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Applied and Environmental Microbiology, July 1999, p. 2895-2900, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Delineation of Key Amino Acid Side Chains and
Peptide Domains for Antimicrobial Properties of Divercin V41, a
Pediocin-Like Bacteriocin Secreted by Carnobacterium
divergens V41
Parwin
Bhugaloo-Vial,1,2
Jean-Paul
Douliez,1
Daniel
Mollé,3
Xavier
Dousset,2
Patrick
Boyaval,3 and
Didier
Marion1,*
Unité de Biochimie et Technologie des
Protéines, INRA, 44316 Nantes Cedex
03,1 Laboratoire de Microbiologie,
ENITIAA, 44072 Nantes Cedex,2 and
Unité de Recherches de Technologie Laitière,
INRA, 35042 Rennes Cedex,3 France
Received 5 August 1998/Accepted 12 April 1999
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ABSTRACT |
Divercin V41 (DV41) is a class IIa bacteriocin produced by
Carnobacterium divergens V41. This antilisterial peptide is
homologous to pediocin PA-1 and contains two disulfide bonds. To
establish the structure-activity relationships of this specific family
of bacteriocin, chemical modifications and enzymatic hydrolysis were performed on DV41. Alteration of the net charge of this cationic bacteriocin by succinylation and acetylation revealed that, in a
certain range, the electrostatic interactions were surprisingly not
necessary for the activity of DV41. Cleavage of DV41 by endoproteinase Asp-N released two fragments N1[1-17] and N2[18-43] corresponding to the conserved hydrophilic N-terminal and the variable hydrophobic C-terminal sequences, respectively. Inhibitory assays showed that only
the C-terminal fragment was active, and after trypsin cleavage at Lys42
or disulfide reduction it lost its inhibitory activity. These results
suggested that both hydrophobicity and folding imposed by the
Cys25-Cys43 disulfide bond were essential for antilisterial activity of
the C-terminal hydrophobic peptide. Chemical oxidation of tryptophan
residues by N-bromosuccinimide demonstrated that these residues were
crucial for inhibitory activity since modification of any one of them
rendered DV41 inactive. On the contrary, only the modification of all
the three tyrosine residues caused a total loss of antilisterial
activity. These latter results strengthened previous results suggesting
that the N-terminal domain containing the YGNGV consensus sequence was
not involved in the binding of DV41 to a potential specific receptor on
listerial cells.
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INTRODUCTION |
Lactic acid bacterium (LAB)
bacteriocins are antimicrobial peptides which could be useful as
natural and nontoxic food preservatives. These compounds could also be
considered for other applications in human health and may provide new
approaches for dealing with antibiotic-resistant bacteria
(22). LAB bacteriocins can be generally divided in two main
classes, the lantibiotics and nonlantibiotic peptides. Among the
latter, the class IIa bacteriocins (17) are cationic
heat-stable and membrane-active peptides, containing one or two
disulfide bonds. Members of this group are amphiphilic with a
hydrophilic conserved NH2-terminal domain and a hydrophobic variable COOH-terminal of equivalent size. The structure of leucocin A,
a class IIa member with one disulfide bond, has been recently obtained
by nuclear magnetic resonance (12). The hydrophilic domain
forms a three-strand antiparallel 
-sheet stabilized by an
intramolecular disulfide bond, while the C-terminal domain forms an
amphipathic helix in lipid micelles. This structure is compatible with
the pore-forming properties of pediocin-like bacteriocins (5).
If it is now accepted that the membrane is the primary target of
bacteriocins, it is still not known how the peptide inserts into
membrane to form a structured pore. In the first step of adsorption
onto membranes, the necessity of a specific receptor is still discussed
(4, 5). As for other membrane active peptides and proteins
(8, 9, 19, 29), this process could only require the highly
hydrophobic and cationic character of the bacteriocin. A preliminary
response can be done by changing chemically, enzymatically, or
genetically the chemical structure of some amino acid side chains. For
example, Fleury et al. (11) and Quadri et al.
(24) have recently shown from synthetic mesentericin Y105
and recombinant carnobacteriocin B2 mutants, respectively, that
changing just a residue in both the N- and the C-terminal domains
induces important and often complete loss of the inhibitory activity of
the corresponding bacteriocins. Chen et al. (3) in studies
with synthetic peptides have suggested that the N-terminal is involved
in the electrostatic interaction of class IIa bacteriocins with anionic
membrane phospholipids of target cells.
As a part of these structure-function studies we have performed, for
the first time, different chemical and enzymatic modifications on
divercin V41 (DV41), a class IIa bacteriocin containing two disulfide
bonds. DV41 is a 43-amino-acid peptide of 4,509 Da with two disulfide
bonds, produced by Carnobacterium divergens V41 (21,
23) (Fig. 1). The results
highlighted the respective roles of the conserved N-terminal and
variable C-terminal domains, as well as of some amino acid side chains.
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FIG. 1.
Amino acid sequence of divercin V41 from C. divergens V41 (21). The modified residues are in
boldface letters. N1 and N2 are the resulting fragments obtained after
endoproteinase Asp-N digestion, and T1 and T3 are the resulting
fragments after trypsin digestion. An asterisk indicates the cysteine
residue involved in the disulfide bridge that maintains the two
fragments.
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MATERIALS AND METHODS |
Chemicals and enzymes.
Tetranitromethane (TNM),
dithiothreitol (DTT), hydroxylamine, and trifluoroacetic acid (TFA)
were purchased from Sigma-Aldrich Fine chemicals (St. Quentin
Fallavier, France). Iodoacetamide, succinic anhydride, and
N-bromosuccinimide (NBS) were from Merck (Darmstadt,
Germany). Acetic anhydride was from Prolabo (Gradignan, France).
Sequencing-grade endoproteinase Asp-N and trypsin were from Boehringer
Mannheim (Meylan, France). High-performance liquid chromatography
(HPLC)-grade acetonitrile (ACN) was purchased from SDS (Marseille, France).
Bacterial strains, culture conditions, and production of DV41 and
inhibitory activity.
C. divergens V41 isolated from fish
viscera (ENITIAA, Nantes, France) and Listeria innocua F
(IFREMER, Nantes, France) were maintained at 
80°C in MRS broth
(6) containing 15% (vol/vol) glycerol. Before use, the
strain was cultivated twice for 24 h at 30°C in MRS broth
(Biokar, Beauvais, France).
C. divergens V41 was grown on a Tween-deficient MRS medium.
Temperature was maintained at 20°C, and pH was regulated at 6.5 by
automatic addition (SET 2M; SGI, Toulouse, France) of 6 N NaOH (23). Inhibitory activity was tested against L. innocua F, grown in Elliker medium (Biokar, Beauvais, France)
containing 10 g of agar per liter. The 10 µl of DV41 or modified
DV41 was spotted onto agar plates containing the indicator strain.
Bacterial activity was monitored by monitoring the appearance of an
inhibition zone (1).
DV41 was purified from the supernatant of a 2-liter culture of
C. divergens V41 by using Triton X-114 phase-partitioning technique
(
2). Briefly, Triton X-114 was added to the culture medium
at up to 2% (wt/vol) at 4°C. After heating at 35°C and phase
separation,
the lower detergent-rich phase was removed, diluted 10 times with
water and loaded onto a carboxymethyl cellulose C200
(Amicon,
Beverly, Mass.) column (20 by 2.5 cm). The excess of the
nonionic
detergent was washed out by elution with deionized water. DV41
was eluted with a gradient of from 100% deionized water to 100%
0.7 M
NaCl in deionized water. Fractions containing DV41 were
detected by
absorbance at 280 nm and by the inhibition spot assay.
DV41 was
desalted and purified by preparative reversed-phase HPLC
(RP-HPLC) as
described
below.
The protein concentration was determined by the bicinchoninic acid
procedure as described by the supplier with bovine serum
albumin as a
standard (Pierce Europe, Oud Beijerland, The
Netherlands).
Chemical modifications.
DV41 was reduced and alkylated
according to the method of Yan et al. (30). Then, 200 µg
of peptide was dissolved in 0.1 M Tris-HCl (pH 8.5) buffer containing 6 M guanidine chloride. A 50 M excess of DTT was added. The reaction was
allowed to proceed overnight at room temperature under a nitrogen
atmosphere. Then, iodoacetamide was added (10 times the mass of DTT
used), and the mixture was placed in the dark for 1 h under nitrogen.
DV41 was succinylated according to the method of Klotz (
18).
A total of 100 mol of succinic anhydride per mol of amino group
(
-NH
2 and NH
2-terminal) was added in small
aliquots to a vigorously
stirred solution of DV41. The pH was
maintained at 8.0 to 8.5
by the addition of 1 M NaOH with a pH stat
(645 Multi-Dosimat).
The reaction was allowed to proceed during 1 h at room
temperature.
Acetylation of DV41 was carried out according to the method of Riordan
and Vallee (
25). A 3 M excess of acetic anhydride
was added
per mol of NH
2 group. A basic pH (ca. 10) was maintained
by
the addition of 1 M NaOH with a pH
stat.
Tryptophan residues were oxidized by the addition of NBS at room
temperature according to the procedure described by Spande
et al.
(
28). Oxidation of tryptophan to oxindolealanine was
followed by measuring the absorbance of these chromophores on
a Cary E1
spectrophotometer (Varian) and by the decrease of the
intensity of
tryptophan fluorescence on a Fluoromax spectrofluorimeter
(SPEX,
Edison, N.J.). DV41 (11 µmol) was solubilized in a 50 mM
sodium
acetate solution (pH 4.5), and small aliquots (1 µl) of
10 mM NBS in
the same buffer were added. For spectrophotometry,
a scan was recorded
at from 240 to 350 nm to monitor the increase
of absorbance at 250 nm
(oxindolealanine), while the absorbance
at 280 nm corresponding to
tryptophan residues decreased. For
spectrofluorimetry, the emission
spectra of tryptophan residues
were recorded from 300 to 400 nm with an
excitation wavelength
set at 295
nm.
Nitration of tyrosine residues was performed as described by Sokolovsky
et al. (
27). Aliquots from a freshly prepared stock
solution
of 25 mM TNM in 96% ethanol were mixed thoroughly with
pure DV41 (20 to 30 µM) solubilized in 0.1 M NH
4HCO
3 (pH
8.0).
Nitration proceeded for 60 min at 25°C.
Enzymatic cleavage of DV41.
Endoproteinase Asp-N was used as
recommended by the supplier. The lyophilized enzyme was suspended in 50 µl of distilled water, resulting in a buffer concentration of 10 mM
Tris-HCl (pH 7.5). DV41 was solubilized in a 50 mM sodium phosphate
buffer (pH 8.0). The enzyme was added in a ratio of 1:50 (wt/wt).
Digestion was allowed to proceed at 37°C for 18 h.
For the cleavage with trypsin, the lyophilized enzyme was solubilized
in deionized water containing 0.01% (vol/vol) TFA. The
peptide was
dissolved in a 20 mM ammonium acetate buffer (pH 8),
and the enzyme was
added in a ratio of 1:100 (wt/wt). Digestion
was allowed to proceed at
37°C for 12
h.
Purification of modified DV41 and endoproteinase peptides.
After nitration, excess reagent was eliminated by using batch
ion-exchange chromatography. Carboxymethyl cellulose C200 gel was added
to the reacting medium, and adsorption was allowed to take place for 30 min at room temperature. The gel was then washed with deionized water,
and finally the modified DV41 was eluted by using 0.1 M Tris-HCl (pH 8)
buffer containing 0.5 M NaCl.
All of the modified and cleaved peptides except acetylated DV41 were
finally purified by analytical RP-HPLC. The samples were
loaded on a
C
18 Nucleosyl column (250 by 4.6 mm, 5-µm-diameter
particles, 300 Å; CIL, Bordeaux, France). Elution was performed
at
50°C with a linear gradient from 100% deionized water with
0.06%
TFA (solvent A) to 100% ACN with 0.04% TFA (solvent B) in
50 min at 1 ml/min. Peptides detected by absorbance at 220 nm
were collected
manually. To prevent from cross-contamination,
only a fraction of the
corresponding absorbance peaks (generally
near the top of the peak) was
collected.
To purify acetylated DV41, semipreparative RP-HPLC was performed at
50°C. The sample was loaded on a C
18 Nucleosyl column
(250 by 10 mm, 300 Å, 5-µm-diameter particles; CIL). After the
loading, the salt was washed away by elution with 100% solvent
A for
10 min. Then, the peptides were subjected to a linear gradient
from
80% solvent A to 100% solvent B in 30 min at 3 ml/min and
were
detected by their absorbance at 280
nm.
Mass spectrometry and amino acid sequencing.
Molecular mass
was determined by using an API-III Plus mass spectrometer (Sciex,
Thornhill, Canada) equipped with an atmospheric pressure ionization
source (Electro-Spray mass spectrometer [ES-MS]). The sample analysis
was carried out either by an on-line coupling between MS and RP-HPLC
(LC-MS) or by using infusion pump syringe at a flow rate of 5 µl/min.
RP-HPLC columns (Symmetry C18; Waters Corp., Milford,
Mass.) were eluted at a flow rate of 250 µl/min with a split to the
MS ionization source that was set at a flow rate of 30 µl/min. The
instrument scale for the mass-to-charge (m/z) ratio was
calibrated with the ions of the ammonium adduct of polypropylene
glycol. Scan data were obtained with Tune 2.5, and mass calculation was
done with Biomultiview 1.2 (Software package Sciex).
The N-terminal amino acid sequence of DV41 was obtained by Edman
degradation performed on a model 447A gas-phase sequencer
equipped with
an on-line 120A phenythiohydantion amino acid analyzer
(Applied
Biosystems, Foster City, Calif.).
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RESULTS |
Endoproteinase cleavages of DV41.
Endoproteinase cleavages
were performed on nonreduced DV41. The endoproteinase Asp-N cleaves at
the N-terminal end of the aspartic and cysteic acids. Since DV41
possesses only one aspartic residue at position 18, two peptides were
obtained, N1 and N2, which were eluted in RP-HPLC at 25 and 38% ACN,
respectively (Fig. 2A). ES-MS revealed
that N1 stood for the NH2-terminal fragment, 1-17, while N2
corresponded to the C-terminal fragment, 18-43 (Fig. 1 and Table
1). N2 and the native DV41 were eluted at
the same retention time. Since ES-MS revealed trace amounts of DV41 in
the N2 peak, N1 and N2 peptides were first separated on the basis of
their net charge by use of a batch cation exchanger. N1 and especially
DV41, as positively charged peptides (net charge +3), were retained on
the cation exchanger, while N2, with a null net charge, was not. The
unbound material was desalted and purified by RP-HPLC, yielding pure N2
for biological inhibitory assays. Both N1 and the native DV41 were
retained on the ion exchanger and further eluted with 0.1 M Tris (pH 8)
containing 0.5 M NaCl. Finally, RP-HPLC afforded peptide N1.
Antimicrobial assays revealed that N1 was totally inactive, whereas N2
still displayed activity against L. innocua (Table 1).
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FIG. 2.
RP-HPLC of endoproteinase digests. (A) Asp-N digest of
divercin V41. N1 and N2 correspond to fragments 1-17 and 18-43, respectively. (B) RP-HPLC of tryptic digest of divercin V41. T1 and T3
correspond to fragments 1-14-15-43 and 3-43, respectively, and T2
corresponds to DV41.
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Trypsin specifically cleaves peptide bonds at the carboxyl side chains
of lysine and arginine. DV41 displayed only four potential
cleavage
sites at Lys2, Lys13, Lys14, and Lys42 (Fig.
1). Three
main peaks were
obtained by RP-HPLC after 12 h of incubation of
DV41 with trypsin
(Fig.
2B). One of them (T2) displayed the same
retention time as DV41,
and ES-MS confirmed that T2 corresponded
to the nonhydrolyzed
bacteriocin (Table
1). At an enzyme/DV41
mass ratio of 1:100, trypsin
hydrolysis was not complete. At higher
enzyme/DV41 ratios, i.e. 1:50
and 1:20, we only observed an increase
of T1 and T3 in regard to T2,
and no other cleavage sites were
obtained (results not shown).
According to ES-MS, T3 was shown
to be the 3-43 fragment, indicating
that DV41 was cleaved at Lys2
(Table
1). ES-MS showed that tryptic
peptide T1 had a molecular
mass of 4,527 Da, which corresponded to the
mass of DV41 plus
18 Da (Table
1). This revealed that the bacteriocin
was indeed
cleaved into two fragments that were still attached by a
disulfide
bridge (Fig.
1). Amino acid sequencing of T1 yielded two
subsequences,
TKYYG and XWVD. X corresponded to a blank cycle in Edman
degradation
and was interpreted as a cysteine residue involved in a
disulfide
bond. These results suggested that the cleavage site was at
Lys14.
Therefore, T1 stood for fragments 1-14 and 15-43 linked by the
disulfide bond Cys10-Cys15. T1 and T3 were still able to inhibit
the
growth of
L. innocua. These results are in good agreement
with those obtained with Asp-N cleavage, since both T1 and T2
contained
the active N2[18-43]
fragment.
When trypsin was added to an Asp-N hydrolysate, two new main peaks were
collected by analytical HPLC (results not shown).
The first peak, with
a molecular mass of 1,672 Da (Table
1),
corresponded to peptide
3-13-15-17, in other words, to peptide
N1 cleaved at Lys2, Lys13, and
Lys14. As expected, this peptide
was not active since N1 was not
active. The second peptide, with
a molecular mass of 2,534 Da (Table
1), corresponded to the fragment
N2 [18-43] cleaved at Lys42. This
fragment was composed of peptide
18-42 linked to Cys43 by the
Cys25-Cys43 disulfide bond. This
peptide showed no inhibitory activity
(Table
1). These results
suggested that the folding imposed by the
disulfide bond Cys25-Cys43
was essential for the activity of the N2
peptide.
Role of disulfide bonds on the antimicrobial activity of DV41.
To determine the contribution of the disulfide bridges Cys10-Cys15 and
Cys25-Cys43 (Fig. 1) to the antimicrobial activity of DV41, they were
reduced by DTT and alkylated with iodoacetamide. This alkylating agent
was chosen because it does not change the net charge of the peptide as
does iodoacetic acid or hydrophobicity as does 4-vinylpyridine. The
alkylation was complete, as confirmed by ES-MS (Table
2), and the corresponding peptide was
totally unable to inhibit growth of L. innocua. Therefore,
it can be deduced that disulfide bonds were mandatory for antilisterial
activity.
Modification of the net charge of DV41.
Acylation of DV41 was
performed to show whether its cationic nature (net charge +3) was
essential for inhibitory activity. First, succinylation yielded
negatively charged succinyl lysine together with the N-terminal and two
serine residues, as revealed by ES-MS (Table 2). Serine residues were
restored by treating the succinylated DV41 with 0.5 M hydroxylamine for
1 h. As a consequence, the threonine residues were also converted
to hydroxamic acid. In both cases, the succinylated DV41 was not active
against L. innocua. Second, DV41 was acetylated by acetic
anhydride neutralizing the lysine positive charge. In this case, only
the four lysine residues and the NH2 terminus were modified
(Table 2). The acetylated peptide conserved its inhibitory activity.
Modification of the aromatic side chains.
LC-MS revealed the
presence of different compounds after the addition of NBS (Fig.
3). W2 corresponded to DV41 with three oxidized tryptophan residues, while W3 and W4 corresponded to DV41 with
one oxidized tryptophan (Table 2). The proportions of W3 and W4 were
similar, and the slight difference of their retention times indicated
that each corresponded to DV41 modified at different tryptophan
positions. W5 corresponded to unmodified DV41. Two other compounds, W1
and W6, were observed by LC-MS with molecular masses of about 843 and
66,000 Da, respectively (Fig. 3 and Table 2). W6 might correspond to an
aggregate of covalently oligomerized DV41 (modified and native) by
disulfide bond rearrangement. These unknown compounds were not further
characterized. Even with just one oxidized tryptophan residue, no
inhibitory activity was observed. Therefore, each tryptophan residue
was essential to antimicrobial activity.
Finally the tryrosine residues were modified by nitration with TNM.
Three main peaks, Y1, Y2, and Y3, were collected by RP-HPLC,
and ES-MS
allowed us to classify them as DV41 with one, two, and
three modified
residues (Fig.
4 and Table
2). Only the
peptide
with all the three nitrated tyrosine residues did not exhibit
any inhibitory activity against
L. innocua.
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FIG. 4.
RP-HLPC of nitrated DV41 with 25 mM TNM in 96% ethanol
at 25°C for 60 min. Y1, Y2, and Y3 represent modified DV41 with 1, 2, and 3 nitrotyrosine derivatives, respectively.
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DISCUSSION |
While some studies have been done by using amino acid substitution
and or by truncating the peptide sequence (11, 24), no
similar data are available for class IIa bacteriocins containing two
disulfide bonds, such as pediocin PA-1. In this study, we used DV41, a
bacteriocin which is highly homologous to pediocin PA-1 and which has
been recently characterized (21).
The cationic nature of membrane-active peptides is generally essential
for their insertion in membranes, probably because they interact
strongly with anionic membrane phospholipids, as demonstrated with
nisin (7) and pediocin PA-1 (2). Upon succinylation, DV41 is transformed into a highly anionic peptide (net
charge of 7) and loses its activity, probably because repulsive forces impair interaction with the anionic phospholipids of the membrane. However, acetylated DV41 still displays inhibitory activity. This observation could be contradictory since acetylated DV41 is
anionic (net charge, 2) and succeeds in interacting with the membrane. In the case of membrane-active cationic peptides or proteins,
acetylation of lysine leads generally to a loss of membrane toxicity
(13, 14). However, Gallagher et al. (12) reported similar results on leucocin A, a class IIa bacteriocin with just one
disulfide bond. In DV41, as in leucocin A, most of the acetylated residues are found in the N-terminal hydrophilic part. The inhibitory activity of acetylated DV41 is in agreement with Asp-N cleavage, which
shows that the C-terminal hydrophobic domain, i.e., fragment 18-43, with a null net charge is still active. Furthermore, it is worth noting
that acetylation of lysine also increases peptide hydrophobicity. These
results provide the first evidence that the hydrophobicity of DV41 and
especially of the C-terminal peptide is more important than the
cationic character of the bacteriocin for the inhibitory activity. This
is in agreement with the oxidation of the hydrophobic tryptophan
residues which induced a loss of inhibitory activity in DV41. Similar
results have been obtained with Trp37-deleted mesentericin Y105
(11) and Phe33-Ser33 substitution in the C-terminal of
carnobacteriocin B2 (24). These results could suggest that
increased hydrophobicity of C-terminal domain would drive insertion in
target membrane to such a degree that electrostatic interaction would
no longer be rate limiting. However, for mesentericin Y105, the
C-terminal end alone does not display any antibacterial activity
(11). The hydrophobicity of the C terminus of DV41 is not
sufficient to maintain antimicrobial activities since the cleavage at
Lys42 by trypsin induced a loss of antilisterial activity of the Asp-N
18-43 fragment. This cleavage and disulfide reduction are detrimental
to the folding of the C-terminal part. Therefore, it is obvious that in
the case of DV41, both folding and hydrophobicity are needed for the
expression of inhibitory activity. These results strengthen the
previous hypothesis that the disulfide bond found in the C terminus is
mandatory to the activity of class IIa bacteriocin containing two
disulfide bonds (5).
The importance of the C-terminal hydrophobic part on the inhibitory
activity has already been addressed by Fimland et al. (10).
However, in the case of mesentericin Y105, Fleury et al. (11) have shown that the N terminus is also essential to the bacteriocin activity. It has been suggested that, in addition to its
role in the recognition by a receptor, it could also play a role in the
electrostatic interaction with anionic lipids of the target membrane
(3). In the case of DV41, these electrostatic bonds are not
necessary since acetylation still maintains an inhibitory activity.
Furthermore, the trypsin cleavages which either remove the two residues
at N terminus or break the loop stabilized by the disulfide bond
Cys10-Cys15 generates peptide fragments which are still able to inhibit
the growth of L. innocua. The latter cleavage by trypsin is
in agreement with previous observations suggesting that the disulfide
bond in the N-terminal conserved hydrophilic domain is not essential to
the antilisterial activity (5). Only the modification of
aromatic tyrosine and especially tryptophan residues are detrimental to
the activity of DV41. However, it was necessary to modify all three
tyrosine residues to obtain a complete loss of inhibitory activity. On
the contrary, replacing Tyr3 by phenylalanine in carnobacteriocin B2
(24) and truncated mesentericin Y105 (fragment 4-37)
(11) yielded peptides with a drastic decrease in activity.
Therefore, our results are in agreement with previous results
(3) providing evidence that the conserved YGNGV sequence is
not directly involved in membrane recognition. The formation of the
bulky tyrosine nitroderivatives probably impairs peptide-peptide
interaction and therefore impairs proper oligomerization of the
bacteriocin within the membrane to form a functional pore.
We have previously postulated that the tryptophan residues of the
N-terminal domain could play a major role in the interaction and
orientation of bacteriocin on the membrane surface (1). The
present study further supports the importance of these residues for the
inhibitory activity of class IIa bacteriocins. This is not surprising
since tryptophan residues are known to play an essential role in the
adsorption and orientation of amphiphilic peptides and proteins in
membranes due to their capacity to form both hydrogen and hydrophobic
bonds with the polar and nonpolar groups of polar lipids (9, 16,
20, 26). In particular, Trp16 and Trp19 at the joining part of
the N- and C-terminal domains could play a major role in the proper
orientation of these domains within the membrane (1). In the
case of the anionic acetylated DV41, both tryptophan residues and
peptide hydrophobicity could promote membrane insertion to such a
degree that electrostatic interaction would no longer be rate limiting.
Finally, this structure-function study shows that, in the case of DV41,
N-terminal conserved peptide is not necessary in order to have an
antimicrobial activity. However, when this domain is present its
structural integrity and some key residues such as tryptophan are
essential for maintaining the inhibitory activity. This domain could
facilitate and could be necessary for a proper orientation of the
peptide at the surface of membranes and for a subsequent anchoring of
the hydrophobic C-terminal within the hydrophobic core of membrane
bilayers to form a functional pore. However, the hydrophobicity of the
C-terminal domain and especially the presence of a disulfide bond which
imposes folding constraints on this domain are essential for a proper
orientation of DV41 within membranes. At the moment, it is not possible
to generalize these conclusions to the other class IIa bacteriocins
with two disulfide bonds, but these results open new perspectives for
further studies with peptide variants provided by peptide synthesis or site-directed mutagenesis.
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ACKNOWLEDGMENTS |
This work was supported by the French Ministry of Agriculture
(DGER) and by the Region Pays de la Loire.
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FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
Biochimie et Technologie des Protéines, INRA, B.P. 71627, 44316 Nantes Cedex 03, France. Phone: (33) (0) 240-67-50-56. Fax: (33) (0)
240-67-50-25. E-mail: marion{at}nantes.inra.fr.
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Applied and Environmental Microbiology, July 1999, p. 2895-2900, Vol. 65, No. 7
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
