicrobial peptide dermaseptin [51] This alternative
Fig 3 Comparison of the change in relative response (DRU) measured
by SPR for biotin-loaded PtdEtn/PA (7 : 3, w/w) liposomes coupled to
the surface of a sensor chip and treated with various concentrations of (A)
CB, (B) CB1, or (C) CB3 DRU, Difference between RU at the time of
association and the time after the dissociation: i.e the total capacity of
peptides bound to the immobilized liposomes.
Trang 6model further suggests that, as a result of this mechanism of
disruption, the membrane decomposes into fragments after
a threshold concentration of peptide has been reached [52]
In this study, we have demonstrated the distinction between
these two models of membrane disruption, using closely
related but structurally different chimaeric peptides from
within the same general family by microscopic,
thermo-dynamic and biosensor analysis of specific peptide–lipid
interactions
Microscopic analysis of the effects of each of the
peptides on LUVs reveals differences in their respective
activities in real-time Interestingly, despite their differences
in structure, the peptides CB (one amphiphilic helix and
one hydrophobic helix) and CB1 (two amphiphilic helices)
appeared to have a similar overall effect on the liposome
integrity, except that a longer lag time preceded visible
effects in the case of CB and the vesicles were more rapidly
disrupted by CB1 than by the parent peptide These
observations may suggest that the more hydrophilic and
cationic CB1 is more rapidly attracted to the anionic
bilayer and that the amphiphilic helix is the predominant
cause of membrane disruption when it is present Overall,
the observable effect of these peptides resembles that of
phospholipase A2from cobra (Naja naja) venom [53] In
contrast, the peptide CB3 (two hydrophobic helices)
showed a markedly different disruptive effect on the
liposome bilayer, particularly evidenced by loss of the
spherical liposome structure and the formation of many
hair-like microtubules Overall, these microscopic
observa-tions are consistent with previous suggesobserva-tions that the
peptides CB and CB1 may act by pore formation, whereas
CB3 may cause gross membrane destabilization
DSC is a thermodynamic technique for probing the
nature, stoichiometry, location, and aggregation state of
peptides in their lipid-bound state by analysis of the thermal
transitions that these peptide–lipid interactions generate and
has been shown to provide valuable information for the
analysis of materials that destabilize membrane structure [54,55] DSC analysis of the thermotrophic phase behaviour
of membranes exposed to CB and CB1 revealed that their effect is strongly concentration-dependent; low concentra-tions (1 lM) lowered and broadened the DSC endotherm, whereas higher concentrations (> 20 lM) resulted in endotherms with two shoulders, one lower and one higher than that observed for untreated liposomes Multiple peaks
in phase-transition endotherms obtained for model mem-branes treated with membrane disrupters such as human defensin, magainin, viral fusion peptides, staphylococcal d-lysin and gramicidin S have been reported previously [55–59] However, in each case, both peaks were at lower temperatures than for untreated lipids The observation of endotherm peaks at higher temperatures in this study suggests that aggregation (or pore formation) of the peptides (CB or CB1 at higher concentration, 20 lM) may occur within the lipid bilayers of liposome The stronger interactions between peptide pore and the lipids causes the higher binding-endothermic effect However, at lower concentration (1 lM), the peptides (CB or CB1) disrupt liposomes into smaller species (without pore formation) which causes smaller effects of phase transition than that of liposome alone (see light microscopic results) By contrast, CB3 had significantly less effect on the endotherm, and higher concentrations (50 lM) were required to obtain multiple peaks similar to the endotherms obtained with CB
or CB1, suggesting that CB3 binds less efficiently to the liposomes and has a lower tendency to aggregate at the membrane
SPR has been used to study a variety of biological interactions; here we applied the technique to study peptide–lipid interactions in greater detail by immobilizing liposomes through an avidin–biotin interaction To reduce any non-specific interactions between the applied peptides and the chip surface, we optimized the derivatization of the chip surface and tested liposome-free chips for
Fig 4 Microcalorimetric determination of the effects of (A) CB, (B) CB1, and (C) CB3on the phase-transition temperature of PtdEtn/PA (7 : 3, w/w) liposomes Thick solid lines indicate the DSC curves for liposomes alone (PtdEtn/PA ¼ 7 : 3, 5 mgÆmL)1), and dashed lines show the DSC curves for peptides CB and CB1 at 20 l M and CB3 at 50 l M in the absence of liposomes Dotted and thin solid lines represent the thermal curves for CB and CB1 with liposomes (PtdEtn/PA ¼ 7 : 3, 5 mgÆmL)1) at 1 l M and 20 l M (respectively) and CB3 with liposomes (PtdEtn/PA ¼ 7 : 3,
5 mgÆmL)1) at 20 l M and 50 l M (respectively).
Trang 7nonspecific binding; in each case non-specific interactions
were concentration-dependent and minimal, suggesting
that the effects observed in the presence of liposomes
result primarily from specific peptide–liposome inter-action(s) SPR analysis with higher concentrations of the peptides, CB and CB1, reveals a net increase in mass One
Fig 5 Microscopic observations of the effect of CB, CB1 and CB3on GUVs (PtdEtn/PA at 7 : 3) A single, isolated vesicle was treated with a 50 l M
concentration of the indicated peptide by application to the side of the vesicle-containing drop and was followed microscopically for the period indicated For CB, A–D were obtained at time (min) 0, 6, 20 and 24, respectively For CB1, E–H were obtained at time (min) 0, 1, 3 and 4, respectively For CB3, I–L were obtained at time (min) 0, 5, 8 and 15, respectively.
Trang 8explanation for these observations could be that higher
peptide concentrations favour peptide aggregation and
that these aggregates then bind to the liposome surface
without causing their destruction Alternatively, as
observed in the DSC analyses, two populations of peptide
may exist at higher concentrations: a predominant
proportion that is aggregated and interacts directly with
the liposome surface, and a second minor population of
free peptides that may interact with liposomes in a
disruptive manner Thus, the mass increase observed at
higher peptide concentrations would be the sum total of
the positive RU values generated by the major
popula-tion By contrast, treatment of surface-bound liposomes
with the peptide CB3 had a markedly different effect: at
each concentration tested, the peptide disrupted liposome
integrity (seen as a loss of mass) Moreover, liposome
disruption was even more pronounced at higher peptide
concentrations, and there was a short lag in CB3-induced
liposome disruption Initially the peptide appeared to bind
to the liposome, as indicated by the short period of mass
increase, and binding was followed by mass loss,
indica-ting liposome destruction This lag phase probably
indicates that CB3 accumulates on the membrane until
a critical concentration is reached, after which the peptides
may coalesce to co-operatively cause membrane
destruc-tion Together these observations suggest that CB3
functions by a markedly different mechanism from that
used by CB and CB1
In summary, on the basis of liposomal lysis as a
function of time observed by the change in SPR, the
thermotrophic phase transition of the final states of
interactions between liposomes and peptides, and the time
course of morphological change in giant liposome
observed by microscopy, CB and CB1, having at least
one amphipathic a-helix, appear to follow the
pore-forming lysis model In contrast, CB3, having no
amphipathic a-helix, appears to have a completely
different lysis mechanism, following the carpet-like lysis
model These proposed lysis pathways are also supported
by our previous studies using spin-label electron spin
resonance [39] The report indicated that the lysis action
of CB1 is related to its capacity to bind to the lipid
bilayers In contrast, there is no evidence of binding for
CB3 CB1 was located in the lipid bilayers by measuring
the collision rate with chromium oxalate in solution [39]
Results from electron spin resonance power saturation
measurements suggested that the N-terminal a-helix of
CB1 is located on the surface of the lipid bilayers,
whereas the C-terminal a-helix of CB1 is embedded
below the surface of the lipid bilayers These conclusions
were further supported by the observed relationship
between the partition distribution of peptides bound to
liposomes at different PA/phosphatidylcholine ratios and
the amounts of free peptides [39]
An understanding of the modes of action of these
peptides should help in the design of more potent and more
specific antimicrobial peptides
Acknowledgements
This work was partially supported by the National Science Council
(Taiwan) (grant NSC 91-2311-B-001-065).
References
1 Steiner, H., Hultmark, D., Engstrom, A., Bennich, H & Boman, H.G (1981) Sequence and specificity of two antibacterial proteins involved in insect immunity Nature (London) 292, 246–248.
2 Zasloff, M (1987) Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor Proc Natl Acad Sci USA 84, 5449–5453.
3 Selsted, M.E., Novotny, M.J., Morris, W.L., Tang, Y.Q & Cullor, J.S (1992) Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils J Biol Chem 267, 4292–4295.
4 Jack, R.W., Tagg, J.R & Ray, B (1995) Bacteriocins of gram-positive bacteria Microbiol Rev 59, 171–200.
5 Severina, E., Severin, A & Tomasz, A (1998) Antibacterial effi-cacy of nisin against multidrug-resistant Gram-positive pathogens.
J Antimicrob Chemother 41, 341–347.
6 Putsep, K., Branden, C.-I., Boman, H.G & Normark, S (1999) Antibacterial peptide from H pylori Nature (London) 398, 671–672.
7 Li, P., Chan, H.C., He, B., So, S.C., Chung, Y.W., Shang, Q., Zhang, Y.-D & Zhang, Y.-L (2001) An antimicrobial peptide gene found in the male reproductive system of rats Science 291, 1783–1785.
8 Lehrer, R.L., Barton, A., Daher, K.A., Harwig, S.S., Ganz, T & Selsted, M.E (1989) Interaction of human defensins with Escherichia coli Mechanism of bactericidal activity J Clin Invest.
84, 553–561.
9 Park, C.B., Kim, H.S & Kim, S.C (1998) Mechanism of action of the antimicrobial peptide buforin II: buforin II kills microorga-nisms by penetrating the cell membrane and inhibiting cellular functions Biochem Biophys Res Commun 244, 253–257.
10 Friedrich, C.L., Moyles, D., Beveridge, T.J & Hancock, R.E.W (2000) Antibacterial action of structurally diverse cationic peptides
on gram-positive bacteria Antimicrob Agents Chemother 44, 2086–2092.
11 Bowles, D.J (1990) Defense-related proteins in higher plants Annu Rev Biochem 59, 873–907.
12 Broekaert, W.F., Terras, F.R.G., Cammue, B.P.A & Osborn, R.W (1995) Plant defensins: novel antimicrobial peptides as components of the host defense system Plant Physiol 108, 1353– 1358.
13 Wijaya, R., Neumann, G.M., Condron, R., Hughes, A.B & Polya, G.M (2000) Defense proteins from seed of Cassia fistula include a lipid transfer protein homologue and a protease inhibitory plant defensin Plant Sci 159, 243–255.
14 Mangoni, M.L., Miele, R., Renda, T.G., Barra, D & Simmaco,
M (2001) The synthesis of antimicrobial peptides in the skin of Rana esculenta is stimulated by microorganisms FASEB J 15, 1431–1432.
15 Wade, D., Andreu, D., Mitchell, S.A., Silveira, A.M., Boman, A., Boman, H.G & Merrifield, R.B (1992) Antibacterial peptides designed as analogs or hybrids of cecropins and melittin Int J Pept Protein Res 40, 429–436.
16 Bro¨tz, H., Josten, M., Wiedemann, I., Schneider, U., Go¨tz, F., Bierbaum, G & Sahl, H.-G (1998) Role of lipid-bound peptido-glycan precursors in the formation of pores by nisin, epidermin and other lantibiotics Mol Microbiol 30, 317–327.
17 Breukink, E., Wiedemann, I., van Kraaij, C., Kuipers, O.P., Sahl, H.-G & de Kruijff, B (1999) Use of the cell wall precursor lipid II
by a pore-forming peptide antibiotic Science 286, 2361–2364.
18 Wiedemann, I., Breukink, E., van Kraaij, C., Kuipers, O.P., Bierbaum, G., de Kruijff, B & Sahl, H.-G (2001) Specific binding
of nisin to the peptidoglycan precursor lipid II combines pore formation and inhibition of cell wall biosynthesis for potent antibiotic activity J Biol Chem 276, 1772–1779.
Trang 919 Fimland, G., Jack, R.W., Jung, G., Nes, I.F & Nissen-Meyer, J.
(1998) The bactericidal activity of pediocin PA-1 is specifically
inhibited by a 15-mer fragment that spans the bacteriocin from
the center toward the C terminus Appl Environ Microbiol 64,
5057–5060.
20 Staubitz, P., Peschel, A., Nieuwenhuizen, W.F., Otto, M., Gotz,
F., Jung, G & Jack, R.W (2001) Structure–function relationships
in the tryptophan-rich, antimicrobial peptide indolicidin J Pept.
Sci 7, 552–564.
21 Wade, D., Boman, A., Wahlin, D., Drain, C.M., Andreu, D.,
Boman, H.G & Merrifield, R.B (1990) All-D amino
acid-con-taining channel-forming antibiotic peptides Proc Natl Acad Sci.
USA 87, 4761–4765.
22 Hancock, R.E.W (1997) Peptide antibiotics Lancet 349, 418–422.
23 Boman, H.G & Broekaert, W.F (1998) Peptide antibiotics come
of age Immunologist 6/6, 235–238.
24 Jaynes, J.M., Julian, G.R., Jeffers, G.W., White, K.L & Enright,
F.M (1989) In vitro cytocidal effect of lytic peptides on several
transformed mammalian cell lines Pept Res 2, 157–160.
25 Lichtenstein, A.K (1991) Mechanism of mammalian cell lysis
mediated by peptide defensins Evidence for an initial alteration of
the plasma membrane J Clin Invest 88, 93–100.
26 Lehrer, R.I., Lichtenstein, A.K & Ganz, T (1993) Defensins:
antimicrobial and cytotoxic peptides of mammalian cells Annu.
Rev Immunol 11, 105–128.
27 Chen, H.M., Wang W., Smith, D & Chan, S.C (1997) Effects
of the anti-bacterial peptide cecropin B and its analogs, cecropins
B-1 and B-2, on liposomes, bacteria, and cancer cells Biochim.
Biophys Acta 1336, 171–179.
28 Christensen, B., Fink, J., Merrified, R.B & Mauzerall, D (1988)
Channel-forming properties of cecropins and related model
com-pounds incorporated into planar lipid membranes Proc Natl
Acad Sci USA 85, 5072–5076.
29 Vaz-Gomes, A., De Waal, A., Berden, J.A & Westerhoff, H.V.
(1993) Electric potentiation, cooperativity, and synergism of
magainin peptides in protein-free liposomes Biochemistry 32,
5365–5372.
30 Silvestro, L., Gupta, K., Weiser, J.N & Axelsen, P.H (1997)
The concentration-dependent membrane activity of cecropin A.
Biochemistry 36, 11452–11460.
31 Merrifield, R.B., Merrifield, E.L., Juvvadi, P., Andreu, D &
Boman, H.G (1994) Design and synthesis of antimicrobial
pep-tides In Antimicrobial Peptides: Ciba Foundation Symposium 186
(Marsh, J & Goode, J.A., eds), pp 5–20 Wiley, Chichester, UK.
32 Puny, Y., Rapaport, D., Mor, A., Nicolas, P & Shai, Y (1992)
Interaction of antimicrobial dermaseptin and its fluorescently
labeled analogues with phospholipid membranes Biochemistry 31,
12416–12423.
33 Sailam, S & Arunkumar, A.I., Yu, C & Chen, H.M (2000)
Conformational study of a custom antibacterial peptide cecropin
B1: implications of the lytic activity Biochim Biophys Acta 1479,
275–285.
34 Sailam, S., Arunkumar, A.I., Yu, C & Chen, H.M (2001)
Crumpled structure of the custom hydrophobic lytic peptide,
cecropin B3 Eur J Biochem 268, 4278–4284.
35 Wang, W., Smith, D., Moulding, K & Chen, H.M (1998) The
dependence of membrane permeability by the anti-bacterial
pep-tide cecropin B and its analogs, CB-1 and CB-3 on liposomes of
different composition J Biol Chem 273, 27438–27448.
36 Chan, S.C., Yau, W.L., Wang, W., Smith, D., Sheu, F.S & Chen,
H.M (1998) Microscopic observations of the different
morpho-logical changes by the anti-bacterial peptides on Klebsiella
pneu-moniae and HL-60 leukemia cells J Pept Sci 4, 413–425.
37 Chan, S.C., Hui, L & Chen, H.M (1998) Enhancement of the
cytolytic effect of anti-bacterial cecropin by microvilli of the cancer
cells Anticancer Res 18, 4467–4474.
38 Wang, W., Smith, D & Chen, H.M (1999) The effect of pH on the structure, binding and model membrane lysis by cecropin B and analogs Biochim Biophys Acta 1473, 418–430.
39 Hung, S.C., Wang, W., Chan, S.I & Chen, H.M (1999) Mem-brane lysis by the custom anti-bacterial peptides cecropins B1 and B3: a spin-label electron spin resonance study on the phospholipid bilayers Biophys J 77, 3120–3133.
40 Chen, H.M., Smith, D & Wang, W (2000) Liposome disruption detected by SPR at lower concentration of a peptide antibiotic Langmuir 16, 9959–9962.
41 Hui, L., Leung, K & Chen, H.M (2001) The combinative effects
of antibacterial peptide cecropin A and antibcancer agents on leukaemia cell Anticancer Res 21, 3247–3250.
42 Chen, H.M., Clayton, A., Wang, W & Sawyer, W.H (2001) Kinetics of membrane lysis by custom lytic peptides and peptide orientation in membrane Eur J Biochem 268, 1659–1669.
43 Wu, Y., Huang, H.W & Olah, G.A (1990) Method of oriented circular dichroism Biophys J 57, 797–806.
44 Vogel, H (1987) Comparison of the conformation and orientation
of alamethicin and melittin in lipid membranes Biochemistry 26, 4562–4572.
45 Ludtke, S.J., He, K., Wu, Y & Huang, H.W (1994) Cooperative membrane insertion of magainin correlated with its cytolytic activity Biochim Biophys Acta 1190, 181–184.
46 Akashi, K., Miyata, H., Itoh, H & Kinosita, K Jr (1996) Pre-paration of giant liposomes in physiological conditions and their characterization under an optical microscope Biophys J 71, 3242–3250.
47 Marassi, F.M., Opella, S.J., Juvadi, P & Merrifield, R.B (1999) Orientation of cecropin A helices in phospholipid bilayers determined by solid-state NMR spectroscopy Biophys J 77, 3152–3155.
48 Bechinger, B (1999) The structure, dynamics and orientation of antimicrobial peptides in membranes by multidimensional solid-state NMR spectroscopy Biochim Biophys Acta 1462, 157–183.
49 Smith, R., Separovic, F., Milne, T.J., Whittaker, A., Bennett, F.M., Cornell, B.A & Makriyannis, A (1994) Structure and orientation of the pore-forming peptide melittin in lipid bilayers.
J Mol Biol 241, 456–466.
50 Juvvadi, P., Vunnam, S & Merrifield, R.B (1996) J Am Chem Soc 118, 8989–8997.
51 Pouny, Y., Rapaport, D., Mor, A., Nicolas, P & Shai, Y (1992) Interaction of antimicrobial dermaseptin and its fluorescently labeled analogues with phospholipid membranes Biochemistry 31, 12416–12423.
52 Oren, Z & Shai, Y (1998) Mode of action of linear amphipathic alpha-helical antimicrobial peptides Biopolymers 47, 451–463.
53 Wick, R., Anglova, M.I., Walde, P & Luisi, P.I (1996) Micro-injection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations Chem Biol 3, 105–111.
54 Zhang, Y.-P., Lewis, R.N.A.H., Hodges, R.S & McElhaney, R.N (2001) A differential scanning calorimetric and31P NMR spectroscopic study of the effect of transmembrane-helical pep-tides on the lamellar-reversed hexagonal phase transition of phosphatidylethanolamine model membranes Biochemistry 40, 474–482.
55 Lohner, K., Latal, A., Lehrer, R.I & Ganz, T (1997) Differential scanning microcalorimetry indicates that human defensin, HNP-2, interacts specifically with biomembrane mimetic systems Bio-chemistry 36, 1525–1531.
56 Lohner, K., Staudegger, E., Prenner, E.J., Lewis, R.N.A.H., Kriechbaum, M., Degovics, G & McElhaney, R.N (1999) Effect
of staphylococcal lysin on the thermotropic phase behavior and vesicle morphology of dimyristoylphosphatidylcholine lipid bilayer model membranes Differential scanning calorimetric, 31 P nuclear magnetic resonance and Fourier transform infrared
Trang 10spectroscopic, and X-ray diffraction studies Biochemistry 38,
16514–16528.
57 Prenner, E.J., Lewis, R.N & McElhaney, R.N (1999) The
inter-action of the antimicrobial peptide gramicidin S with lipid bilayer
model and biological membranes Biochim Biophys Acta 1462,
201–221.
58 Matsuzaki, K., Sugishit, K.-I., Ishibe, N., Ueha, M., Nakata, S.,
Miyajima, K & Epand, R.M (1998) Relationship of membrane
curvature to the formation of pores by magainin 2 Biochemistry
37, 11856–11863.
59 Holopainen, J.M., Saily, M., Caldentey, J & Kinnunen, P.K (2000) The assembly factor P17 from bacteriophage PRD1 inter-acts with positively charged lipid membranes Eur J Biochem.
267, 6231–6238.