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J Biol Chem, Vol. 274, Issue 13, 8405-8410, March 26, 1999
From Positively charged antimicrobial peptides with
membrane-damaging activity are produced by animals and humans as
components of their innate immunity against bacterial infections and
also by many bacteria to inhibit competing microorganisms.
Staphylococcus aureus and Staphylococcus
xylosus, which tolerate high concentrations of several
antimicrobial peptides, were mutagenized to identify genes responsible
for this insensitivity. Several mutants with increased sensitivity were
obtained, which exhibited an altered structure of teichoic acids, major
components of the Gram-positive cell wall. The mutant teichoic acids
lacked D-alanine, as a result of which the cells carried an
increased negative surface charge. The mutant cells bound fewer
anionic, but more positively charged proteins. They were sensitive to
human defensin HNP1-3, animal-derived protegrins, tachyplesins, and
magainin II, and to the bacteria-derived peptides gallidermin and
nisin. The mutated genes shared sequence similarity with the
dlt genes involved in the transfer of D-alanine into teichoic acids from other Gram-positive bacteria. Wild-type strains bearing additional copies of the dlt operon
produced teichoic acids with higher amounts of D-alanine
esters, bound cationic proteins less effectively and were less
sensitive to antimicrobial peptides. We propose a role of the
D-alanine-esterified teichoic acids which occur in many
pathogenic bacteria in the protection against human and animal defense systems.
Antimicrobial peptides play an important role in the defense of
insects, vertebrates, and humans against pathogenic microorganisms (1)
and have accordingly been designated "host defense peptides." Substances such as defensins from the granules of phagocytes, epithelial surfaces, and skin (2), protegrins from porcine leukocytes
(3), tachyplesins from the hemocytes of the horseshoe crab (4), and
magainins from amphibian skin (5) share an amphiphilic cationic
structure and a membrane-damaging activity by forming pores or
disintegrating the cytoplasmic membrane bilayer. Peptides with similar
structure and activity are also produced by many Gram-positive bacteria
and include the nonribosomally synthesized gramicidins and polymyxin B
(6), ribosomally synthesized peptides, such as lactococcin A or
pediocin PA-1 (7), and lantibiotics, which contain the
posttranslationally formed thioether amino acid lanthionine (8).
Because of their unique structural features and biotechnological
importance, lantibiotics such as nisin, subtilin, Pep 5, epidermin, and
gallidermin have been extensively investigated (8, 9).
Staphylococcus aureus, a major human pathogen, tolerates
high concentrations of several host defense peptides and lantibiotics. Strains resistant to defensin-like platelet microbicidal proteins have
been shown to be more virulent than sensitive ones (10). The mechanisms
responsible for the resistance phenotype are unknown. The broad range
of antimicrobial peptides tolerated distinguishes them from the highly
specific self-protection systems of lantibiotic-producing staphylococcal strains (11, 12).
Gram-positive bacteria are not protected by an outer membrane. The
S. aureus cell wall is instead formed by a thick
peptidoglycan fabric and by polymers of alternating phosphate and
alditol groups called teichoic acids. These polymer chains are either
covalently connected to the peptidoglycan (wall teichoic acids,
WTA)1 (13) or to membrane
glycolipids (lipoteichoic acids, LTA) (14). Teichoic acids of the
various Gram-positive species are highly variable in the use of alditol
groups (glycerol or ribitol) and in modifications of the alditol with
glycosyl residues or D-alanine. The highly charged teichoic
acids are essential for viability and seem to be involved in the
control of cell shape, autolytic enzymes, and magnesium ion
concentration within the cell envelope (13, 15).
In an attempt to understand the mechanisms by which staphylococci
resist antimicrobial peptides, we isolated gallidermin-sensitive Staphylococcus xylosus and Staphylococcus aureus
mutants, analysis of which revealed that the absence of
D-alanine esters from teichoic acids leads to increased
sensitivity toward cationic antimicrobial peptides.
Growth Conditions, Transposon Mutagenesis, and DNA Sequence
Analysis--
S. aureus Sa113 (16), S. xylosus
C2a (17), and Escherichia coli DH5
DNA was sequenced by cycle sequencing (18) on a DNA sequencer 4000 L
(LI-COR Inc., Lincoln, NE) using the Thermo Sequenase fluorescent-labeled prime cycle sequencing kit (Amersham Pharmacia Biotech, Uppsala, Sweden). Tn917-specific primers were used
to directly sequence genomic DNA upstream and downstream from the transposon insertion sites. Remaining sequence gaps were filled in by
primer walking. To perform amino acid sequence similarity searches and
to determine the degree of identity of two given sequences the program
BLAST 2.0 with the nonredundant protein data base and program BLAST 2 Sequences of the National Center for Biotechnology Information
(Bethesda, MD) were used, respectively. Multiple sequence alignments
were accomplished using the Higgins-Sharp algorithm of the program
MacDNASIS Pro (Hitachi Software Engineering, San Bruno, CA).
Construction of Plasmids and Homologous Recombination--
In
order to replace the dltA gene of S. aureus Sa113
by a spectinomycin resistance gene, DNA fragments of 1.5-kilobase pair flanking dltA were amplified by PCR, cloned into the
SmaI site of pUC18 according to standard methods (18), and
then sequenced. The upstream and downstream fragments were isolated
from the resulting plasmids by cleavage with
SphI/SacI and BamHI/EcoRI,
respectively, and inserted into the polylinker of the
temperature-sensitive shuttle plasmid pBT2 (17) together with a 1277-bp
SacI-BamHI fragment encoding the spectinomycin
adenyltransferase gene (spc) from Tn554 (20), as
shown in Fig. 1. The resulting plasmid pBT
Plasmid pRBdlt1 was constructed by ligation of a 4655-bp PCR fragment
bearing the dltABCD operon of S. xylosus C2a,
together with 393 bp upstream of the dltA start codon with
the putative promoter region and 185 bp downstream of the
dltD stop codon with the terminator structure into the
SmaI site of pUC18. After sequence analysis the fragment was
isolated by BamHI-EcoRI cleavage from the
resulting plasmid, cloned into the multiple cloning site of the shuttle
vector pRB473 (22), and transformed into S. xylosus C2a and
S. aureus Sa113 by electroporation (21). Direct
transformation of S. aureus and S. xylosus dlt
mutants was unsuccessful, probably as a result of the altered surface
charge. pRBdlt1 was therefore introduced into mutant protoplasts by
fusion with wild-type protoplasts bearing the plasmid and subsequent
selection for spectinomycin (S. aureus) or erythromycin
(S. xylosus) resistance and the plasmid-encoded chloramphenicol resistance. Protoplast fusion was carried out as
described previously (23). The resulting clones were verified by
restriction fragment analysis of the plasmid and sequencing of the DNA
flanking the chromosomal resistance determinant.
Isolation of WTA and LTA--
Bacteria were grown overnight in
500 ml of BM broth containing 0.25% (S. xylosus) or 0.3%
glucose (S. aureus), harvested by centrifugation, and washed
in 100 ml of sodium acetate buffer (20 mM, pH 4.6). Cells
were disrupted in the same buffer using glass beads and a Disintegrator
S (Biomatik GmbH, Rodgau, Germany) as described previously (24).
In order to isolate WTA, 500-µl aliquots of the crude cell extracts
were diluted 4-fold in sodium acetate buffer containing 2% SDS,
sonicated for 15 min, and then vigorously shaken for 1 h at
60 °C. The cell walls were sedimented by centrifugation, subjected
to repeated washings with sodium acetate buffer to remove the
SDS, and finally resuspended in 1 ml of sodium acetate buffer. WTA was
extracted by diluting 250 µl of purified cell walls 4-fold in 5%
trichloroacetic acid and incubating at 60 °C for 4 h. The peptidoglycan was removed by centrifugation.
LTA was isolated from 250 µl of the crude cell extract after 2-fold
dilution with sodium acetate buffer by extraction with 500 µl of
aqueous phenol (vigorous shaking for 1 h at 60 °C) and subsequent hydrophobic interaction chromatography. The chromatographic purification was carried out essentially as described by Koch et
al. (25). Briefly, the aqueous layer was further diluted by
addition of 250 µl of sodium acetate buffer, mixed with 500 µl of
octyl-Sepharose 4 Fast Flow (Amersham Pharmacia Biotech), which had
been equilibrated with sodium acetate buffer, and incubated for 15 min
at room temperature. The suspension was applied to Ultrafree-MC
centrifugal filter units with 0.45 µm pore size (Millipore, Bedford,
MA), and the solvent was removed by centrifugation for 1 min at
340 × g. After three washing steps, each with 500 µl of 15% 1-propanol in sodium acetate buffer, the lipoteichoic acids were eluted by resuspending the gel in 500 µl of 50% 1-propanol in
sodium acetate buffer and incubating for 15 min at room temperature. After collecting the eluate by centrifugation, the elution procedure was repeated, and eluates were combined.
Analysis of D-Alanine and
Phosphorus--
D-Alanine was analyzed according to an
established method (26). WTA and LTA samples were adjusted to pH 9-10
with NaOH to a final volume of 100 µl and were incubated for 1 h
at 37 °C to hydrolyze the D-alanine esters. Tris-HCl
(200 µl, 0.2 M, pH 8.4) containing 2.5 mg of
D-amino acid oxidase/ml (1.3 units/mg; Sigma) was added,
and samples were incubated for 1 h at 37 °C. The reaction was
stopped with 100 µl of 30% trichloroacetic acid, and the
precipitated protein was removed by centrifugation.
2,4-Dinitrophenylhydrazine (100 µl of a 0.1% solution prepared in 2 M HCl) was added to the supernatant and incubated for 5 min. After addition of 200 µl of 2.5 M NaOH, the
A525 was determined. The amount of phosphorus in
WTA and LTA samples was determined according to Chen et al. (27).
Isolation of Defensin HNP1-3 from Human Neutrophils--
Human
neutrophil peptides HNP1-3 were isolated from human peripheral blood
neutrophils as described previously (28). To improve the yield, blood
from a patient who had been stimulated with granulocyte
colony-stimulating factor was used (kindly provided by Dr.
Handgretinger, Children's Hospital, University of Tübingen). The
defensin peptides were highly enriched from the granules of the
granulocytes by intensive extraction with 5% acetic acid and further
purified by RP-HPLC yielding 5.1 mg of defensin peptides from 500 ml of
blood. The lyophilized samples were stored at 4 °C and dissolved in
0.01% acetic acid at a concentration of 1 mg/ml. The product was
composed of the three defensin variants HNP1, HNP2, and HNP3, which
differ only in the first amino acid. The purity and quality was
confirmed by ESI-MS yielding the expected masses.
Synthesis, Folding, Purification, and Characterization of
Protegrins and Tachyplesins--
Protegrins 3 and 5 (29) and
tachyplesins 1 and 3 (4) were synthesized by standard methods for
multiple parallel synthesis of peptides (30) using a Fmoc
(9-fluorenylmethylcarbonyl protecting group) strategy. All peptides
were synthesized as peptide-amides. After lyophilization, the crude,
linear peptides were obtained in purities ranging from 75-82% as
determined by RP-HPLC and ESI-MS. Folding of the raw peptides was
achieved by the method of Aumelas et al. (29) after Tam
et al. (31). Briefly, crude linear peptides were dissolved
in 30% aqueous isopropyl alcohol (0.8 mg/ml) and then added slowly and
with stirring to folding buffer (25% dimethyl sulfoxide, 10%
isopropyl alcohol, 0.1 M Tris-HCl, final pH 6.8) at a ratio
of 1.5 ml of peptide solution to 4 ml of folding buffer (final peptide
concentration about 0.22 mg/ml). The reaction was allowed to proceed in
capped glass vials with gentle stirring for 24 h and was followed
by the periodic removal of 150 µl samples which were acidified by
addition of 10 µl of trifluoroacetic acid and then characterized by
RP-HPLC. No precipitation of the peptides was observed in any of the
samples, and after 24 h disulfide bond formation appeared to have
ceased. The purity of the crude, folded product was estimated to range
from 56-69%, as judged by RP-HPLC. Crude, folded preparations were
stored frozen at
Peptides were purified by semipreparative RP-HPLC using appropriate
acetonitrile gradients in 0.1% trifluoroacetic acid. After lyophilization, the folded peptides were characterized by RP-HPLC and
ESI-MS; in each case purity was judged to exceed 95%, while masses
showed a loss of 4 Da, suggesting oxidation of the peptides to form the
respective disulfide bridges. Disulfide bond formation was also
confirmed by a previously described method (32). Briefly, samples (5 µg) of each of the four peptides was alkylated with 4-vinylpyridine
both in the presence and absence of a reducing agent
(2-mercaptoethanol), purified by RP-HPLC, and then analyzed by ESI-MS.
In no case were free thiol groups detected, suggesting that all of the
cysteinyl thiol functions were oxidized and were involved in disulfide bonds.
Analysis of Minimal Inhibitory Concentrations--
Gallidermin
was kindly provided by Dr. Karl Thomae GmbH, Biberach, Germany. Nisin,
synthetic ([A8,13,18]magainin II amide, gramicidin D, and
polylysine (average molecular mass of 3.97 kDa) were purchased from
Sigma. In a serial dilution test, LB broth (1% tryptone, 0.5% yeast
extract, 0.5% NaCl) containing increasing concentrations of the
antimicrobial peptides were inoculated with 1/100 volume of
precultures. The cultures were incubated until the stationary phase was
reached, and the turbidity of the cultures was monitored at 590 nm.
Since the activity of human defensins is very sensitive to the salt
concentration (28), LB broth without NaCl and with only half of the
amount of tryptone and yeast extract was used. The tested strains all
grew well in this medium.
Analysis of Green Fluorescent Protein, Cytochrome c, and
Gallidermin Binding--
Bacteria were grown to stationary phase in BM
broth and harvested by centrifugation. In order to analyze the
interaction with anionic and cationic proteins, cells were washed twice
with sodium phosphate buffer (100 mM, pH 7) for incubation
with green fluorescent protein (CLONTECH) and
gallidermin. For incubation with cytochrome c (Merck,
Darmstadt, Germany), MOPS buffer (20 mM, pH 7) was used. The cells were resuspended in the same buffers to a final
A578 of 7 (green fluorescent protein and
cytochrome c) or A578 of 4 (gallidermin), incubated for 10 min with 0.5 µg of protein/ml (green
fluorescent protein and gallidermin) or 0.5 mg/ml (cytochrome c), and subsequently removed by centrifugation. The amount
of green fluorescent protein in the supernatant was analyzed
fluorometrically (excitation at 395 nm, emission monitored at 509 nm).
Cytochrome c was quantitated photometrically at 530 nm, the
absorption maximum of the prosthetic group. The amount of gallidermin
in the supernatant was determined by RP-HPLC analysis using a linear
gradient from 30 to 60% acetonitrile in 0.1% trifluoroacetic acid
over 20 column volumes on a Spherisorb ODS2 column (Grom Analytik,
Herrenberg, Germany).
Identification and Sequence Analysis of the S. aureus and S. xylosus dlt Operons--
S. aureus Sa113 and the
coagulase-negative S. xylosus C2a reveal high innate
tolerances toward several antimicrobial peptides (see below). To
investigate the resistance mechanism, S. xylosus C2a was
mutagenized with Tn917 as described under "Experimental Procedures," and the resulting transposon insertion mutants were analyzed for reduced growth on agar plates containing the antimicrobial peptide gallidermin. The nucleotide sequence upstream and downstream of
the transposon from seven clones whose growth was specifically reduced
in the presence of gallidermin was determined. In all seven mutants,
the transposon had integrated into the same determinant of 4600 bp,
which encodes four open reading frames, arranged in an operon-like
structure, and followed by a typical terminator (Fig.
1). The open reading frames showed
sequence similarity with the Lactobacillus casei and
Bacillus subtilis dltABCD operons, which are responsible for
esterification of teichoic acids with D-alanine (33-35).
The transposon had integrated into the S. xylosus dltA
(mutant XG13), dltB (XG4, XG12, XG9, XG14), or
dltD genes (XG24) or into the putative promoter region
(XG16) (Fig. 1). Growth rates in the absence of gallidermin and
microscopic appearance of the mutants were indistinguishable from those
of the wild-type strain.
The S. xylosus dlt operon showed similarity to a DNA
sequence from S. aureus KAN96, which has recently been
deposited to the GenBankTM.2 We
sequenced and completed the respective DNA sequence from S. aureus Sa113 and found an identical organization to that in
S. xylosus (Fig. 1). In both organisms, the dltA
and dltB genes and the dltC and dltD
genes overlap by 4 bp each, while the dltB and dltC genes are separated by 17 bp. The dltA,
dltB, dltC, and dltD genes encode
proteins of 485, 404, 78, and 391 amino acids (S. aureus)
and 487, 404, 78, and 382 amino acids (S. xylosus),
respectively. The protein sequences of the two organisms are 61-89%
identical and 30-62% identical to the corresponding proteins of
B. subtilis and L. casei. A further,
less-pronounced sequence identity in the C-terminal 270 amino acids of
the putative membrane proteins DltB was found to AlgI of
Pseudomonas aeruginosa (28%), which is involved in
substitution of the exopolysaccharide alginate with acetyl groups (36),
and to a related gene product from Helicobacter pylori
(24%). Three domains in the C-terminal portions of the proteins are
particularly well conserved as shown in Fig. 2A. All four DltD proteins
bear hydrophobic stretches at the N termini resembling a signal
peptide. Putative signal peptidase I cleavage sites are, however, only
weakly conserved (Fig. 2B).
The dlt operons are preceded in S. aureus and
S. xylosus by orf1, which shows up to 44%
identity to hydroxyacid dehydrogenases from various organisms.
orf1 and dltA are separated by noncoding regions
of 573 bp (S. aureus) and 865 bp (S. xylosus), respectively.
Disruption of the S. aureus dlt Operon and Analysis of the
D-Alanine Content in LTA and WTA--
The dltA
gene of S. aureus Sa113 was replaced by a spectinomycin
resistance gene (spc) by homologous recombination producing the gallidermin-sensitive strain AG1. WTA and LTA of S. aureus and S. xylosus wild-type strains and mutants
were isolated, and the molar ratios of D-alanine to
phosphorus were determined as shown in Table
I. In the wild-type strains, 75%
(S. aureus) and 95% (S. xylosus) of the alditol
phosphate residues in LTA were esterified with D-alanine,
while only 51% (S. aureus) and 15% (S. xylosus)
were esterified in WTA. In the S. aureus dlt mutant AG1 and
in the S. xylosus mutants XG13, XG4, and XG24, bearing the
transposon in the dltA, dltB, and dltD
genes, respectively, no D-alanine was detected in LTA or
WTA, indicating that the pathway for D-alanine
incorporation was inactivated by the spectinomycin resistance gene and
transposon insertions.
When the mutant strains S. aureus AG1 and S. xylosus XG13 were complemented with plasmid pRBdlt1 bearing the
dlt operon, normal or slightly increased amounts of
D-alanine were found in LTA and WTA. Transformation of the
wild-type strains with pRBdlt1 resulted in an increase of
D-alanine in LTA and WTA by 5-18% (Table I).
Sensitivity toward Antimicrobial Peptides--
The minimal
inhibitory concentrations of gallidermin and of several other
membrane-damaging antimicrobial peptides, which were isolated or
synthesized as described under "Experimental Procedures" were
determined for the S. aureus and S. xylosus
wild-type and mutant strains. The mutants were sensitive to a variety
of antimicrobial peptides that bear a positive net charge (Table II). The sensitivity of the S. aureus mutant AG1 to defensin HNP1-3 from human neutrophils and
to protegrins 3 and 5 from porcine leukocytes was at least 10-23-fold
higher. Factors of 7-12 were determined with tachyplesin 1 and 3 from
hemocytes of the horseshoe crab and to a variant of magainin II from
clawed frog skin. The tolerance toward the lanthionine-containing
peptides gallidermin from Staphylococcus gallinarum and
nisin from Lactococcus lactis was 8-50-fold decreased; very
similar results were obtained with the S. xylosus strains
(Table II).
The increased sensitivity of dlt mutants seemed to be
restricted to cationic peptides, since no considerable differences were observed in the inhibitory concentrations of the neutral peptide gramicidin D from Bacillus brevis. Furthermore, the mutants
were not sensitive to cationic polylysine, indicating that cationic properties are not sufficient for activity of a peptide against S. aureus and S. xylosus strains lacking
D-alanine esters in their teichoic acids.
The mutant strains AG1 and XG13 revealed normal sensitivities to
gallidermin and nisin after complementation with the plasmid pRBdlt1.
Wild-type strains bearing pRBdlt1 revealed increased tolerances against
most tested cationic peptides, suggesting a direct correlation between
the tolerance to cationic peptides and the D-alanine
content of the teichoic acids.
Binding Studies with Anionic Green Fluorescent Protein and the
Cationic Proteins Cytochrome c and
Gallidermin--
D-Alanine esters modulate the teichoic
acid net charge by introducing positively charged amino groups to the
negatively charged backbone (37). To determine whether the lack of
D-alanine caused an alteration in the overall charge of the
cell envelope, the capacity of the wild-type and dlt mutant
cells to bind negatively or positively charged proteins was compared.
The cells were incubated at pH 7 with either negatively charged green
fluorescent protein (calculated pI 5.8), positively charged cytochrome
c (calculated pI 10.0), or positively charged gallidermin.
The bacteria were subsequently removed by centrifugation and the
amounts of these substances remaining in the supernatants were
determined (Fig. 3). The mutants bound
lower amounts of anionic green fluorescent protein and higher amounts
of cationic cytochrome c and gallidermin than the wild-type
strains, while wild-type strains bearing pRBdlt1 revealed the opposite
behavior (with the exception of green fluorescent protein binding by
S. aureus Sa113 bearing pRBdlt1). These observations are in
agreement with the proposed higher negative charge of the cell surface
of the mutants and the lower negative charge of wild-type strains
containing additional copies of the dlt operon.
The dltABCD genes of S. aureus, S. xylosus, L. casei, and B. subtilis are
similar in sequence and organization. Studies in L. casei
have demonstrated a role of DltA as a
D-alanine-D-alanyl carrier protein ligase
(Dcl), which activates D-alanine by hydrolysis of ATP and
transfers it to the phosphopantetheine cofactor of a specific
D-alanine carrier protein (Dcp), which is encoded by dltC (33, 34) (Fig. 4). The
hydrophobic DltB is indispensable for D-alanine
incorporation into teichoic acids and may be involved in the transfer
of activated D-alanine across the cytoplasmic membrane (35)
(Fig. 4). The essential role of DltD and the presence of a putative
N-terminal signal peptide suggest an involvement in the transfer of
D-alanine from the membrane carrier to teichoic acids (Fig.
4).
The increased sensitivity of S. aureus and S. xylosus
dlt mutants toward a variety of membrane-active antimicrobial
peptides provides evidence for a role of the D-alanine
substituents in the protection of the bacteria against these
substances. Inactivation of dlt genes caused considerably
lower minimal inhibitory concentrations of (i) host defense peptides
with Cationic properties are necessary for the initial interaction of
membrane-damaging peptides with the negatively charged membrane surface
(38). Reduction of the negative cell envelope charge by incorporation
of D-alanine may thus be regarded as a multiple drug
protection mechanism. A similar observation has been made with
Gram-negative bacteria whose resistance to the cationic peptides polymyxin B and protamine was caused by a reduction of the anionic nature of the lipopolysaccharide (39). Bacteria have to cope with
antimicrobial peptides in many environments. The production of
bacteriocin-like molecules such as gallidermin or nisin is a prevalent
strategy among microorganisms to inhibit the growth of competing
strains (7, 8) and, in higher organisms, to support the innate immunity
against bacterial infections (1). For bacteria such as staphylococci,
which live in intimate contact with humans and animals, mechanisms
protecting against host defense peptides are of particular benefit.
Accordingly, a correlation between the capacity of S. aureus, coagulase-negative staphylococci, and streptococci to
cause endocarditis, and the in vitro resistance to
defensin-like platelet microbicidal proteins has been demonstrated (10). Understanding the mechanisms by which bacteria are able to
circumvent the human defense systems has a great impact on the
treatment of infections and the search for new antimicrobial agents. To
our knowledge, the study presented here describes the first mechanism
conferring resistance to host defense peptides in a Gram-positive
organism. D-Alanine-esterified teichoic acids occur in many
Gram-positive human pathogens including staphylococci, streptococci,
enterococci, and listeria (14), and our study raises the question
whether the D-alanine esters or further resistance systems
against host defense peptides contribute to the virulence and
persistence of these bacteria.
We thank Vera Augsburger for technical
assistance, Reinhold Brückner for providing transposon mutants,
and Rupert Handgretinger for providing human peripheral blood neutrophils.
*
This work was supported by grants from the Deutsche
Forschungsgemeinschaft (GO 371/3-1 and SFB 323).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF032440 (S. xylosus C2a dlt operon) and AF101234 (S. aureus Sa113 dlt operon).
§
To whom correspondence should be addressed: Tel.: 49-7071-297-5938;
Fax: 49-7071-29-5937; E-mail: mikrogen{at}uni-tuebingen.de.
2
The partial nucleotide sequence of the S. aureus KAN96 dlt operon has been deposited in the
GenBankTM data base under GenBankTM accession
number D86240.
The abbreviations used are:
WTA, wall teichoic acid(s);
LTA, lipoteichoic acid(s);
PCR, polymerase chain reaction;
RP-HPLC, reversed-phase high-performance liquid chromatography;
ESI-MS, electrospray ionization mass spectrometry;
MOPS, 4-morpholinepropanesulfonic acid;
bp, base pair(s).
Inactivation of the dlt Operon in
Staphylococcus aureus Confers Sensitivity to Defensins,
Protegrins, and Other Antimicrobial Peptides*
§,,
,
Mikrobielle Genetik, Universität
Tübingen, Waldhäuser Straße 70/8, 72076 Tübingen,
Germany, ¶ Organische Chemie, Universität
Tübingen, 72076 Tübingen, Germany, ECHAZ
Microcollections®, Sindelfingerstraße 3, 72070 Tübingen,
Germany, and Physiologische Chemie, Universität
Tübingen, Ob dem Himmelreich 7, 72074 Tübingen,
Germany
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(18) were grown in BM
broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl, 0.1%
K2HPO4, 0.1% glucose) unless otherwise noted.
Plasmid pTV1ts, used for transposon mutagenesis, is composed of a
temperature-sensitive replicon, a chloramphenicol resistance gene, and
the transposon Tn917, which mediates erythromycin resistance (19). To generate a mutant library, S. xylosus (pTV1ts) was grown overnight at 30 °C in BM broth containing 5 µg of
erythromycin/ml and 20 µg of chloramphenicol/ml. The culture was
subsequently diluted 100-fold in BM broth containing 2.5 µg of
erythromycin/ml and incubated for 14 h at 42 °C to select for
transposon insertion mutants. This culture was diluted again in the
same way and grown for another 14 h at 42 °C. Appropriate
amounts of the bacterial suspension were spread on BM agar plates
containing 2.5 µg of erythromycin/ml and incubated at 37 °C.
Mutant clones (4000) were transferred to BM agar plates containing 3 µg of gallidermin/ml and monitored for impaired growth on gallidermin.
dlt1 was transformed into
S. aureus Sa113 by electroporation (21). By incubation at
42 °C and subsequent screening for spectinomycin-resistant clones
without the plasmid-encoded chloramphenicol resistance, mutant AG1,
which carries the spc gene instead of dltA in the chromosome, was identified. The recombination procedure has been described recently in detail (17). The proper integration of spc was verified by direct sequencing of the genomic DNA at
the borders of the PCR-derived regions. A 1448-bp fragment comprising the dltA gene and additional 10 bp at the 5' end was
deleted; 18 bp of the dltA 3' end bearing the ribosomal
binding site of dltB were retained.
20 °C prior to purification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Organization of dlt genes in
S. xylosus and S. aureus and
disruption by transposon or resistance gene insertions. Sites of
Tn917 integration in S. xylosus mutants are
indicated by triangles. The dlt operon of
S. aureus was disrupted by replacing the dltA
gene with the spectinomycin resistance gene spc as shown in
the lower part of the figure. The spc gene and
PCR fragments flanking dltA were cloned using the
restriction sites indicated to produce the integration vector
pBT dlt1. The symbol T indicates a transcriptional
terminator structure.
View larger version (39K):
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Fig. 2.
Sequence alignments of DltB and DltD.
A, three well conserved regions of the DltB proteins of
S. aureus (Sa), S. xylosus
(Sx), B. subtilis (Bs), and L. casei (Lc) are compared with the homologous AlgI
proteins of P. aeruginosa (Pa) and H. pylori (Hp). B, alignment of the N-terminal
portions of DltD proteins from S. aureus (Sa),
S. xylosus (Sx), B. subtilis
(Bs), and L. casei (Lc). Conserved
amino acids are indicated below the sequence. Similar amino acids are
indicated by +. Putative signal peptidase cleavage sites, as calculated
according to von Heijne (40), are indicated by underlining
the three preceding amino acids (the B. subtilis DltD
contains two possible cleavage sites).
D-Alanine content of S. aureus and S. xylosus teichoic
acids
Activity of antimicrobial peptides against wild-type strains S. aureus
Sa113/S. xylosus C2a and dlt mutants S. aureus AG1/S. xylosus XG13
View larger version (39K):
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Fig. 3.
Interaction of S. aureus and
S. xylosus strains with negatively and positively
charged proteins. S. aureus Sa113 and S. xylosus C2a wild-type strains (black columns,
a), dlt mutants S. aureus AG1 and
S. xylosus XG13 (white columns, b),
and wild-type strains bearing additional copies of the dlt
operon on the plasmid pRBdlt1 (gray columns, c)
were incubated at neutral pH with anionic green fluorescent protein,
cationic cytochrome c, or cationic gallidermin. The samples
were then centrifuged, and the amounts of protein in the supernatants
were determined (the capacities of wild-type strains bearing pRBdlt1 to
bind gallidermin were not determined).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (77K):
[in a new window]
Fig. 4.
Schematic representation of the putative
pathway of D-alanine transfer into teichoic acids. A
teichoic acid molecule is depicted as a chain of alternating alditol
(Ato) and phosphate (P) residues.
D-Alanine (D-Ala) is activated in the cytoplasm
by DltA (A) via ATP hydrolysis and the release of
pyrophosphate and is coupled to the phosphopantetheine prosthetic group
of the D-alanine carrier protein DltC (C) (33,
34). The hydrophobic protein DltB (B) is likely to be
involved in the transfer of D-alanine across the
cytoplasmic membrane, and DltD (D), which bears a putative
N-terminal signal peptide, is assumed to catalyze the esterification of
teichoic acid alditol groups with D-alanine resulting in
the introduction of positive charges into the otherwise negatively
charged teichoic acids.
-sheet structure and disulfide bridges, such as human defensin
HNP1-3, protegrins from porcine leukocytes, and tachyplesins from
horseshoe crab hemocytes; (ii) the linear peptide magainin II from
amphibian skin; and (iii) the bacteria-derived lantibiotics gallidermin
and nisin, which contain thioether bridges. Since the common structural
feature of these molecules is a positive net charge, and since the
sensitivity toward neutral gramicidin D was the same in the wild-type
and the dlt mutants, we propose that the basis for increased
sensitivity is an altered electrostatic interaction of the peptides
with the mutant cells. The teichoic acid backbone is highly charged by deprotonized phosphate groups, and esterification with
D-alanine causes a reduction of the net negative charge by
introduction of basic amino groups (Fig. 4). Accordingly, the S. aureus and S. xylosus dlt mutants, whose teichoic acids
are devoid of D-alanine, bound lower amounts of negatively
charged green fluorescent protein but higher amounts of positively
charged cytochrome c and gallidermin, while strains with
increased D-alanine content showed the opposite behavior.
Increased accumulation of antimicrobial peptides in the vicinity of the
cytoplasmic membrane is therefore very likely to be the basis for the
higher sensitivity of the dlt mutants.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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A. S. Bayer, P. McNamara, M. R. Yeaman, N. Lucindo, T. Jones, A. L. Cheung, H.-G. Sahl, and R. A. Proctor Transposon Disruption of the Complex I NADH Oxidoreductase Gene (snoD) in Staphylococcus aureus Is Associated with Reduced Susceptibility to the Microbicidal Activity of Thrombin-Induced Platelet Microbicidal Protein 1 J. Bacteriol., January 1, 2006; 188(1): 211 - 222. [Abstract] [Full Text] [PDF]
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R. A. Dorschner, B. Lopez-Garcia, A. Peschel, D. Kraus, K. Morikawa, V. Nizet, and R. L. Gallo The mammalian ionic environment dictates microbial susceptibility to antimicrobial defense peptides FASEB J, January 1, 2006; 20(1): 35 - 42. [Abstract] [Full Text] [PDF]
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C. Weidenmaier, A. Peschel, V. A. J. Kempf, N. Lucindo, M. R. Yeaman, and A. S. Bayer DltABCD- and MprF-Mediated Cell Envelope Modifications of Staphylococcus aureus Confer Resistance to Platelet Microbicidal Proteins and Contribute to Virulence in a Rabbit Endocarditis Model Infect. Immun., December 1, 2005; 73(12): 8033 - 8038. [Abstract] [Full Text] [PDF]
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C. M. A. Linde, S. Grundstrom, E. Nordling, E. Refai, P. J. Brennan, and M. Andersson Conserved Structure and Function in the Granulysin and NK-Lysin Peptide Family Infect. Immun., October 1, 2005; 73(10): 6332 - 6339. [Abstract] [Full Text] [PDF]
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V. Dartois, J. Sanchez-Quesada, E. Cabezas, E. Chi, C. Dubbelde, C. Dunn, J. Granja, C. Gritzen, D. Weinberger, M. R. Ghadiri, et al. Systemic Antibacterial Activity of Novel Synthetic Cyclic Peptides Antimicrob. Agents Chemother., August 1, 2005; 49(8): 3302 - 3310. [Abstract] [Full Text] [PDF]
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K. Ouhara, H. Komatsuzawa, S. Yamada, H. Shiba, T. Fujiwara, M. Ohara, K. Sayama, K. Hashimoto, H. Kurihara, and M. Sugai Susceptibilities of periodontopathogenic and cariogenic bacteria to antibacterial peptides, {beta}-defensins and LL37, produced by human epithelial cells J. Antimicrob. Chemother., June 1, 2005; 55(6): 888 - 896. [Abstract] [Full Text] [PDF]
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A. Resch, R. Rosenstein, C. Nerz, and F. Gotz Differential Gene Expression Profiling of Staphylococcus aureus Cultivated under Biofilm and Planktonic Conditions Appl. Envir. Microbiol., May 1, 2005; 71(5): 2663 - 2676. [Abstract] [Full Text] [PDF]
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M. Pietiainen, M. Gardemeister, M. Mecklin, S. Leskela, M. Sarvas, and V. P. Kontinen Cationic antimicrobial peptides elicit a complex stress response in Bacillus subtilis that involves ECF-type sigma factors and two-component signal transduction systems Microbiology, May 1, 2005; 151(5): 1577 - 1592. [Abstract] [Full Text] [PDF]
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H. Zhang, K. Morikawa, T. Ohta, and Y. Kato In vitro resistance to the CS{alpha}{beta}-type antimicrobial peptide ASABF- is conferred by overexpression of sigma factor sigB in Staphylococcus aureus J. Antimicrob. Chemother., May 1, 2005; 55(5): 686 - 691. [Abstract] [Full Text] [PDF]
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J. Xue, I. Hunter, T. Steinmetz, A. Peters, B. Ray, and K. W. Miller Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in Listeria innocua, Identified by Screening for Pediocin AcH Resistance Appl. Envir. Microbiol., March 1, 2005; 71(3): 1283 - 1290. [Abstract] [Full Text] [PDF]
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R. Leonardi, S. Chohnan, Y.-M. Zhang, K. G. Virga, R. E. Lee, C. O. Rock, and S. Jackowski A Pantothenate Kinase from Staphylococcus aureus Refractory to Feedback Regulation by Coenzyme A J. Biol. Chem., February 4, 2005; 280(5): 3314 - 3322. [Abstract] [Full Text] [PDF]
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K. M. Mason, R. S. Munson Jr., and L. O. Bakaletz A Mutation in the sap Operon Attenuates Survival of Nontypeable Haemophilus influenzae in a Chinchilla Model of Otitis Media Infect. Immun., January 1, 2005; 73(1): 599 - 608. [Abstract] [Full Text] [PDF]
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M. Sieprawska-Lupa, P. Mydel, K. Krawczyk, K. Wojcik, M. Puklo, B. Lupa, P. Suder, J. Silberring, M. Reed, J. Pohl, et al. Degradation of Human Antimicrobial Peptide LL-37 by Staphylococcus aureus-Derived Proteinases Antimicrob. Agents Chemother., December 1, 2004; 48(12): 4673 - 4679. [Abstract] [Full Text] [PDF]
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M. A. Campos, M. A. Vargas, V. Regueiro, C. M. Llompart, S. Alberti, and J. A. Bengoechea Capsule Polysaccharide Mediates Bacterial Resistance to Antimicrobial Peptides Infect. Immun., December 1, 2004; 72(12): 7107 - 7114. [Abstract] [Full Text] [PDF]
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V. Vadyvaloo, S. Arous, A. Gravesen, Y. Hechard, R. Chauhan-Haubrock, J. W. Hastings, and M. Rautenbach Cell-surface alterations in class IIa bacteriocin-resistant Listeria monocytogenes strains Microbiology, September 1, 2004; 150(9): 3025 - 3033. [Abstract] [Full Text] [PDF]
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P. Joseph, A. Guiseppi, A. Sorokin, and F. Denizot Characterization of the Bacillus subtilis YxdJ response regulator as the inducer of expression for the cognate ABC transporter YxdLM Microbiology, August 1, 2004; 150(8): 2609 - 2617. [Abstract] [Full Text] [PDF]
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M. J. Franklin, S. A. Douthit, and M. A. McClure Evidence that the algI/algJ Gene Cassette, Required for O Acetylation of Pseudomonas aeruginosa Alginate, Evolved by Lateral Gene Transfer J. Bacteriol., July 15, 2004; 186(14): 4759 - 4773. [Abstract] [Full Text] [PDF]
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J. M. Voyich, K. R. Braughton, D. E. Sturdevant, C. Vuong, S. D. Kobayashi, S. F. Porcella, M. Otto, J. M. Musser, and F. R. DeLeo Engagement of the Pathogen Survival Response Used by Group A Streptococcus to Avert Destruction by Innate Host Defense J. Immunol., July 15, 2004; 173(2): 1194 - 1201. [Abstract] [Full Text] [PDF]
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S. Nouaille, J. Commissaire, J. J. Gratadoux, P. Ravn, A. Bolotin, A. Gruss, Y. Le Loir, and P. Langella Influence of Lipoteichoic Acid D-Alanylation on Protein Secretion in Lactococcus lactis as Revealed by Random Mutagenesis Appl. Envir. Microbiol., March 1, 2004; 70(3): 1600 - 1607. [Abstract] [Full Text] [PDF]
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A. Gravesen, B. Kallipolitis, K. Holmstrom, P. E. Hoiby, M. Ramnath, and S. Knochel pbp2229-Mediated Nisin Resistance Mechanism in Listeria monocytogenes Confers Cross-Protection to Class IIa Bacteriocins and Affects Virulence Gene Expression Appl. Envir. Microbiol., March 1, 2004; 70(3): 1669 - 1679. [Abstract] [Full Text] [PDF]
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M. Cao and J. D. Helmann The Bacillus subtilis Extracytoplasmic-Function {sigma}X Factor Regulates Modification of the Cell Envelope and Resistance to Cationic Antimicrobial Peptides J. Bacteriol., February 15, 2004; 186(4): 1136 - 1146. [Abstract] [Full Text] [PDF]
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U. Pag, M. Oedenkoven, N. Papo, Z. Oren, Y. Shai, and H.-G. Sahl In vitro activity and mode of action of diastereomeric antimicrobial peptides against bacterial clinical isolates J. Antimicrob. Chemother., February 1, 2004; 53(2): 230 - 239. [Abstract] [Full Text] [PDF]
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F. C. Neuhaus and J. Baddiley A Continuum of Anionic Charge: Structures and Functions of D-Alanyl-Teichoic Acids in Gram-Positive Bacteria Microbiol. Mol. Biol. Rev., December 1, 2003; 67(4): 686 - 723. [Abstract] [Full Text] [PDF]
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C. Heilmann, G. Thumm, G. S. Chhatwal, J. Hartleib, A. Uekotter, and G. Peters Identification and characterization of a novel autolysin (Aae) with adhesive properties from Staphylococcus epidermidis Microbiology, October 1, 2003; 149(10): 2769 - 2778. [Abstract] [Full Text] [PDF]
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D. M. Hoover, Z. Wu, K. Tucker, W. Lu, and J. Lubkowski Antimicrobial Characterization of Human {beta}-Defensin 3 Derivatives Antimicrob. Agents Chemother., September 1, 2003; 47(9): 2804 - 2809. [Abstract] [Full Text] [PDF]
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S. Katzif, D. Danavall, S. Bowers, J. T. Balthazar, and W. M. Shafer The Major Cold Shock Gene, cspA, Is Involved in the Susceptibility of Staphylococcus aureus to an Antimicrobial Peptide of Human Cathepsin G Infect. Immun., August 1, 2003; 71(8): 4304 - 4312. [Abstract] [Full Text] [PDF]
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K. Midorikawa, K. Ouhara, H. Komatsuzawa, T. Kawai, S. Yamada, T. Fujiwara, K. Yamazaki, K. Sayama, M. A. Taubman, H. Kurihara, et al. Staphylococcus aureus Susceptibility to Innate Antimicrobial Peptides, {beta}-Defensins and CAP18, Expressed by Human Keratinocytes Infect. Immun., July 1, 2003; 71(7): 3730 - 3739. [Abstract] [Full Text] [PDF]
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G. Bell and P.-H. Gouyon Arming the enemy: the evolution of resistance to self-proteins Microbiology, June 1, 2003; 149(6): 1367 - 1375. [Abstract] [Full Text] [PDF]
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E. Wahlstrom, M. Vitikainen, V. P. Kontinen, and M. Sarvas The extracytoplasmic folding factor PrsA is required for protein secretion only in the presence of the cell wall in Bacillus subtilis Microbiology, March 1, 2003; 149(3): 569 - 577. [Abstract] [Full Text] [PDF]
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 M. R. Yeaman and N. Y. Yount Mechanisms of Antimicrobial Peptide Action and Resistance Pharmacol. Rev., March 1, 2003; 55(1): 27 - 55. [Abstract] [Full Text] [PDF]
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T. Theis, M. Wedde, V. Meyer, and U. Stahl The Antifungal Protein from Aspergillus giganteus Causes Membrane Permeabilization Antimicrob. Agents Chemother., February 1, 2003; 47(2): 588 - 593. [Abstract] [Full Text] [PDF]
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B. Said-Salim, P. M. Dunman, F. M. McAleese, D. Macapagal, E. Murphy, P. J. McNamara, S. Arvidson, T. J. Foster, S. J. Projan, and B. N. Kreiswirth Global Regulation of Staphylococcus aureus Genes by Rot J. Bacteriol., January 15, 2003; 185(2): 610 - 619. [Abstract] [Full Text] [PDF]
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S. A. Kristian, M. Durr, J. A. G. Van Strijp, B. Neumeister, and A. Peschel MprF-Mediated Lysinylation of Phospholipids in Staphylococcus aureus Leads to Protection against Oxygen-Independent Neutrophil Killing Infect. Immun., January 1, 2003; 71(1): 546 - 549. [Abstract] [Full Text] [PDF]
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M. Durr and A. Peschel Chemokines Meet Defensins: the Merging Concepts of Chemoattractants and Antimicrobial Peptides in Host Defense Infect. Immun., December 1, 2002; 70(12): 6515 - 6517. [Full Text] [PDF]
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T. Koprivnjak, A. Peschel, M. H. Gelb, N. S. Liang, and J. P. Weiss Role of Charge Properties of Bacterial Envelope in Bactericidal Action of Human Group IIA Phospholipase A2 against Staphylococcus aureus J. Biol. Chem., November 27, 2002; 277(49): 47636 - 47644. [Abstract] [Full Text] [PDF]
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H. Ulvatne, H. H. Haukland, O. Samuelsen, M. Kramer, and L. H. Vorland Proteases in Escherichia coli and Staphylococcus aureus confer reduced susceptibility to lactoferricin B J. Antimicrob. Chemother., October 1, 2002; 50(4): 461 - 467. [Abstract] [Full Text] [PDF]
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