From the Laboratoire de Chimie Biologique et
Unité Mixte de Recherche 8576 du CNRS, Université des
Sciences et Technologies de Lille,
59655 Villeneuve d'Ascq Cedex, France and the
¶ Département Récepteurs et Protéines
Membranaires, Unité Propre de Recherche 9050 du CNRS, Ecole
Supérieure de Biotechnologie de Strasbourg, rue Sébastien
Brant, 67400 Illkirch, France
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ABSTRACT |
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The binding of lactoferrin, an iron-binding
glycoprotein found in secretions and leukocytes, to the outer membrane
of Gram-negative bacteria is a prerequisite to exert its bactericidal
activity. It was proposed that porins, in addition to
lipopolysaccharides, are responsible for this binding. We studied the
interactions of human lactoferrin with the three major porins of
Escherichia coli OmpC, OmpF, and PhoE. Binding experiments
were performed on both purified porins and porin-deficient E. coli K12 isogenic mutants. We determined that lactoferrin binds
to the purified native OmpC or PhoE trimer with molar ratios of
1.9 ± 0.4 and 1.8 ± 0.3 and Kd
values of 39 ± 18 and 103 ± 15 nM,
respectively, but not to OmpF. Furthermore, preferential binding of
lactoferrin was observed on strains that express either OmpC or PhoE.
It was also demonstrated that residues 1-5, 28-34, and 39-42 of
lactoferrin interact with porins. Based on sequence comparisons, the
involvement of lactoferrin amino acid residues and porin loops in the
interactions is discussed. The relationships between binding and
antibacterial activity of the protein were studied using E. coli mutants and planar lipid bilayers. Electrophysiological
studies revealed that lactoferrin can act as a blocking agent for OmpC
but not for PhoE or OmpF. However, a total inhibition of the growth was
only observed for the PhoE-expressing strain (minimal inhibitory
concentration of lactoferrin was 2.4 mg/ml). These data support the
proposal that the antibacterial activity of lactoferrin may depend, at least in part, on its ability to bind to porins, thus modifying the
stability and/or the permeability of the bacterial outer membrane.
Lactoferrin (Lf)1 is an
80-kDa iron-binding glycoprotein found in various biological
secretions, mainly in milk (1), and in polymorphonuclear leukocytes
(2). The biological roles of Lf include antibacterial activities
through mechanisms not yet clearly elucidated. The ability of Lf to
tightly chelate two ferric ions allows the protein to limit the iron
availability to bacteria and ultimately causes bacteriostasis (3, 4).
Moreover, Lf and an Lf-derived peptide called lactoferricin (Lfcin) (5) (residues 1-47 and 18-41 for human (hLf) and bovine (bLf) Lfs, respectively) were shown to bind to Gram-negative bacteria, including Escherichia coli and to release
lipopolysaccharides (LPS) from the outer membrane (6-8). Stable
complexes are formed between free LPS and Lf (9-12), but the mechanism
of membrane destabilization is not clearly elucidated. During the last
few years, Naidu and co-workers (13-15) reported potential
interactions of either hLf or bLf with porins, the major pore-forming
proteins of the outer membrane of various Gram-negative bacteria. In
particular, the binding of Lf to porins OmpF and OmpC in E. coli was reported (13). However, the biochemical evidence of
interactions between Lf and porins was mainly based upon Western blot
analyses using SDS-extracted and heat-denatured porin monomers. Because
porins are potential receptors for bacteriophages and colicins (16, 17), it is likely that Lf may use porins as anchoring sites at the
surface of the outer membrane of bacteria. One can also hypothesize
that the binding of Lf to porins either facilitates the destabilization
of the bacterial outer membrane or limits the permeability of the membrane.
In the present paper, we have investigated whether hLf specifically
binds to porins and how it does. The binding characteristics of hLf to
porins OmpC, OmpF, and PhoE in E. coli were studied. Purified native porins and porin-deficient isogenic mutants of E. coli K12 were used. The porin-binding site of hLf was located using various hLf-derived proteins. Finally, a possible relationship between hLf binding to porins and the antibacterial activity of the
protein was investigated with E. coli porin mutants and
through electrophysiological studies in a planar lipid bilayer system.
Bacterial Strains--
E. coli K12 strains
constitutively expressing OmpC only, EC1234; OmpF only, EC1233; PhoE
only, EC1230; or no major porins at all, EC1231 (18) were kindly
provided by Prof. J. Tommassen (Utrecht, The Netherlands) and were
routinely grown in Luria-Bertani (LB) medium at 37 °C with aerobic shaking.
Purification of Porins--
Native OmpC, OmpF, and PhoE porin
trimers were extracted and purified to homogeneity from EC1234, EC1233,
and EC1230 E. coli K12 strains, respectively, as described
previously (19). Purity of porin samples was verified by
SDS-polyacrylamide gel electrophoresis (20) followed by Coomassie Blue
staining. No LPSs were detected by the Limulus amoebocyte lysate assay
(QCL1000, BioWhittaker).
Preparation of Lfs and Lf-derived Proteins--
Native hLf was
prepared according to Spik et al. (21). The iron-saturated
and apo forms of hLf were prepared as described previously (22, 23).
Mild tryptic digestion of hLf gave the N-terminally deleted proteins
hLf Expression and Purification of Recombinant hLfs--
Nonmodified
recombinant hLf (rhLf), rhLf Radiolabeling of hLf--
Human Lf was labeled either with
125I or with 59Fe. Radioiodination of hLf
(100-200 µg) was carried out with 0.3 mCi of 125I in the
presence of two Iodo-Beads (Pierce), according to the manufacturer's
instructions. After 10 min of incubation on ice, free iodine was
removed by gel filtration on a Sephadex G-25 PD-10 column (Amersham
Pharmacia Biotech). Specific radioactivity was typically between
700,000 and 1,000,000 cpm/µg of protein. For 59Fe
labeling, iron was first removed from hLf as described previously (22).
Human Lf was then incubated 30 min at room temperature in a 0.25 mM sodium nitrilotriacetate, 0.1 M Tris/HCl,
0.1 M sodium bicarbonate, pH 8.2, solution containing an
appropriate amount of 59Fe (33 µg/ml of carrier-free
59FeCl3 in 0.1 M of HCl, 0.1 mCi/ml, Amersham
Pharmacia Biotech). Finally, free iron was eliminated by passing the
solution through a Dowex 1 × 8 column (Bio-Rad) and desalting on
a PD-10 column (Amersham Pharmacia Biotech). A typical specific
radioactivity of about 5,000 cpm/µg of 59Fe-hLf was
obtained. To avoid radiation damage, the radiolabeled proteins were
used immediately for the binding experiments.
Binding of hLf to Purified Porins--
Purified porins OmpC,
OmpF, and PhoE were diluted in a 75 mM Tris/HCl, pH 6.8, buffer at a concentration of 46 nM. Denaturation of porins
was achieved by boiling aliquots of the porin solutions at 95 °C for
10 min in the presence of 1% SDS (w/v). Fifty µl of the porin
solutions (0.9 pmol of porin trimer) were then slot blotted to a
0.45-µm nitrocellulose membrane (Schleicher & Schuell) using a
Bio-Dot SF Microfiltration Apparatus (Bio-Rad). After drying, excess
sites on the membrane were blocked with a phosphate-buffered saline/2%
Tween 20 (w/v) solution for 45 min, and the membrane was washed with
phosphate-buffered saline/0.05% Tween 20 (w/v) (washing solution). It
was then incubated for 1 h with washing solution containing 125 nM unlabeled or radiolabeled hLf. For the optimal binding
pH experiments, incubations were performed using 125 nM
unlabeled hLf in the washing solution at pH values ranging from 5.0 to
8.0 with 0.5 pH unit increments. For Scatchard analysis (28),
nitrocellulose strips were incubated with various hLf concentrations
ranging from 0 to 250 nM in phosphate-buffered saline, pH
7.3. When experiments were performed with radiolabeled proteins,
membranes were washed three times with the washing solution and dried,
and the slots were cut for further counting on a Compugamma LKB-Wallac
Study of the hLf Porin-binding Site--
Purified native OmpC
and PhoE trimers (0.9 pmol) and 0.09-3.12 pmols samples of hLf-derived
proteins were immobilized on nitrocellulose paper that was blocked and
washed as described above. Blots were then incubated with 125 nM of each of the hLf-derived proteins for 1 h at room
temperature and immunostained as previously mentioned. Quantitation of
porin-bound proteins was performed with Quantiscan software in
comparison with the blotted protein standards.
Binding of hLf to E. coli Porin-deficient
Mutants--
Radioiodinated proteins are generally used for cell
binding studies. However, because we have demonstrated that
125I-hLf was unable to bind to purified porins, two
different methods allowed us to compare the binding parameters of hLf
to mutant strains EC1230, EC1231, EC1233, and EC1234. First, the
affinity constants were estimated by flow fluorocytometry. In this
method, bacterial strains were grown 3-5 h in LB medium at 37 °C to
an A600 of 0.5 (5 × 108
cells/ml), washed in M9 medium supplemented with essential amino acids,
pH 7.3 (29), harvested, and resuspended in M9 medium containing 0.2%
hTf (w/v) to minimize nonspecific binding to plastic vials and to
bacteria. Aliquots of a 200-µl cell suspension containing 106 cells were incubated with hLf concentrations ranging
from 0 to 250 nM for 45 min. After washing, bacterial cells
were incubated for 45 min in M9 medium containing 0.2% (w/v) bovine
serum albumin and purified rabbit fluorescein isothiocyanate-labeled
anti-hLf antibodies prepared in our laboratory. Green fluorescence
bound to bacteria was then measured using a Becton-Dickinson FACScan Flow Cytometer. Bacteria were gated for forward and side angle light
scatters, and 10,000 events of the gated population were analyzed. The
1024 fluorescence channels were set on a logarithmic scale, and the
mean fluorescence intensity was determined. The affinity constants of
hLf to E. coli mutant strains were calculated by Scatchard
plot analysis using the Biosoft Enzfitter software.
The lack of internal hLf standard in cytometry experiments led to
59Fe-hLf binding studies to estimate the total number of
hLf-binding sites on bacteria. For this, 108 bacterial
cells in 1 ml of M9 medium were incubated with 250 nM
59Fe-hLf for 45 min. Then, cells were washed three times
with M9 medium, and pellets were counted on a Compugamma LKB-Wallac
Planar Lipid Bilayer Assays--
All experiments were carried
out using double quartz-distilled water, reagent grade chemicals, and a
septum pressed between two Teflon cups. Schindler (30) technique
bilayers were formed across a 200-µm hole in a 12-µm-thick Teflon
septum pretreated with a solution of 2% n-hexadecane in
n-hexane. Conductance measurements and the criteria for
bilayer formation were as described (19). The trans
compartment was held to virtual earth. The sign of the membrane
potential referred to that on the cis side of the membrane, and the values quoted, therefore, refer to
Vcis-Vtrans. 50-85 pmols
of OmpC, OmpF, or PhoE native purified porins were then added to the
subphase on the cis side of the preformed bilayers with the
aqueous solution stirred by a magnetic bar. The membrane current was
amplified with a Burr Brown current-voltage converter with an
operational amplifier and feedback resistors ranging from 108 to 109 ohms. Solutions of hLf or bLf at
final concentrations of 56-560 nM in a 10 mM
Tris acetate, 5 mM CaCl2, 0.1 M NaCl, pH 7.0, were incubated in the cis compartment at a 50 mV potential
to favor interactions of hLf with porins. Recordings were filtered at 1 kHz with a EG and G low-pass filter and recorded on a Racal FM tape
recorder. All experiments were performed at room temperature in a
Faraday chamber.
Minimal Inhibitory Concentration Assays--
Bacterial strains
EC1230, EC1231, EC1233, and EC1234 were grown 3-5 h in LB medium at
37 °C to an A600 of 0.5 (5 × 108 cells/ml) and then diluted in 1% Bacto-tryptone (w/v)
(Difco) to obtain 2 × 105 bacteria/ml. Five µl of
this cell suspension were added to 250 µl of 1% Bacto-tryptone (w/v)
buffered at pH 7.3 with 20 mM sodium phosphate and
containing iron-saturated hLf at concentrations of 0.12, 0.5, 1.2, 2.4, 3.6, and 16 mg/ml that was previously sterilized by filtration on 0.1 µm sterile filters (Millipore). Incubations were performed in 5-ml
sterile glass tubes at 37 °C for 18 h under gentle shaking.
Bacteria were then diluted and grown for 24 h at 37 °C on LB
agar plates for counting. The sample without hLf served as the negative control.
Binding Parameters of Lf to Purified Porins--
We tested whether
hLf was able to bind to nitrocellulose-immobilized porins, either in
the SDS- and heat-denatured monomeric form or in the native trimeric
form. Binding studies were performed with 125 nM unlabeled
and iron-saturated hLf followed by immunostaining. As shown in Table
I, hLf bound to native trimeric OmpC and,
in a lesser extent, to denatured monomeric OmpC with hLf/porin molar ratios of 1.9 and 0.8, respectively. Human Lf bound to native trimeric
PhoE in a way similar to that of OmpC trimers with a molar ratio of
1.8, but no binding was observed when PhoE porins were denatured
(monomeric form). Concerning OmpF, a very low binding of hLf to either
denatured or native forms of the porin was noted. The molar ratio was
about 0.1. Similar results were obtained when either nonsaturated (apo)
or 59Fe-saturated hLf was used as a probe (data not shown).
However, the binding of 125I-labeled hLf was only observed
on denatured porins OmpC and PhoE with molar ratios as low as 0.2 and
0.1, respectively (data not shown).
Because hLf exhibited significant binding to the OmpC and PhoE porins
in the native trimeric form, further studies were performed only with
these two porins. The binding of iron-saturated hLf to native porins
OmpC and PhoE was estimated at pH values ranging from 5.0 to 8.0. Maximal binding of hLf to OmpC and PhoE occurred around pH 6.6 and 7.5, respectively (data not shown). On the basis of these results, an
intermediate pH value of 7.3 was used to determine the hLf binding
parameters to porins according to the Scatchard method (28). As
shown in Fig. 1, the binding of hLf to porins was both concentration-dependent and saturable at
concentrations ranging from 10 to 250 nM. Calculated
dissociation constants (Kd) were 39 ± 18 and 103 ± 15 nM, and hLf/porin trimer molar ratios were 1.9 ± 0.1 and 1.8 ± 0.2 for OmpC and PhoE,
respectively.
Location of the hLf Porin-binding Site--
To localize the
porin-binding site of hLf, the binding of eight hLf-derived proteins to
native OmpC and PhoE was assayed. Furthermore, hTf and bLf were used to
evaluate the specificity of hLf binding. Table
II shows that none of the N- and
C-terminal fragments or the N2 fragment of hLf significantly bound to
OmpC or PhoE. Furthermore, the absence of residues 1-3, 1-4, and 1-5 in hLf strongly decreased the binding of the protein to PhoE. The
binding of hLf Effect of hLf on the Permeability of Porins--
The effect of hLf
on the channel conductance of OmpC, OmpF, and PhoE integrated in
azolectin bilayers was studied. Porin trimers were incorporated into
these bilayers by injecting detergent-solubilized protein into the
bathing solution (cis compartment). The appearance of
channels was shown by the stepwise increases in current when voltage
clamped at 50 mV. When the potential across the bilayer was raised from
80 to 200 mV, downward current steps confirmed the closure of
individual monomers. Depending on the experiment, conductance
measurements revealed 1-4 porin trimers integrated into the bilayers.
Fig. 2a illustrates a
representative experiment where four OmpC trimers were integrated in
the membrane and incubated with 56 nM hLf. After a time lag
of 160 s, injection of hLf in the cis compartment
increased the noise of the current trace. The enlargment shown in the
inset of Fig. 2a demonstrates that this noise
consisted of 25 pA current fluctuations because of the fast closing and
opening of porin channels. These fluctuations are of the same amplitude
as those observed in the absence of hLf. Raising the hLf concentrations
to 560 nM significantly increased the frequency but not the
amplitude of the current fluctuations (Fig. 2b). Fluctuation
did not occur when the polarity of the membrane potential was reversed
(Fig. 2b). When bLf was used instead of hLf, a blocking
effect on OmpC was also observed but at a lower frequency (Fig.
2c). In contrast to OmpC, neither PhoE (Fig. 2d) nor OmpF porins (data not shown) exhibited significant conductance alterations in the presence of 56-560 nM hLf
concentrations. These results show that hLf and, to a lower extent, bLf
induce open-closed transitions of the OmpC pores, whereas PhoE and OmpF
are unaffected.
Binding of hLf to E. coli Porin-deficient Mutants--
To assess
the role of OmpC or PhoE in binding hLf to the bacterial cell surface,
the binding parameters of hLf to the isogenic E. coli K12
strains EC1230 (PhoE+), EC1231 (no major porins), EC1233 (OmpF+), and
EC1234 (OmpC+) were investigated. Because we observed that
59Fe-hLf, but not 125I-hLf, was able to bind to
purified porins, the radioiron-labeled protein was used as a probe.
However, because of its low specific radioactivity (about 5000 cpm/µg), 59Fe-hLf was unsuitable to perform Scatchard
analysis at low ligand concentrations. Therefore, we carried out flow
cytofluorometry studies using immunorevealed hLf to estimate the
dissociation constants (Kd) of the ligand. Then,
the number of binding sites/bacterium was compared through binding
experiments with radioiron-labeled hLf. As reported in Table
III, lower Kd were
calculated for the strains expressing OmpC and PhoE than for the
strains expressing OmpF or no major porins at all. Moreover, 40-50,000
more binding sites/bacterium were calculated for both OmpC+ and PhoE+
strains compared with the two other strains (Table III).
Effect of hLf on the Growth of E. coli Mutant Strains--
Because
preferential binding of hLf occurred on OmpC and PhoE either in the
isolated form or present on E. coli K12, we investigated whether this phenomenon would affect the growth of bacteria. To avoid
iron deprivation of the medium by hLf and subsequent bacteriostasis as
described previously (3, 4) and because holo and apo hLf are able to
bind to porins in the same manner, iron-saturated hLf was used for
these experiments. Minimal inhibitory concentration assays were
performed on mutant strains with hLf concentrations up to 16 mg/ml
(Fig. 3). As shown in Fig. 3a,
the growth of only one strain, EC1230 (PhoE+), was totally inhibited by
hLf at concentrations above 2.4 mg/ml. No clear growth inhibition was
evidenced for the other strains (Fig. 3,
b-d).
Porins were pointed out as potential targets for the binding of Lf
to the surface of Gram-negative bacteria (13). However, the target
porins were not clearly identified. Furthermore, the mechanisms by
which Lf binds to porins and, hence, exerts its antibacterial activity
have not been thoroughly investigated. Here we show that hLf binds with
high affinity to purified native OmpC and PhoE trimers but not to OmpF.
This binding also occurs when OmpC and PhoE porins are present on the
surface of E. coli K12 mutant strains. Indeed, whereas
binding of hLf to OmpF-expressing cells was not significantly greater
than the porin-deficient cells, 40,000-50,000 extra binding sites were
present on OmpC- and PhoE-expressing strains. Furthermore, 2 and 3.5 times higher affinities, respectively, were observed on these mutant
strains. Because the E. coli strains differ only by the
expression of the major porins (18), it can be concluded that OmpC and
PhoE porins constitute functional hLf-binding sites at the surface of
bacteria. However, hLf bound to approximately 70,000 sites on EC1231
(no major porin). Thus, other binding sites may exist on E. coli K12.
According to x-ray diffraction studies, porins are organized as
trimeric complexes, and each monomer is folded as a
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3N (residues 4-692) and hLf
4N (residues
5-692) (24), the 30-kDa N-terminal (residues 4-283) and 50-kDa
C-terminal (residues 284-692) fragments, as well as the 18-kDa
N-terminal domain 2 (residues 91-255) of hLf (25). Human
serotransferrin (hTf) and bLf were purchased from Sigma and Biopole
(Brussels, Belgium), respectively. All chemicals used were of the
highest analytical grade.
5N, a mutated rhLf, in which
sequence 1GRRRR5 was deleted, and rhLf-EGS, a
rhLf whose sequence 28RKVRGPP34 was replaced by
EGS (the 365-367 C-terminal counterpart of sequence 28-34) were
produced in a baculovirus expression system as previously reported (12,
24, 26). The rhLf-SAST corresponds to rhLf in which
39KRDS42 was substituted by residues SAST, the
372-375 C-terminal counterpart of sequence 39-42. For this
purpose, a mutagenic oligonucleotide 5'-GTCAGCTGCATATCAGCATCAACCCCCATCCAGTG-3' was synthesized by
Eurogentec (Seraing, Belgium). The template for the mutagenesis was the
phage M13-mp11, containing a 346-base pair
EcoRI-AccI fragment of the coding sequence cloned
into a pBluescript SK plasmid (27). The mutation was confirmed by DNA
sequence analysis, and the mutated EcoRI-AccI was
ligated back into pBluescript SK with the complementary part of the
full-length cDNA of hLf as formerly described (27). Finally, the
mutated cDNA was subcloned into pVL1392 (PharMingen), and the
rhLf-SAST was produced in the baculovirus expression system and
purified on a SP-Sepharose fast flow column, as described previously
(26). The purity of the mutant protein was verified by 7.5%
SDS-polyacrylamide gel electrophoresis followed by Coomassie Blue
staining. The N-terminal amino acid sequence analysis was performed
with the Edman degradation procedure, using an Applied BioSystem 477 protein sequencer.
-radiation counter. When immunochemical staining was required,
washed membranes were first incubated with rabbit polyclonal anti-hLf
antibodies (1/1000 diluted whole antiserum produced in our laboratory)
in a washing solution for 40 min, washed again, and incubated for 40 min with goat peroxidase-labeled anti-rabbit IgG antibodies (dilution
1/2500 in washing solution) (Biosys). Staining was achieved by using
the Diaminobenzidine Peroxidase Substrate Tablet Set (Sigma) according
to the manufacturer's instructions. All incubations were carried out
at room temperature. Samples containing 6.25, 3.12, 1.56, 0.75, 0.37, 0.19, and 0.09 pmols of either unlabeled or 125I- or
59Fe-labeled hLf dissolved in 75 mM Tris/HCl,
pH 7.3, were also applied to nitrocellulose paper as standards for
further quantification. Quantification of hLf bound to porins was
performed by densitometry analysis using a Hewlett-Packard 4C scanner
and Biosoft Quantiscan software. Scatchard plot analysis was performed
using Biosoft Enzfitter software.
-radiation counter. Nonspecific binding of hLf to bacteria was
determined by adding a 100-fold excess of cold iron-saturated hLf to
the incubation mixture. All incubations were performed at room temperature.
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ABSTRACT
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DISCUSSION
REFERENCES
Binding of hLf to purified OmpC, OmpF, and PhoE porin trimers
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Fig. 1.
Binding of hLf to purified native OmpC and
PhoE porin trimers. Curves show the binding of hLf to 0.9 pmol of
OmpC ( ) and PhoE (
) porin trimers immobilized onto nitrocellulose
and immunostained as described under "Experimental Procedures." The
inset shows a Scatchard plot (28) analysis of the data and
the calculated binding parameters Kd and
n (hLf/porin trimer molar ratio). Values are mean ± S.E. of three separate experiments conducted in duplicate.
3N, hLf
4N, and
rhLf
5N to PhoE was only 26, 24, and 9% of that of intact
hLf, respectively. Similar results were obtained with OmpC, except that
the deletion of residues 1-3 (hLf
3N) from hLf did not
affect the binding of the protein. The binding of hLf
4N
and rhLf
5N to OmpC was 42 and 7% of the native hLf
control, respectively. Furthermore, Table II shows that rhLf-EGS and
rhLf-SAST, which correspond to hLf modified at sequences 28-34 and
39-42, respectively, also exhibited low binding capacities to both
OmpC and PhoE. Lastly, whereas the binding of hTf to the porins was not
detectable, bLf bound to OmpC and PhoE in a way similar to that of
hLf.
Binding of hLf-derived proteins to purified OmpC, OmpF, and PhoE
porin trimers
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Fig. 2.
Effect of hLf and bLf on the conductance of
porins OmpC and PhoE integrated in a planar lipid bilayer system.
A, after activation of four OmpC porin trimers, hLf (56 nM final concentration) was injected in the cis
compartment at 100 mV applied potential. After a short time lag, the
current trace became noisy. The inset shows that this noise
was because of single channel fluctuations. Upon reversal of the
membrane potential to 100 mV, the noise disappeared. B,
increasing ten times the hLf concentration increased the noise.
C, same experiment as in b but with 560 nM bLf. Three OmpC porin trimers were activated.
D, in this experiment, only one PhoE trimer was activated.
Because PhoE is more sensitive to high voltage than OmpC, closing
events of PhoE single channels occurred. However, the injection of hLf
(56 or 560 nM) did not affect the conductance. The applied
voltage was +110 mV.
Binding parameters of hLf to porin-deficient E. coli K12 strains
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Fig. 3.
Minimal inhibitory concentration assays of
hLf on strains (A) EC1230, (B)
EC1231, (C) EC1233, and (D)
EC1234. Methodology is as described under "Experimental
Procedures." Results are means ± S.E. of three experiments in
duplicate. CFU, colony-forming units.
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-barrel built by
16 anti-parallel
-strands with short turns aligning the channel at
the periplasmic side and long loops extending into the medium (31).
Most loops, i.e. L1, L2, and L4-L8, are exposed at the cell
surface and exhibit reactivities to various phages and colicins
(32-34). A striking feature of porin loops is the presence of several
acidic amino acid patterns, e.g. sequence 290DDED293 of OmpC in L7 (Fig.
4). Because of its basic charge (pI
8.5-9), hLf could interact in a nonspecific manner with negatively
charged regions of porins. Nevertheless, the binding of hLf to purified OmpC and PhoE trimers cannot be attributed to simple electrostatic interactions because of the following: (i) hLf binding occurs with a
high affinity and (ii) no significant hLf binding to OmpF was noted
despite the presence of acidic patterns in its amino acid sequence. As
shown in Fig. 4, all the surface-exposed loops of OmpC are either
shorter or longer than the OmpF counterparts. When calculated using the
Alignp program from the Fasta package (35), sequence identities between
OmpC and OmpF range from 35.3% for L6 to less than 10% for L4 and L1.
It can thus be assumed that hLf binds to structural motifs that are
present on OmpC but not on OmpF. L1 of OmpC, which poorly matches with
the OmpF sequence, is 57.1% identical to L1 of PhoE and contains a
similar number of amino acid residues (Fig. 4). Therefore, L1 may
participate in the binding of hLf.
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Fig. 4.
Comparison of the sequences of the external
loops of porins OmpC, PhoE, and OmpF. Conserved residues in the
three porins are boxed. Residues identical in OmpC and PhoE
but different in OmpF are shaded. Alignments and numberings
of OmpC, PhoE, and OmpF sequences are taken from Jeanteur et
al. (56).
The N-terminal sequence 2RRRR5 and a loop
28RKVRGPP34 of hLf interact with many target
molecules of mammalian cells such as receptors (36-39), DNA (11), and
proteoglycans (11, 24, 40-42). It was also hypothesized that another
loop region 39KRDS42, which inhibits platelet
aggregation (43), is a part of the hLf receptor (36). These three
regions are present in human Lfcin, an antibacterial peptide consisting
of residues 1-47 of hLf (5). Moreover, 2RRRR5
and 28RKVRGPP34 were previously shown to bind
with a high affinity to the lipid A moiety of LPS (9-12). Our results
demonstrate that residues 2-5, but also loops 28-34 and 39-42 of
hLf, are involved in the interactions with porins OmpC and PhoE. First,
as shown in Table II, the C-terminal fragment (residues 284-692), the
N-terminal fragment (residues 4-283), thus lacking
1GRR3, nor the N2 fragment (91-255) isolated
from hLf significantly bound to purified porins. Furthermore,
sequential removal of the N-terminal amino acid residues
1GRRRR5 of hLf gradually but strongly decreased
the binding of hLf to porins. As it could be expected from the
estimated binding parameters of hLf to purified porins, the
participation of residues 2-5, 28-34, and 39-42 in binding to either
OmpC or PhoE is not identical. Whereas the modification of loop 28-34
inhibited binding to both porins to a similar extent, modification of
loop 39-42 inhibited more efficiently the binding of hLf to PhoE. It
was also observed that removing residues 1-3 from hLf altered its
binding to PhoE but not to OmpC. Fig. 5
shows that three residues of hLf within regions 1-5, 28-34, and
39-42 (Arg4, Lys29, and Arg40) are
identical to bLf, but different from hTf. These basic residues are
potential candidates for porin binding. However, because iodination of
hLf prevents the binding to porins but no tyrosine residue lies in the
1-50 region, other amino acids may interact with porins. This is
supported by our finding that both hLf and bLf alter the permeability
of OmpC and thus probably fit closely to several surface-exposed loops
of the porin channel.
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Porins have traditionally been described as permanently open pores (44). High voltages allow the closure of pores, but the physiological relevance of this effect is still unclear (45). Polyamines such as cadaverine, which is normally associated with the outer membrane of E. coli, were shown to promote closures of porins and therefore could be regulators of OmpC and OmpF activity in vivo (46). We show that Lf, in a similar way to polyamines, induces open-closed transitions of the OmpC channel and thereby decreases the total amount of ion flux across the planar membrane. The effect is more pronounced with hLf than with bLf and is observed only at positive voltage. This is in agreement with the basic nature of the hLf region predicted to interact with the porin. At positive voltage, the electric field in the pore mouth will promote plugging of the pore by positively charged ligands, whereas negative potentials will repel them. No evidence of interactions of hLf with PhoE could be obtained from conductance studies. This apparent contradiction with direct binding studies can be resolved considering that the PhoE channel is anion selective, whereas OmpC porin is cation selective. The channel lumen of PhoE contains an additional lysine residue (31) that may prevent direct interaction of hLf with the constriction of the pore as hLf may do with OmpC. This hypothesis supports the idea that there is more than one contact site between hLf and porins.
The relationships between bactericidal activity and binding of Lf in
numerous Gram-negative bacteria have been previously reported (7, 8),
but the mechanism(s) by which hLf exerts its activity at the membrane
level was not clearly elucidated. Both lactoferrin and its
pepsin-derived peptide fragment, Lfcin (5), cause membrane disruption
thus leading to the release of LPS (47). Affinity sites for
Ca2+ and Mg2+ on the pyrophosphoryl- and/or
phosphodiester groups of the ketodeoxyoctulonic acid-lipid A region and
on the ketodeoxyoctulonic acid trisaccharide unit of LPS play an
important role in the assembly or maintenance of the organization of
the outer membrane (48). Therefore, it has been hypothesized that Lf or
Lfcin, in a manner similar to EDTA, may chelate divalent cations or
bind to the cation-binding sites of LPS (47, 49). Evidence of direct
interactions between either Lf or Lfcin and LPS strongly supports the
second hypothesis (10, 12, 50). Lf was shown to interact with the lipid
A moiety of LPS (9). Elsewhere, Lfcin and Lfcin-derived basic helicoidal peptides bear similarities with several classes of natural
antimicrobial peptides, which also interact with the cation-binding sites of LPS (50-52). However, the mode of action of these peptides is
still much debated. It was proposed that cationic amphipathic -helical peptides form ion channels through membrane bilayers (53).
The basic amphipathic region of Lfcin (residues 21-31 in hLf) adopts
an
-helical structure, which is critical for its antimicrobial
potency (50, 54). Furthermore, the homologous region in bovine Lfcin
appears able to adopt a helical or sheet-like conformation similar to
what has been proposed for the prion proteins and Alzheimer's peptides
(52). It was hypothesized that membrane interactions alone are capable
of inducing a similar
-to-
-transition in the N-terminal region of
intact Lf (52). Lf has a less potent bactericidal effect than Lfcin and
its derived peptides (5, 50, 54). This may have been explained by the
following: (i) the degree to which the peptides can interact with the
bacterial membrane, as a result of their increased flexibility and (ii) their ability to enter the outer membrane and to reach the cytoplasmic membrane (50, 54). Another explanation is that the carbohydrate moiety
of LPS may prevent or limit the binding of Lf, a larger molecule than
Lfcin, to the outer membrane. We detected about 105 hLf
bound to one E. coli cell, whereas a 10-fold higher binding of Lfcin was reported (55). In the present work, we show that OmpC and
PhoE provide anchoring sites on E. coli that may facilitate the accessibility of hLf to the outer membrane. As illustrated in Fig.
6, tight interactions between Lf and OmpC
probably limit ion and nutrient fluxes through the outer membrane but
do not lead to a marked growth inhibition. In contrast, Lf binds to
PhoE in a way that does not limit the permeability of the porin but permits an antibacterial effect. We postulate that preliminary binding
of Lf to PhoE with a slightly lower affinity than to OmpC allows
further interactions of the protein with the outer membrane thus
facilitating its destabilization (Fig. 6).
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In conclusion, our findings show that hLf may use OmpC and PhoE porins
of E. coli as high affinity anchoring sites. They open the
way to investigating the nature and physiological relevance of hLf
binding to porins and other outer membrane proteins of pathogenic bacteria.
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ACKNOWLEDGEMENTS |
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We thank Dr. Marie-Odile Husson for her useful advice, Monique Benaïssa for her technical assistance, and Joyce M. Gardiner for her secretarial work.
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FOOTNOTES |
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* This work was supported in part by the Ministère de l'Enseignement et de la Recherche Scientifique (ACC SV5, Interface Chimie-Biologie) and the Centre National de la Recherche Scientifique.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: Health and Environment Unit, Laval University, Medical Research Center, CHUQ, Faculty of Medicine, 2705, Blvd. Laurier, Sainte-Foy, Quebec, Canada G1V 4G2.
Present address: Institut de Biochimie de l'Académie
Roumaine, Splaiul Independentei 296, 77.700 Bucarest 17, Rumania.
** To whom correspondence should be addressed. Tel.: 33 3 20 33 72 38; Fax: 33 3 20 43 65 55; E-mail: Dominique.Legrand{at}univ-lille1.fr.
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ABBREVIATIONS |
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The abbreviations used are:
Lf, lactoferrin;
hLf, human lactoferrin isolated from milk;
rhLf, recombinant hLf;
hLf3N and hLf
4N, milk hLf lacking the
first three or four N-terminal residues;
rhLf
5N, rhLf
lacking the first five N-terminal residues;
Lfcin, lactoferricin;
bLf, bovine lactoferrin isolated from milk;
hTf, human
transferrin;
LPS, lipopolysaccharides.
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REFERENCES |
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