From the Institut für Medizinische
Mikrobiologie und Immunologie der Universität Bonn, D-53105 Bonn,
Germany, the § Department of Biochemistry of Membranes,
Center for Biomembranes and Lipid Enzymology, Institute of
Biomembranes, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The
Netherlands, and the ¶ NIZO Food Research, Microbial
Ingredients Section, P.O. Box 20, 6710 BA, Ede, The Netherlands
Received for publication, July 28, 2000, and in revised form, September 27, 2000
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ABSTRACT |
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Unlike numerous pore-forming amphiphilic peptide
antibiotics, the lantibiotic nisin is active in nanomolar
concentrations, which results from its ability to use the lipid-bound
cell wall precursor lipid II as a docking molecule for subsequent pore
formation. Here we use genetically engineered nisin variants to
identify the structural requirements for the interaction of the peptide with lipid II. Mutations affecting the conformation of the N-terminal part of nisin comprising rings A through C, e.g.
[S3T]nisin, led to reduced binding and increased the peptide
concentration necessary for pore formation. The binding constant for
the S3T mutant was 0.043 × 107
M The antimicrobial peptide nisin is produced by numerous strains of
Lactococcus lactis and inhibits a broad range of
Gram-positive bacteria (1, 2). It belongs to the lantibiotics, a group of antimicrobial peptides that is characterized by the presence of
intramolecular rings formed by the thioether amino acids lanthionine and 3-methyllanthionine (3, 4). Nisin has had a long history as a
potent and safe food preservative, although recent insight into the
molecular mechanism of its bactericidal activity also make it
interesting for biomedical applications (5, 6). Generally, the
nisin-type subgroup of lantibiotics comprises elongated cationic
peptides that have the capacity to adopt amphiphilic structures. Such
peptides are assumed to kill microbes by disturbing the integrity of
the energy-transducing membrane. Indeed, early experiments demonstrated
that nisin or related lantibiotics induced rapid efflux of ions or
cytoplasmic solutes such as amino acids and nucleotides. The
concomitant depolarization of the cytoplasmic membrane resulted in an
instant termination of all biosynthetic processes (7, 8). Structural
analysis in the presence of micelles indicated that the hydrophilic
groups of the peptide interact with the phospholipid headgroups, and
the hydrophobic side chains are immersed in the hydrophobic core of the
membrane (9, 10). The wedge model as proposed by Driessen et
al. (11) takes into account such structural data and proposes that
the peptides insert into the membrane without losing contact with the
membrane surface, resulting in the formation of a short-lived pore.
Whereas the wedge model may illustrate results obtained with model
membranes, a number of effects observed with intact living cells remain
unexplained; in particular, the fact that nisin acts on model membranes
at micromolar concentrations whereas in vivo minimal
inhibitory concentration
(MIC)1 values are in the
nanomolar range. The discrepancies were explained by the finding that
nisin and epidermin use lipid II, the bactoprenol-bound precursor of
the bacterial cell wall as a docking molecule for subsequent pore
formation (12). The specificity of the nisin-lipid II interaction and
the resulting high level activity of nisin was demonstrated in a
comparative study with the pore-forming amphiphilic defense peptide
magainin 2 (5). We found that, in contrast to magainin, the activity of
nisin is enhanced by a factor of 103 when lipid II is
available for targeted pore formation. The fact that nisin specifically
binds to lipid II was particularly remarkable, because previously
mersacidin and actagardine from the type B class of lantibiotics also
had been shown to form a complex with the cell wall
precursor. In this case however, binding only blocked the precursor from incorporation into polymeric peptidoglycan (13, 14).
We therefore assumed that lantibiotics may have defined lipid II
binding motifs that could represent interesting lead compounds for the
development of novel antibiotics. In particular, these lantibiotics are
active against the Gram-positive bacteria that are most prominent with
regard to resistance development, e.g.
Staphylococci and Enterococci.
Here we studied the interaction between lipid II and a number of nisin
mutant peptides to identify the structural elements of the nisin
molecule that are important for the interaction with lipid II and
subsequent pore formation. Surprisingly, we identified nisin mutant
peptides that were unable to form pores but retained considerable
antimicrobial activity through blocking lipid II from incorporation
into peptidoglycan. Thus nisin is a dual-function antibiotic with two
killing mechanisms combined into one molecule.
Chemicals and Materials--
All chemicals were of analytical
grade or better. The phospholipids
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and
1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG) were
purchased from Avanti Polar Lipids, Inc. and stored at Nisin Z and Nisin Mutants--
Nisin Z (16) and
prepeptide nisin Z with the N-terminal leader sequence of
subtilin (17) as well as all mutant peptides (18) were produced in SPYS
medium (3% sucrose, 1% peptone (Difco), 1% yeast extract (Difco),
0.2% NaCl, 0.002% MgSO4, and 1%
KH2PO4, pH 6.8). Peptides were purified from
supernatants by hydrophobic-interaction chromatography using Fractogel
TSK butyl 650-M (Tosohaas), followed by preparative reversed phase-HPLC
using acetonitrile and 0.1% trifluoroacetic acid as eluents. Mutations
were verified by mass spectroscopy. Nisin Z is a natural variant of
nisin A that only differs in one single residue (Asn instead of His at
position 27) and possesses almost identical antimicrobial properties
(19, 20). The [ Lipid II Synthesis--
Lipid II was synthesized in
vitro using membrane preparations of Micrococcus
luteus ATCC 4698. Membranes were isolated from lysozyme-treated
cells by centrifugation (40,000 × g), washed twice in 50 mM Tris-HCl, 10 mM MgCl2, pH 7.5 and stored under liquid nitrogen. For extraction and purification, the
protocol elaborated by Brötz et al. (14) for
[14C]lipid II was applied. The unlabeled lipid II
generated for the present study was detected by exploiting its ability
to form a complex with mersacidin. Aliquots of the respective
chromatographic fractions were added to a mersacidin activity test
(12), and those fractions that were able to antagonize mersacidin
activity were collected. The lipid II concentration in stock solutions was determined by quantitative analysis of amino acids and amino sugars
using the o-phthaldialdehyde (OPA) derivatization technique (22). Lipid II samples were hydrolyzed in sealed tubes with 6 N HCl at 110 °C for 18 h. After evaporation of HCl,
amino acids and sugars were taken up in 200 mM borate
buffer, pH 9.5 and derivatized with OPA. The derivatives were separated
by reversed-phase C18 HPLC chromatography using 50 mM phosphate buffer, pH 6.5 (buffer A) and
methanol/tetrahydrofuran (97:3) as eluent B. Fluorescence of the
derivatives was recorded at 450 nm (excitation at 340 nm) and
quantitated using appropriate standards. Purified lipid II was stored
in chloroform/methanol (1:1, v/v) at Preparation of Vesicles--
Large unilamellar vesicles for the
binding, CF efflux, and potassium efflux experiments were prepared by
the extrusion technique (23) and treated as described previously (24).
Vesicles were made of DOPC with or without 0.065 mol % lipid II
(referring to the total amount of lipids). Phospholipid was determined
as inorganic phosphate after treatment with perchloric acid (25).
CF-loaded vesicles were prepared with 50 mM CF and then
diluted in 1.25 ml of K+ buffer (50 mM Mes-KOH,
pH 6.0, 100 mM K2SO4) in a final
concentration of 25 µM phospholipid (on a phosphorous
base). After addition of wild-type or mutant nisin, the induced leakage
was monitored for 3 min. The increase of fluorescence intensity was
measured at 515 nm (excitation at 492 nm) on a SPF 500C
spectrophotometer (SLM instruments Inc.) at 20 °C. The nisin-induced
CF leakage was expressed relative to the total amount of CF released
after lysis of the vesicles by addition of 10 µl of 20% Triton
X-100.
Potassium-loaded vesicles were prepared with KCl buffer (150 mM KCl, 30 mM Mes, 20 mM Tris, pH
6.5). The vesicles were added to 5 ml of buffer (30 mM Mes,
20 mM Tris, pH 6.5, 150 mM choline chloride) in
a final concentration of 25 µM phospholipid. Nisin or
nisin mutant-induced leakage was monitored with a K+
selective electrode (15-K, Philips) and a reference electrode (R44/2,
Philips). The total amount of K+ in the vesicles was
determined after addition of 20 µl of a 20% solution of
N,N-dimethyldodecylamine N-oxide (Fluka). Leakage was expressed relative to the total amount of K+. The
electrodes were calibrated by titrating known amounts of potassium.
Radioactive Labeling--
Radioactive labeling of nisin and
binding experiments were performed as described previously by Breukink
(24). Nisin Z, [S3T]nisin Z and [ In Vitro Peptidoglycan Synthesis--
A crude membrane fraction
was prepared from Escherichia coli TB1 by disruption of
exponentially growing cells with a Branson Sonifier 250 (Danburry CT)
twice for 3-5 min, 50% cooling phase and power level 5. Membranes
were collected by centrifugation (40,000 × g), then
washed and resuspended in buffer (50 mM Tris HCl, pH 7.5),
to give a protein concentration of 8 mg/ml.
UDP-N-acetylmuramylpentapeptide (UDP-MurNAc-pentapeptide)
was isolated from Staphylococcus simulans 22. To accumulate
the murein precursor, exponentially growing cells were supplemented
with 5 µg/ml vancomycin and incubated for 30 min. After
centrifugation, cells were resuspended in distilled water (0.1 g/ml)
and extracted by stirring the suspension slowly into 2.5 volumes of
boiling water. After 15 min, the suspension was cooled, the cell debris
removed (48,000 × g) and the supernatant lyophilized.
The lyophilized material was then dissolved in water and brought to pH
2 with 20% H3PO4. After acidification,
precipitated material was removed by centrifugation, and the
supernatant fractionated on reversed-phase C18 HPLC under
isocratic conditions (50 mM
NaH2PO4, pH 5.2). The UDP-MurNAc-pentapeptide
was identified by mass spectrometry. For cell wall biosynthesis assays
(35), it was applied in 0.2 mM concentrations together with
membranes (160 µg of protein), 0.05 mM
[14C]UDP-GlcNAc and 15-60 µM nisin
peptides in a total volume of 60 µl of membrane suspension buffer (50 mM Tris-HCl, pH 8, 10 mM MgCl2, 1 mM mercaptoethanol). Incubation proceeded for 45 min at
32 °C before the reaction was stopped by heating (100 °C, 30 s). Aliquots were analyzed by paper chromatography (paper GB 002, Schleicher und Schüll, Dassel, Germany) in isobutyric acid/1 M NH4OH (5:3, v/v). Polymeric peptidoglycan
does not migrate from the starting point, and the respective area was
cut out and counted in a Activity of Wild-type Nisin Z on Lipid II-containing Unilamellar
Vesicles--
Previous studies had shown that nisin in low
concentrations can induce rapid efflux of CF from multilamellar
liposomes when supplemented with 0.1 mol % lipid II. In the absence of
lipid II, such liposomes were not affected by nisin concentrations as high as 1.25 µM (12). To characterize in detail the lipid
II effect, we used unilamellar vesicles doped with 0.065 mol % of the
cell wall precursor. At 5 nM nisin, significant dye release was observed, and ~60% release was achieved with 50 nM
nisin (Fig. 1A). Because dye
efflux reached a plateau within less than a minute, we plotted the
percentage of dye released after 1 min for a closer inspection of the
concentration dependence of the nisin pore formation. Fig.
1B demonstrates that upon the incorporation of lipid II, the
sensitivity of DOPC (1,2 dioleoyl-sn-glycero-3-phosphocholine) liposomes increased by
three orders of magnitude. CF release started at about 1 nM
of the lantibiotic, whereas more than 1 µM nisin was
necessary to affect DOPC liposomes without lipid II. In accordance with
previous studies in the absence of lipid II (24), membranes containing
the negatively charged lipids DOPG were considerably more sensitive
(Fig. 1B) than the DOPC liposomes. However, the difference
in sensitivity disappeared after incorporation of 0.065 mol % lipid II
(Fig. 1C). Although the overall activity of nisin toward
lipid II-containing liposomes was relatively independent from the
phospholipid composition, slightly higher release rates were obtained
with DOPC as the bulk phase constituent. Under the test conditions
applied, the maximum marker release was achieved with nisin
concentrations in the range of 0.05 to 0.1 µM (Fig. 1C). Application of higher concentrations reduced the dye
release efficiency with the DOPC liposomes and with the DOPC/DOPG
mixture. With DOPG vesicles, the dye-release rate increased again at 1 µM, which agrees well with the results depicted in Fig.
1B, because it coincides with the onset of the marker
release activity in the absence of lipid II.
Similar to the dependence of pore formation on peptide concentration,
dye release was also dependent on the lipid II concentration in
liposomes (Fig. 1D). In a lipid II titration series in which the nisin concentration was kept constant at 10 nM or 100 nM, efflux started at 0.001 mol % lipid II,
i.e. for an average liposome of 400 nm in diameter and
7 × 105 bulk lipid molecules (of 0.70 nm2), only seven lipid II molecules per vesicle were
sufficient to promote dye release. With increasing peptide
concentrations, the release activity reached an optimum, in this case
at ~0.1 mol %, and decreased with greater lipid II concentrations.
The presence of an optimum in the curves obtained in both titration
series suggests that there is an optimal lipid II to peptide ratio,
which may be in the range of 1:1, e.g. at 0.1 mol % lipid
II (optimum in Fig. 1D) the ratio is 1.0:0.4 (lipid II to
nisin) for 10 nM nisin, or 1:4 for 100 nM
nisin, respectively.
We also replaced the negatively charged CF with K+ to
detect indications for ion selectivity of the pore. Interestingly, the efflux rates did not significantly differ with both markers
(e.g. Fig. 2; 90%
K+ release after 1 min with 100 nM nisin). The
absence of ion selectivity of nisin pores formed in lipid II-doped
membranes stands in contrast to data obtained without lipid II (24) and
suggests that lipid II not only enhances the pore formation activity of
nisin but also has an influence on the pore architecture.
Binding Characteristics of Nisin Z to Lipid II-containing
Liposomes--
Membrane poration by cationic amphiphilic peptides is
considered a sequential process that includes binding to the target membrane, insertion into the membrane, and the formation of a functional pore. Detailed studies with various model peptides (27) and
with nisin Z (24) demonstrate that lipids with a net negative charge,
such as DOPG, are important for binding via electrostatic interactions
(24). Nisin, for example, was bound stoichiometrically to one DOPG
molecule per positively charged amino acid residue. In contrast, very
little nisin was bound by pure DOPC liposomes, and the above data (Fig.
1C) illustrate that DOPG is not essential when lipid II
serves as a specific target. We therefore studied the binding behavior
of radiolabeled nisin to DOPC liposomes supplemented with lipid II.
With lipid II present in 0.5 mol %, nisin showed a binding behavior
that was best described by the Langmuir adsorption model with an
apparent minimal binding constant of 2 × 107
M
The data shown in Fig. 3 indicate that nisin binding saturates when
each lipid II molecule is loaded with one nisin Z molecule, indicative
of a 1:1 binding stoichiometry, which agrees well with the optimum
curves obtained in the peptide and lipid II titration experiments (Fig.
1D). A 1:1 stoichiometry requires that all lipid II is
available for binding; however, the procedure for preparation of lipid
II-containing liposomes is very likely to result in a symmetric
distribution of lipid II headgroups with regard to the bilayer;
i.e. only 50% of the incorporated lipid II molecules should
be outwardly orientated. However, recent experiments with C-terminal
His-tagged nisin Z convincingly demonstrate that the lantibiotic is
able to translocate across a negatively charged lipid-containing
membrane (28). In light of these results it seems likely that in the
presence of lipid II, nisin can translocate across the membrane and
thus the inwardly orientated lipid II becomes available for interaction
with nisin.
Structural Requirements for the Lipid II-mediated Pore
Formation--
We used the DOPC/0.065 mol % lipid II vesicle system
to identify the structural elements in the nisin molecule that are
important for the interaction with lipid II and/or subsequent pore
formation. Structure-function studies with nisin and other lantibiotics
had identified various structural features important for the
bactericidal activity in vivo or for activities in in
vitro systems. For instance, the C-terminal part of nisin is
important for binding to membranes with a negative surface charge (24)
and the two C-terminal amino acids (at positions 33 and 34) are not
essential for the activity in vivo (29). In contrast, a
short flexible region in the central part of the molecules was found to
be indispensable not only in nisin and other type A lantibiotics (21)
but also of unmodified peptide antibiotics (30). Moreover, strong hints
for an important role of the N-terminal double ring system for binding
to lipid II came from structural comparison of several type A
lantibiotics in a previous study (12). Based on these considerations we
selected a number of mutants in the respective regions of nisin (Fig.
4) and compared their activities in the
lipid II-containing liposome system to their MIC values for M. flavus and S. thermophilus (Table I).
Mutants with Charge Differences--
Introduction of a charged
residue in position 32 only slightly reduced K+ release
(Fig. 2) and CF release activities (Fig.
5A) at low peptide concentrations, regardless of whether Val-32 was replaced by a Lys or a
Glu residue. Similarly, only minor effects were observed in
vivo. A strong reduction of the activity of nisin in the liposome assay by almost two orders of magnitude was observed when a Lys residue
was introduced into ring C of the peptide (Fig. 5A, M17K), demonstrating that for pore formation a charged residue is not tolerable in the central segment of the molecule. However, in vivo, the M17K peptide lost only 50% of its activity against
M. flavus (Table I), which may be explained by the
particular role of anionic cell wall polymers in the overall activity
of cationic peptides (see "Discussion").
Hinge Region Mutants--
An even more drastic effect on the
activity was found for the hinge region mutants (Fig. 5B).
In [ N-terminal Mutants--
Because nisin-(1-12) covering the
N-terminal rings A and B had been reported to antagonize binding of
wild-type nisin to target cells (31) it was especially interesting to
study peptides mutated in this segment of the lantibiotic. However,
nonconservative mutations in rings A and B, which could have an impact
on the structure of the rings and thus be valuable tools for this
study, are apparently incompatible with the biosynthesis machinery and
could not be produced (21, 32). We tested three peptides in which the
Dhb (i.e. the Thr in the prepeptide) in position 2 was
replaced by Ser, Ala, and Val, respectively. These mutations had little
effect on the MIC and were slightly less active than wild-type nisin Z
on lipid II-containing liposomes (Fig. 5C). In contrast, a
strong effect was observed with [S3T]nisin. The S3T mutation results in the formation of a 3-methyllanthionine ring instead of a lanthionine ring and was found to exert relatively little influence on the activity
of the mutant peptide when tested on pure PG-liposomes without lipid II
(data not shown). However, with a MIC of 39 nM against
M. flavus, this peptide was considerably less active
in vivo.
The apparent discrepancy between the activity of [S3T]nisin in
vivo and in vitro disappeared when lipid II was
incorporated into the liposomes. Similar to the activity of the mutant
peptide against bacterial cells, its dye releasing activity was
strongly reduced in the presence of lipid II (Fig. 5C); the
concentration necessary to release 50% of CF dropped about 75-fold
from 0.015 µM (Fig. 1B) to 1.15 µM for the mutant peptide. The concentration dependence
of the pore formation by the S3T mutant was unchanged as compared with
the wild-type peptide (Fig. 5C, parallel lines), indicating that the affinity of the peptide for lipid II was impaired rather than its pore formation activity. In fact, the binding constant
of this mutant peptide dropped about 50-fold to 0.043 × 107 M
Another interesting mutant that demonstrated the importance of a
correct structure of the N-terminal part of nisin is the T13C peptide.
This mutation results in the formation of a disulfide bond instead of a
thioether, which influences considerably the conformation of ring C
(33). Accordingly, this peptide was almost inactive in vivo
(Table I) and in the liposome assay in the presence of lipid II (Fig.
5C). N-terminal extensions of nisin had also been found to
be detrimental to the activity of the lantibiotic; e.g. the
modified but uncleaved nisin Z precursor peptide was completely
inactive against M. flavus (34). A related precursor in
which the leader peptide of nisin was exchanged for the subtilin leader
segment (17) did not show any activity against lipid II-containing
liposomes as well (Fig. 5C).
Inhibition of in Vitro Peptidoglycan Biosynthesis by Nisin
Peptides--
For [S3T]nisin Z, we observed comparable reductions in
its affinity for lipid II, its pore formation activity on liposomes, and its in vivo activity. In contrast, the hinge region
mutants were completely inactive in pore formation but retained
comparatively high in vivo activity (Table I). Because the
residual activity e.g. of [ In this report we have analyzed the first known case of a pore
formation mechanism by an amphiphilic antibiotic peptide that is based
on specific interaction with a defined integral component of bacterial
cytoplasmic membranes. We demonstrate that the lantibiotic nisin binds
with high affinity to the membrane-bound bacterial cell wall precursor
lipid II, which consists of the bactoprenol-carrier lipid and the
complete monomeric disaccharide-pentapeptide peptidoglycan subunit.
The high potency that derives from such a target-mediated poration may
well explain why nisin is so much more effective against bacteria in
which the docking molecule is available for binding as compared with a
peptide that apparently acts in a nontargeted fashion such as magainin
2 (5). The results reported here and in previous papers on the
molecular mechanisms of activity of nisin in the absence of lipid II
(7, 8, 18, 24, 28, 36) show that nisin can permeabilize membranes via
two different mechanisms. At high concentrations (µM
range) pores can be formed without lipid II, i.e. in a
target-independent fashion. For optimal activity in the absence of
lipid II, membranes should contain 50-60% negatively charged
phospholipids, and the positively charged C terminus of nisin is
important for initial binding (24) and for antimicrobial activity (18).
Pores formed under such conditions are significantly anion-selective
(24); the membrane potential supports pore formation, and the wedge
model (37) may possibly describe mechanistic details. In contrast, when
lipid II is available, nisin can disturb the barrier function of a
bilayer already at nM concentrations and a model taking
into account features elaborated in this study is presented in Fig.
7.
1 compared with 2 × 107
M
1 for the wild-type peptide, and the minimum
concentration for pore formation increased from the 1 nM to
the 50 nM range. In contrast, peptides mutated in the
flexible hinge region, e.g. [
N20/
M21]nisin, were
completely inactive in the pore formation assay, but were reduced to
some extent in their in vivo activity. We found the
remaining in vivo activity to result from the unaltered capacity of the mutated peptide to bind to lipid II and thus to inhibit
its incorporation into the peptidoglycan network. Therefore, through
interaction with the membrane-bound cell wall precursor lipid II, nisin
inhibits peptidoglycan synthesis and forms highly specific pores. The
combination of two killing mechanisms in one molecule potentiates
antibiotic activity and results in nanomolar MIC values, a strategy
that may well be worth considering for the construction of novel antibiotics.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
20 °C in a
9:1 chloroform/methanol solution. Carboxyfluorescein (CF) was purchased
from Kodak and purified as described previously (15). Protein
concentration was determined using the bicinchonic acid protein assay
reagent (Pierce Chemical Corp.) with bovine serum albumin as a standard.
N20/
M21]nisin A, [S3T]nisin Z, [M17K]nisin
Z, and [N20P/M21P]nisin A (21) and the charged mutants [V32E]nisin Z and [V32K]nisin were obtained by site-directed mutagenesis. Mutations of the structural genes nisA or nisZ
were performed by two successive polymerase chain reactions, and
mutated genes were expressed in L. lactis NZ9800 as
described previously (18). Nisin stock solutions were stored at
80 °C in 0.05% acetic acid. The MIC was determined by the
inhibition of growth of the indicator strains Micrococcus
flavus DSM 1790 and Streptococcus thermophilus Rs in a
liquid bioassay (16, 18).
20 °C.
N20/
M21]nisin A were
14C-labeled by reductive methylation of the
-amino group
of the lysine residues according to the method of Dottavio-Martin and Ravel (26). The resulting preparations had specific activities of
4.6 × 104, 8.2 × 104, and 13.3 × 104 dpm/nmol, respectively. The radiolabeled peptides
were stored in 0.05% (v/v) acetic acid at
80 °C. The binding of
the [14C]nisin Z and variants was determined under
equilibrium conditions. Vesicles (160 µM, on a phosphorus
base) made of DOPC with 4% DOPG or with 0.5 mol % lipid II were
incubated at room temperature with 14C-labeled peptide in
0.5 ml of buffer (25 mM Mes, pH 6.0, 50 mM K2SO4) for 5 min. The mixture was then
centrifuged at 430,000 × g for 45 min to spin down the vesicles
with the bound nisin Z. The amount of unbound nisin in the supernatant
and of vesicle-bound nisin in the pellet was determined by
scintillation counting. No phosphate could be detected in the
supernatant after the centrifugation steps, and 90-100% of the
applied radioactivity was recovered. The binding isotherms of the
nisin-lipid II interaction could be well described by the Langmuir
adsorption model assuming that lipid II was the only binding site for
nisin in the context of DOPC vesicles.
-scintillation counter (1900 CA Tri-Carb
scintillation counter, Packard, Zürich).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Activity of nisin against lipid II-containing
liposomes. A, time course of nisin-induced leakage of
CF from DOPC vesicles with 0.065 mol % lipid II. After ~30 s, 0.05 µM nisin Z (A) and 0.005 nisin Z
(B) was added, and the CF efflux was recorded. The 100%
leakage level was determined by addition of Triton X-100 after 180 s. The vesicle concentration was 25 µM on a
Pi basis. B, concentration dependence of nisin
Z-induced CF leakage from DOPC vesicles with 0.065 mol % lipid II
( ), pure DOPG vesicles (
), and pure DOPC vesicles (
). Addition
of nisin in a concentration of 0.015 µM to vesicles with
lipid II results in 50% leakage. A similar extent of leakage from pure
DOPG vesicles was observed when nisin was added in a 100× higher
concentration of 1.4 µM. 50% leakage from pure DOPC
vesicles was not achieved even with a 1000× higher nisin concentration
(10 µM). The CF leakage was determined after 60 s,
and the vesicle concentration was 25 µM on a
Pi basis. C, influence of anionic phospholipids
on nisin Z activity. Vesicles were made of DOPC (
), DOPC/DOPG (1:1)
(
), and DOPG (
), each supplemented with 0.065 mol % lipid II. CF
leakage was determined after 60 s, and the vesicle concentration
was 25 µM on a Pi basis. D, lipid
II concentration dependence of the nisin pore formation activity. DOPC
liposomes were supplemented with increasing amounts of lipid II and
treated with two concentrations of nisin, 10 nM nisin (
)
and 100 nM nisin (
).
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Fig. 2.
Nisin Z and [V32E]- and
[V32K]nisin-induced CF leakage (100 nM, open
bars) and K+ leakage (filled
bars) from DOPC vesicles with 0.065 mol % lipid II after 1 min.
1 (Fig.
3).
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Fig. 3.
Binding behavior of radiolabeled nisin Z to
vesicles made of DOPC and complemented with 0.5 mol % lipid II.
Experimental data ( ); theoretical data for Langmuir type binding
(
) with K = 2 × 107
M
1. In the theoretical analysis it was
assumed that nisin binds only to lipid II.
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Fig. 4.
Primary structure of nisin Z. The
modifications of the nisin mutants used in this study are indicated by
arrows. [V32E]nisin Z and [V32K]nisin Z contain an
unmodified Ser residue on position 33 instead of Dha in
wild-type nisin (dotted arrow). N20/
M21 and N20P/M21P
are modifications of nisin A. Nisin A differs from nisin Z at position
27 (His). Dha, dehydroalanine; Dhb,
dehydrobutyrine; Ala-S-Ala, lanthionine;
Abu-S-Ala,
-methyllanthionine.
Antimicrobial activities of nisin and nisin variants determined as MIC
against Micrococcus flavus and Streptococcus thermophilus
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Fig. 5.
Activity of nisin mutant peptides on the
lipid II-doped liposomes. The CF leakage from DOPC vesicles
containing 0.065 mol % lipid II was determined after 60 s; the
vesicle concentration was kept at 25 µM on a
Pi basis. A, activity of charge mutants:
wild-type nisin ( ), V32E (
), V32K (
), M17K (
).
B, activity of hinge region mutants: wild-type nisin (
),
N20/
M21 (
), N20P/M21P (
), M21G (
). C,
activity of N-terminal mutants: wild-type nisin (
), T2S (
), T2A
(
), T2V (
), S3T (
), T13C (
), and nisin Z prepeptide with
subtilin leader (
).
N20/
M21]nisin, the flexible segment that connects rings C
and D is shortened to just one residue; this peptide did not release
more than 20% of the dye even when tested in concentrations higher
than 1 µM. Replacement of both residues with two prolines
[N20P/M21P]nisin reduced the dye release to a maximum of ~10%.
[M21G]Nisin was slightly less impaired; however, the 10% efflux
obtained at 0.01 µM could not be increased by raising the
concentration of the peptide. Although the in vivo activities of the hinge region mutants were also reduced, the effect
was comparatively less drastic for M. flavus. However, for
S. thermophilus, the MIC values increased by a factor of 25, 40, and 2 for [N20P/M21P]nisin A, [
N20/
M21]nisin A, and
[M21G]nisin Z, respectively.
1. The strong effect of the
incorporation of an additional methyl group points out a crucial role
of the conformation of the N-terminal ring of nisin in the specific
binding of lipid II.
N20/
M21]nisin against
Micrococcus strains or S. thermophilus was in the
same range of concentrations as the activity of mersacidin and
vancomycin (30-70 nM, Ref. 14) we speculated that the
nisin hinge region mutants could exert their antibiotic activity
primarily via inhibition of cell wall biosynthesis. Both mersacidin and vancomycin form a complex with lipid II and thereby block de
novo synthesis of polymeric peptidoglycan (14). We therefore
compared the inhibitory activity of nisin and of the relevant mutant
peptides in an established in vitro cell wall biosynthesis
assay (35) based on isolated E. coli membranes. The
wild-type peptide clearly inhibited the formation of polymeric
peptidoglycan in a concentration-dependent manner (Fig.
6). As expected, the
N20/
M21
deletion mutant was fully inhibitory and may even have a somewhat
stronger effect in this assay, explaining its relatively strong
in vivo activity. In contrast, the S3T mutant had almost no
inhibitory effect, which correlates well with its reduced affinity for
lipid II and with its pore formation activity as depicted in Fig.
5C.
View larger version (25K):
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Fig. 6.
Inhibition of in vitro
peptidoglycan synthesis by nisin Z and mutant peptides. The
amount of polymeric peptidoglycan produced in the presence of the
peptides is given relative to an untreated control. The experiment
shown is representative of three independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (54K):
[in a new window]
Fig. 7.
Model for the lipid II-mediated nisin pore
formation. Based on our results, we propose that nisin first binds
to the outwardly orientated carbohydrate moiety of lipid II in a 1:1
stoichiometry. The N-terminal segment of the lantibiotic is essential
for binding, and a negative surface charge is not necessary. The
C-terminal part of nisin is then assumed to translocate across the
membrane, in accordance with the translocation found in the absence of
lipid II (28). For this step the flexible hinge region between ring
clusters A, B, C and D, E is important. Recent preliminary results with
lipid II-doped black lipid bilayers indicated that the pore stability
increases from the millisecond range to several seconds and that the
pore formation process becomes voltage-independent.2
This suggests that interactions with the lipid II moiety stabilize a
conducting transmembrane orientation of the peptides. Interactions of
-amino groups of lysine side chains with pyrophosphate groups of
lipid II may be helpful in this regard and could also explain the
reduced anion selectivity of the pore. Several nisin/lipid II complexes
are presumed to assemble for a functional pore thus explaining the
rapid efflux from liposomes and living cells of molecules of the size
of carboxyfluorescein, amino acids, and ATP (7). The position of the
lipid moiety of lipid II in the bilayer is unknown but may be of
ultimate importance for nisin pore formation; structural details of the
molecule are given in the bottom of the figure. The
cartoon of nisin points out functionally important segments
of the molecule; for the complete structure of nisin see Fig. 6.
Throughout this study we observed a striking correlation between the activity of nisin in vivo and its activity against lipid II-doped DOPC vesicles in contrast to pure DOPG or DOPC vesicles (Fig. 1B). The most important observation in this regard is that MIC values and the concentrations necessary for activity against lipid II-containing liposomes are in the same range, i.e. nanomolar instead of micromolar against lipid II-free liposomes. Also, high affinity binding to a docking molecule as an initial step of the activity can explain the discrepancy between the activity of some mutants on model membranes and against intact bacterial cells. In this regard, the S3T mutation as described earlier is the most clear example. For the hinge region mutants the correlation between the dye release activity (Fig. 5B) and the in vivo activity (Table I) was less obvious. However, what appears to be a discrepancy at first sight, rather highlights a unique feature of nisin that was not identified in other antibiotics so far. The N terminus seems to be involved in the binding of nisin to lipid II, whereas the hinge region between ring clusters A, B, C and D, E is most important for pore formation (as depicted in Fig. 7). Whereas peptides mutated in this segment lose the pore formation activity, their capacity for binding lipid II is less affected. Therefore, nisin retains the activity to prevent lipid II incorporation into polymeric peptidoglycan, thus acting in a way that is analogous to the antibiotics mersacidin and vancomycin (14). In contrast, the S3T mutation affects initial binding to lipid II and thus equally affects cell wall biosynthesis and the pore forming activity.
For the charged mutants, comparison of the in vivo activity and in vitro liposome results is complicated by the fact that the Gram-positive cell wall is a highly charged anionic polymer that acts as a binding matrix for polycationic peptides. Several studies (e.g. Ref. 38) have demonstrated the importance of the accumulation of cationic peptide antibiotics in the cell wall, which results in highly increased local concentrations of the peptides as compared with the surrounding media. Thus, although pore forming activity of the M17K peptide is affected in vitro, the increase in its net positive charge should lead to higher accumulation. Increased local concentration in addition to effective inhibition of peptidoglycan biosynthesis could explain the relatively small impact on the M17K mutant in vivo activity. In line with this interpretation is the relatively small effect on dye release of the introduction of either a positive or a negative charge in position 32, which in vivo resulted in wild-type activity when a positive charge is introduced (V32K) and a quite significant reduction when the net positive charge is further reduced (V32E).
The identification of a specific target enables for the first time rational design strategies, and a clear picture for structural and functional relationships in nisin is emerging. Although specific target-mediated poration may be detected more often in the future, nisin seems to be a particular case in that two different killing mechanisms are combined in one molecule. For both mechanisms, structural features of the peptide and defined segments in the molecule can now be identified. The results convincingly demonstrate that the interaction of nisin with lipid II is relevant in vivo at least for those bacterial strains, which are of high or intermediate sensitivity toward the lantibiotic. Low level sensitivity, which may start in the micromolar concentration range (corresponding to ~3 µg/ml) could result from lipid II-independent activity as observed with pure DOPG/DOPC liposomes and could explain the low activity of nisin against microorganisms that do not contain lipid II in their membranes, such as yeasts and fungi, as well as its low toxicity in animal model studies (2).
Thus, the accessibility of the docking molecule may be ultimately
relevant for the activity of the lantibiotic. Because cell wall
biosynthesis is a tightly controlled process, and the bactoprenol-bound central carriers in that process are limited in number, one can assume
that these molecules are in intimate contact with proteins involved in
the membrane-associated steps of cell wall formation. Depending on the
type of interactions with such proteins, the access to lipid II may be
restricted to such an extent that even Gram-positive bacteria could
become comparatively insensitive toward nisin as observed for the genus
Listeria (39). There is in fact very little knowledge on how
cell wall synthesis is functionally organized, and how bactoprenol
carriers interact with the enzymes; it is not even clear how lipid II,
in particular the bactoprenol C55 lipid tail (Fig. 7), is
embedded within the membrane, e.g. in a
trans-bilayer fashion or in between the monolayers. We
consider it realistic that the results reported here not only will form
the basis for new strategies to rationally improve nisin and related
lantibiotics, but also will enable us to address fundamental questions
about the structural organization of bacterial cell wall biosynthesis
and the underlying mechanisms by which bacteria can become resistant to
antimicrobial peptides.
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FOOTNOTES |
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* This work was supported by a short term fellowship from the European Molecular Biology Organization (EMBO) (to I. W.), by The Netherlands Foundation for Chemical Research (SON) with financial aid from The Netherlands Foundation of Scientific Research (NWO), the Foundation of Applied Science (STW) (to E. B., and C. V. K.) by the Bundesministerium für Forschung und Technologie (BMBF), and by the BONFOR Programme of the Medical Faculty of the University of Bonn.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present Address: Dept. of Genetics, Inst. of Biomolecular
Sciences and Biotechnology, University of Groningen, P.O. Box 14, 9750 AA Harem, The Netherlands.
** To whom correspondence should be addressed: Institut für Medizinische Mikrobiologie und Immunologie der Universität Bonn, Sigmund-Freud-Str. 25, 53105 Bonn, Germany. Tel:. 49 (228) 287 5704; Fax: 49 (228) 287 4808; E-mail: sahl@mibi03.meb.uni-bonn.de.
Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M006770200
2 I. Wiedemann, R. Benz, and H.-G. Sahl, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: MIC, minimal inhibitory concentration; DOPC, 1,2 dioleoyl-sn-glycero-3-phosphocholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; CF, carboxyfluorescein; OPA, o-phthaldialdehyde; HPLC, high pressure liquid chromatography; Mes, 4-morpholineethanesulfonic acid.
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REFERENCES |
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