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J Biol Chem, Vol. 273, Issue 44, 29143-29149, October 30, 1998
From the Surface proteins of Staphylococcus
aureus are covalently linked to the bacterial cell wall by a
mechanism requiring a COOH-terminal sorting signal with a conserved
LPXTG motif. Cleavage between the threonine and the glycine
of the LPXTG motif liberates the carboxyl of threonine to
form an amide bond with the pentaglycyl cross-bridge in the
staphylococcal peptidoglycan. Here, we asked whether altered
peptidoglycan cross-bridges interfere with the sorting reaction and
investigated surface protein anchoring in staphylococcal
fem mutants. S. aureus strains carrying
mutations in the femA, femB, femAB,
or the femAX genes synthesize altered cross-bridges, and
each of these strains displayed decreased sorting activity.
Characterization of cell wall anchor structures purified from the
fem mutants revealed that surface proteins were linked to
cross-bridges containing one, three, or five glycyl residues, but not
to the Gram-positive bacteria display proteins on their surface as a
means to interact with host tissues and to establish human infections (1, 2). The mechanism of surface protein anchoring to the bacterial
cell wall has recently been established for protein A of
Staphylococcus aureus. After synthesis in the cytoplasm, protein A is first initiated into the secretory pathway by an NH2-terminal signal (leader) peptide (3). A 35-residue
COOH-terminal sorting signal is necessary and sufficient for the
anchoring of protein A and functions to first retain the polypeptide
within the secretory pathway (4). This allows proteolytic cleavage between the threonine and the glycine of the conserved LPXTG
motif (5). The liberated carboxyl of threonine is amide linked to the
amino of the pentaglycyl cross-bridge of the staphylococcal peptidoglycan, thereby tethering the COOH-terminal end of protein A to
the bacterial cell wall (6, 7). This amide bond exchange mechanism
displays striking similarity to the penicillin-sensitive transpeptidation reaction (8), during which the cell wall pentapeptide precursor is cleaved at the peptide bond between
D-alanyl-D-alanine, while the liberated
carboxyl of D-alanyl is amide linked to the free amino of
the peptidoglycan cross-bridge (9), pentaglycyl in S. aureus
(10). Elements involved in transpeptidation and the sorting reaction
are conserved in Gram-positive bacteria (6, 11). Thus, it seems likely
that cell wall sorting is a universal mechanism for the anchoring of
surface proteins (6). If so, sortase, the enzymatic activity that is
thought to catalyze this reaction, might also be found conserved in
many different bacterial species and could provide a target for an
antibacterial therapy that interferes with surface protein anchoring
(6).
Although the presence of free amino groups is a common feature in the
cross-bridges of bacterial peptidoglycans, the overall chemical nature
of this structure varies between different Gram-positive bacteria (11).
In staphylococci, the cross-bridge is composed of five glycyl, whereas
in some streptococci and listerial species it consists of two alanyl
and meso-diaminopimelic acid, respectively (11). Genetic
analysis of staphylococcal methicillin resistance has provided insights
into the synthesis of peptidoglycan cross-bridges. Staphylococcal
strains expressing the penicillin binding protein PBP2a (PBP2') are
resistant to most Biochemical studies on the synthesis of staphylococcal peptidoglycan
revealed that the pentaglycyl cross-bridge is synthesized via
modification of the lipid II precursor
(undecaprenylpyrophosphate-MurNAc(-L-Ala-D-iGln-L-Lys-D-Ala-DAla)-( Here, we asked whether the sorting reaction of surface proteins can
proceed in fem mutant staphylococci. As compared with wild-type cells, the half-life of surface protein precursor molecules was increased in fem mutant strains, suggesting that the
sorting reaction is partially hindered. Characterization of anchor
structures revealed that surface proteins were linked to peptidoglycan
with cross-bridges harboring mono-, tri-, or pentaglycyl as well as tetraglycyl-monoseryl. No surface protein was found attached to the
Bacterial Strains and Plasmids--
Plasmids pHTT4 (7) and
pSeb-Cws-BlaZ1 (5) were
transduced with phages Pulse-Chase Analysis of Seb-Cws-BlaZ
Processing--
Staphylococcal strains harboring pSeb-Cws-BlaZ were
grown overnight in tryptic soy broth supplemented with chloramphenicol (10 µg/ml), diluted 1:20 into fresh medium, and grown with shaking at
37 °C until A600 0.6. Cells from 1 ml of
culture were sedimented by centrifugation at 8000 × g
for 2 min and washed with 1 ml of prewarmed minimal medium (3). The
cells were suspended in 1 ml of minimal medium, and newly synthesized
polypeptide was labeled with 100 µCi of [35S]Promix
(Amersham) for 1 min. Labeling was quenched by the addition of an
excess non-radioactive amino acid (50-µl chase (100 mg/ml casamino
acids, 20 mg/ml methionine)). At timed intervals after the addition of
the chase, 0, 2, 5, and 10 min, 250-µl aliquots were removed, and
protein was precipitated by the addition of 250 µl of 10%
trichloroacetic acid. The precipitate was sedimented by centrifugation
15,000 × g for 10 min, washed with 1 ml of acetone, and dried. Samples were suspended in 1 ml of 0.5 M
Tris-HCl, pH 6.8, and staphylococcal peptidoglycan was digested by
adding 50 µl of mutanolysin (5000 units/ml, Sigma) and incubating
4 h at 37 °C. Proteins were again precipitated with
trichloroacetic acid, washed with acetone, and subjected to
immunoprecipitation with Purification of Anchor Peptides--
Staphylococcal strains
harboring pHTT4 were grown overnight in tryptic soy broth supplemented
with 10 µg/ml chloramphenicol, diluted 1:40 into 4 liters of fresh
medium, and grown with 250 rpm shaking for 5 h at 37 °C. Cells
were sedimented by centrifugation at 8000 × g for 15 min. Pellets were suspended in 100 ml of water, extracted with 100 ml
of ethanol-acetone (1:1), and incubated for 30 min on ice. The cells
were collected by centrifugation, washed with 300 ml of ice-cold water,
and suspended in 30 ml of 0.1 M Tris-HCl, pH 7.5. The
peptidoglycan was digested by adding 210 µg of purified MALDI-MS--
MALDI-MS spectra were obtained on a reflectron
time-of-flight instrument (PerSeptive Biosystems Voyager RP) in the
linear mode. Samples (0.5-1.0 µl) were co-spotted with 1.0 µl of
matrix ( Sorting of Surface Protein Precursor in Staphylococcal fem
Mutants--
Previous work characterized the processing of
Seb-Cws-BlaZ, harboring a central sorting signal flanked by
NH2-terminal Seb and COOH-terminal BlaZ domains (5).
Seb-Cws-BlaZ is exported from the cytoplasm by an
NH2-terminal signal peptide and cleaved between the
threonine and the glycine of its LPXTG motif (5). The
NH2-terminal Seb fragment is linked to the staphylococcal peptidoglycan, whereas the COOH-terminal BlaZ domain remains in the
bacterial cytosol (5). All surface protein that is cleaved at the
LPXTG motif is also anchored to the cell wall (5). Hence, the rate of cleavage at the LPXTG motif of pulse-labeled
Seb-Cws-BlaZ is a measure for the rate of surface protein anchoring in
various staphylococcal fem mutant strains.
Staphylococci were pulse labeled with [35S]methionine for
1 min. At timed intervals during chase, culture aliquots were
precipitated with trichloroacetic acid and washed in acetone. The
staphylococcal peptidoglycan was digested with mutanolysin, and all
protein was again precipitated with trichloroacetic acid prior to
immunoprecipitation with Purification of Surface Proteins from Cell Wall Anchor Structure of Surface Proteins in femB Mutant
Staphylococci--
Anchor peptides purified from the peptidoglycan of
the femB mutant strain UT34-2 were desalted and analyzed by
MALDI-MS. The mass spectrum in Fig. 3
revealed signals at m/z 2123, 2193, 2601, 2642, 2670, 4224, and 4742. The measurement of m/z 2123 was consistent with
the structure of a COOH-terminal peptide linked to
L-Ala-D-iGln-L-Lys(Gly3)-D-Ala, whereas the signal at m/z 2193 was consistent with an anchor
peptide and linked
L-Ala-D-iGln-L-Lys(Gly3)-D-Ala-D-Ala.
Compounds with m/z 2601 and 2670 represented anchor peptides
with linked
MurNAc-[L-Ala-D-iGln-L-Lys(Gly3)-D-Ala]-( Cell Wall Anchor Structure of Surface Proteins in femA Mutant
Staphylococci--
After digestion of the cell wall of S. aureus UK17 (pHTT4) with Cell Wall Anchor Structure of Surface Proteins in femAB
Mutant Staphylococci--
Strain BB308 carries a transposon insertion
in the promoter region of the femAB operon, a mutation which
is known to decrease the expression of both genes (18, 23). In contrast
to the femA and femB mutants analyzed above,
strain BB308 synthesizes cross-bridges containing pentaglycyl,
tetraglycyl-monoseryl (Gly4-Ser1), monoglycyl,
and small amounts of triglycyl (26, 29). When subjected to MALDI-MS,
anchor peptides purified from Cell Wall Anchor Structure of Surface Proteins in femAX Mutant
Staphylococci--
The femAX mutant strain UK31 has been
generated by chemical mutagenesis of strain UK14 (femA)
(29). The peptidoglycan of S. aureus UK31 displayed reduced
peptidoglycan cross-linking as compared with its UK14 parent (63% as
compared with 67% for UK14 and 76% for wild-type strain BB270) (29).
In contrast to the cell wall of strain UK14, in which all cross-bridges
consist of monoglycyl, about half of all peptidoglycan monomer isolated
from S. aureus UK31 contains cross-bridges without glycine,
in which the All bacterial peptidoglycan is synthesized from lipid-linked
precursor molecules (10, 11). Penicillin binding proteins have evolved
to cleave these precursors at the amide bond of
D-alanyl-D-alanine (9). Even when compared with
penicillin binding protein homologs from distantly related species,
these enzymes share significant sequence and structural similarity (9).
In contrast to the conservation of
D-alanyl-D-alanine, the chemical nature of
cross-bridges within bacterial peptidoglycans is highly variable.
Nevertheless, the presence of a free amino group is a common feature
(11), and this element functions as a nucleophile to attack the acyl intermediate formed between the carbonyl of D-alanyl and
the active site serine residue of PBPs. The nucleophilic attack results
in the formation of a peptide bond between D-alanyl and the
peptidoglycan cross-bridge as well as the regeneration of the hydroxyl
at the active site serine (9). In vitro, when tested with
purified pentapeptide substrate and enzyme, several different
nucleophiles can substitute for the free amino of the peptidoglycan
cross-bridge, including hydroxylamine, alanine, glycine, and others
(40). However, in vivo the transpeptidation reaction is
known to proceed with great specificity, cross-linking only neighboring
wall peptides (41). These observations suggest that the fidelity of the
transpeptidation reaction may depend at least in part on a unique
environment in which the free amino of the cross-bridge is the only
available nucleophile.
The sorting reaction of Gram-positive bacteria displays similarity to
the penicillin-sensitive transpeptidation reaction (6). Here, the
substrate for proteolytic cleavage is the LPXTG motif of
surface proteins, which is cleaved between the threonine (T) and the
glycine (G) (5). The nucleophilic amino group donor of the sorting
reaction is identical to that of the transpeptidation reaction,
i.e., the peptidoglycan cross-bridge. Because the
LPXTG motif is found in sorting signals of many different
surface proteins (42), it seems likely that sortase, the enzyme
proposed to catalyze this reaction, is structurally conserved between
different Gram-positive bacteria. Here, we asked whether mutationally
altered cross-bridges of the staphylococcal peptidoglycan can serve as
substrates for the sorting reaction. The rate of surface protein
precursor cleavage at the LPXTG motif was employed to
measure this reaction. S. aureus strains carrying mutations
in the fem genes displayed a decreased rate of precursor
cleavage as compared with the wild-type strains, suggesting that the
altered cross-bridges slowed the anchoring of surface proteins.
We also revealed here the anchor structures of surface proteins
expressed in S. aureus strains that carry mutations in
various fem genes. The results showed that surface protein
was linked to tri- and monoglycyl cross-bridges of peptidoglycan
isolated from femB and femA mutant staphylococci,
respectively. However, no surface protein was found linked directly to
the The loss of We thank Kym F. Faull for suggestions and
discussion during the course of this work and laboratory members for
critically reading this manuscript.
*
This work was supported by United States Public Health
Service Grants AI 33985 and AI 38897.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 abbreviations used are:
Seb, staphylococcal
enterotoxin B; Cws, cell wall sorting signal; BlaZ, staphylococcal
2
W. W. Navarre, H. Ton-That, K. F. Faull, and O. Schneewind, manuscript in preparation.
Anchor Structure of Staphylococcal Surface Proteins
III. ROLE OF THE FemA, FemB, AND FemX FACTORS IN ANCHORING
SURFACE PROTEINS TO THE BACTERIAL CELL WALL*
,
Department of Microbiology and Immunology,
UCLA School of Medicine, Los Angeles, California 90095, § Bayer AG, Pharma Research Antiinfectives I, D-42096
Wuppertal, Germany, and the ¶ Institute of Medical Microbiology,
University of Zürich, CH-8028 Zürich, Switzerland
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-amino of lysyl in muropeptides without glycine. When tested
in a femAB strain synthesizing cross-bridges with mono-,
tri-, and pentaglycyl as well as tetraglycyl-monoseryl, surface
proteins were found anchored mostly to the five-residue cross-bridges
(pentaglycyl or tetraglycyl-monoseryl). Thus, although wild-type
peptidoglycan appears to be the preferred substrate for the sorting
reaction, altered cell wall cross-bridges can be linked to the
COOH-terminal end of surface proteins.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-lactam antibiotics including methicillin
(12-16). Genetic screens designed to identify elements that are also
necessary for methicillin resistance yielded mutations in approximately
ten different fem (aux) genes (17-21). Some of these genes are involved in the synthesis of the pentaglycyl
cross-bridge (22-26) or the amidation of D-iso-glutamyl
within the wall peptide (27, 28). The precise biochemical defect of
other fem mutations is still unknown (29). Presently
available staphylococcal strains harboring mutations in the
femA, femB, and femX genes synthesize altered cell wall cross-bridges with either three glycyl
(femB), one glycyl (femA), or a combination of no
or one glycyl (29). The latter phenotype has been reported for a mutant
with a combination of a femA mutation and a second one
leading to a partial non-functional FemX protein (hereafter called
femAX) (29).
1-4)-GlcNAc) (30-32). Three glycyl tRNA species are dedicated to this
biosynthetic pathway (33-36). After being charged with amino acid,
these tRNAs are thought to serve as substrate in a sequence of
reactions that successively add glycine either directly to the
-amino of lysyl or to the amino of another glycyl (31, 34). It seems
likely that the femA, femB, and femX
genes specify enzymatic activities that catalyze these reactions (29).
In this model, one enzyme, presumably FemX, adds the first glycine to
the -amino side chain of lysyl within lipid II (29). FemA and FemB
are thought to each add two additional glycines, thereby synthesizing
cross-bridges with three and five glycyl, respectively (29). It is
conceivable that each Fem factor might recognize one of the three
glycyl tRNA species (35, 36). If so, one would predict that mutations in any one of the three glycyl tRNA genes should cause the same phenotype as the corresponding fem mutants.
-amino of lysyl, suggesting that the sorting reaction discriminates between certain peptidoglycan cross-bridges.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
11 or 80
into UK17 (femA) (29),
UT34-2 (femB)(25), BB308 (femAB) (18), and UK31
(femAX) strains (29). Briefly, strains were grown in CY
broth containing glycerol phosphate overnight (per liter CY: 10 g
of casamino acids, 10 g of yeast extract, 5 g of glucose,
5.9 g of NaCl; 40 ml of sterile filtered 1.5 M -glycerophosphate were added after autoclaving) (37). Staphylococci (109 colony-forming units) were sedimented by
centrifugation at 7000 × g for 3 min and suspended in
200 µl of phage buffer (1 mM MgSO4, 4 mM CaCl2, 50 mM Tris-HCl, 100 mM NaCl, pH 7.8, and 1 g of gelatin added per liter).
Phage lysate, 200 µl generated from S. aureus OS2 carrying
either pHTT4 or pSeb-Cws-BlaZ, was added for 10 min, and cells were
plated on tryptic soy agar containing 10 µg/ml chloramphenicol.
Escherichia coli BL21(DE3) pLysS, pHTT2 was used to purify
staphylococcal 11 hydrolase as described previously (7).
-BlaZ (38) followed by SDS-PAGE and
PhosphorImager analysis.
11
hydrolase for 16 h at 37 °C. Seb-MH6-Cws was
purified as described previously (7). Briefly, the digested cell wall
was centrifuged at 17,000 × g for 15 min to remove
insoluble material, and the supernatant was subjected to affinity
chromatography. 2 ml of Ni-NTA Sepharose (Qiagen) were washed with
equilibration buffer (50 mM Tris-HCl, 150 mM
NaCl, pH 7.5), loaded with the supernatant of the 11 hydrolase
digest, washed first with 30 ml of wash buffer (50 mM
Tris-HCl, 150 mM NaCl, 10% glycerol, pH 7.5) and then with
30 ml of equilibration buffer. Bound protein was eluted with 0.5 M imidazole in wash buffer. Seb-MH6-Cws was precipitated with 7% trifluoroacetic acid (v/v), washed with acetone, dried under vacuum, and dissolved in 600 µl of 70% formic acid. A
crystal of CnBr was added, and the cleavage reaction was incubated for
16 h at room temperature. The reaction mixture was dried under vacuum, washed with water, and dissolved in 1 ml of buffer A (6 M guanidine hydrochloride, 0.1 M
NaH2PO4, 0.01 M Tris-HCl, pH 8.0).
The sample was subjected to 1 ml Ni-NTA Sepharose column pre-equilibrated with buffer A, washed with 10 ml of buffer A, 10 ml of
buffer B (6 M urea, 0.1 M
NaH2PO4, 0.01 M Tris-HCl, pH 8.0),
and 10 ml of buffer C (same as buffer B, but pH 6.3). Anchor peptides
were eluted with 2 ml of 0.5 M acetic acid, desalted over
C18 cartridge (Analtech), and subjected to MALDI-MS.
-cyano-4-hydroxycinnamic acid at 1 mg/100 µl
CH3CN:water:trifluoroacetic acid (70:30:0.1)) and mass
measured using an external calibration with bovine insulin (7).
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-BlaZ and SDS-PAGE. The amount of
Seb-Cws-BlaZ precursor and BlaZ cleavage fragment were determined by
PhosphorImager analysis. Fig.
1B shows the autoradiogram of
a typical experiment that measured the processing of Seb-Cws-BlaZ in
S. aureus BB308 (femAB). In wild-type cells the
half-life of the precursor was 0.53 min for S. aureus OS2
(spa
, ermr) and 1.4 min for BB270
(Mcr). All S. aureus fem
strains displayed an increased half-life of the Seb-Cws-BlaZ precursor:
2.12 min (UK17 (femA of BB270)), 2.58 min
(UT34-2 (femB:Tn551 of BB270)), 2.38 min (BB308
(femA:Tn551 of BB270)), and 2.25 min (UK31 (femAX
of BB270)). Together, these results suggested that the sorting reaction
of surface proteins is significantly slowed in the fem
mutant staphylococci.
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Fig. 1.
Cleavage of surface protein precursor in
fem mutant staphylococci. A, structure of
Seb-Cws-BlaZ harboring an NH2-terminal signal (leader)
peptide and the sorting signal of protein A, which consists of an
LPXTG motif, hydrophobic domain (shaded box), and
charged domain (boxed RRREL). The sorting signal is fused to
the COOH terminus of Seb and to the NH2 terminus of mature
BlaZ. Seb-Cws-BlaZ precursor (substrate 1) and peptidoglycan precursor
lipid II (substrate 2) are thought to be substrates for the sorting
reaction during which the polypeptide is cleaved between the threonine
and the glycine of the LPXTG motif and amide linked to the
pentaglycyl cross-bridge of lipid II. An intermediate of the surface
protein linked to peptidoglycan precursor may then be incorporated into
the cell wall by transglycosylase and transpeptidase reactions to
generate anchored surface protein (product 1). The COOH-terminal
cleavage fragment of the sorting precursor, BlaZ (product 2), remains
in the bacterial cytosol. B, the rate of Seb-Cws-BlaZ
sorting to the cell wall was measured with pulse-chase experiments that
determined the concentration of precursor (substrate 1) and
COOH-terminal BlaZ cleavage fragment (product 2). The figure shows a
representative autoradiogram of Seb-Cws-BlaZ cleavage in S. aureus BB308 (femAB). [35S]Methionine
pulse-labeled staphylococci were incubated for 0, 2, 5, and 10 min and
precipitated with trichloroacetic acid. After digestion of the
peptidoglycan with mutanolysin, Seb-Cws-BlaZ precursor and BlaZ
cleavage product were immunoprecipitated with anti-BlaZ ( -BlaZ),
separated on SDS-PAGE, and analyzed by PhosphorImager. C,
rate of sorting precursor cleavage in wild-type and fem
mutant S. aureus strains. Results from three independent
experiments such as the one shown in B were quantified and
averaged to determine the time required for 50% precursor cleavage.
The standard deviations of these measurements are indicated in
parentheses.
11 Hydrolase-digested
Peptidoglycan--
To characterize the anchor structure of surface
proteins in fem mutant staphylococci, we employed another
hybrid protein. Seb-MH6-Cws is composed of enterotoxin B
carrying an NH2-terminal signal peptide and a
COOH-terminally fused sorting signal of protein A. At the fusion joint
between Seb and the sorting signal, a methionine followed by six
histidines is inserted. When expressed in staphylococci, this protein
is exported and linked to the bacterial cell wall. 11 hydrolase
cleaves the staphylococcal peptidoglycan at the peptide bonds between
N-acetylmuramyl-L-alanyl (amidase) and
D-alanyl-glycyl.2
After peptidoglycan solubilization with the 11 hydrolase,
Seb-MH6-Cws was affinity purified on nickel Sepharose,
cleaved at methionyl with CnBr, and COOH-terminal anchor peptides were
purified by another round of chromatography on Ni-NTA Sepharose. Fig.
2 shows that purified
Seb-MH6-Cws migrated as two distinct species on SDS-PAGE.
The faster migrating species has
L-Ala-D-iGln-L-Lys(Gly5)d-Ala amide linked to the carboxyl of its COOH-terminal threonine, whereas the slower migrating species carries
MurNAc[L-Ala-D-iGln-L-Lys(Gly5)-D-Ala]-(1-4)-GlcNAc (7). The migration pattern of Seb-MH6-Cws purified from
strains OS2 (wild type), UK17 (femA), UT34-2
(femB), BB308 (femAB), and UK31
(femAX) was indistinguishable on SDS-PAGE.
View larger version (20K):
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Fig. 2.
Purification of cell wall-anchored surface
proteins from 11 hydrolase-digested peptidoglycan.
A, structure of Seb-MH6-Cws harboring an
NH2-terminal signal (leader) peptide with signal peptidase
cleavage site as well as a COOH-terminally fused cell wall sorting
signal consisting of the LPXTG motif, hydrophobic domain
(black box), and positively charged tail (boxed
+). Upstream of the LPXTG motif, a methionine followed by a
histidine tag was inserted that allowed purification of the recombinant
Seb-MH6-Cws protein on Ni-NTA Sepharose. Surface proteins
were solubilized from the staphylococcal peptidoglycan by treatment
with 11 hydrolase, purified on Ni-NTA Sepharose, separated on 12%
SDS-PAGE, and stained with Coomassie Brilliant Blue. The SDS-PAGE shows
Seb-MH6-Cws purified from S. aureus strains OS2
(wild type, WT), UK17 (femA), UT34-2 (femB),
BB308 (femAB), and UK31 (femAX). The migration of
molecular size markers is indicated in kDa. B, structure of
the staphylococcal peptidoglycan. Arrows point to the
peptide cleavage sites for 11 hydrolase. Glyn indicates
the variable number of glycyl within the cross-bridges of
staphylococcal fem mutants.
1-4)-GlcNAc and
MurNAc-[L-Ala-D-iGln-L-Lys(Gly3)-D-Ala-D-Ala]-(1-4)-GlcNAc, respectively. The ion with m/z 2642 was explained as anchor
peptide linked to
O-6-acetyl-MurNAc-[L-Ala-D-iGln-L-Lys(Gly3)-D-Ala]-(1-4)-GlcNAc in which the muramic acid is N,O-6-diacetylated, a
modification present in approximately half of all staphylococcal
muramoyl residues (39). Compounds with m/z 4224 and 4742 represented anchor peptides in which the methionine at position 251 had
not been cleaved, resulting in additional upstream peptide sequence
(NH2-VDSKDVKIEVYLTTKKGTMHHHHHHAQALPET-anchor structure) (7). Taken together, the results indicated that all
detectable anchor peptides were linked to the peptidoglycan of the
femB mutant S. aureus strain UT34-2 via
triglycyl cross-bridges.
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Fig. 3.
Cell wall anchor structure of surface
proteins in femB mutant staphylococci. A,
11 hydrolase-solubilized Seb-MH6-Cws was purified on
nickel-NTA Sepharose and cleaved with CnBr, and the COOH-terminal
anchor peptides were isolated by a second affinity chromatography step.
The samples were desalted over C18 column and analyzed by MALDI-MS. The
numbers indicate the m/z values of the identified
ions. B, the calculated mass of predicted anchor peptide
structures (Calc m/z) in the peptidoglycan of
femB mutant strain UT34-2 was compared with the observed
ion signals (Obs m/z).
11 hydrolase, anchor peptides were
purified and analyzed by MALDI-MS. The spectrum shown in Fig.
4 revealed ions at m/z 2009, 2080, 2486, 2528, 2557, and 2599. Observation of the signal with
m/z 2009 was consistent with the calculated mass of anchor peptide linked to
L-Ala-D-iGln-L-Lys(Gly1)-D-Ala.
The compound with m/z 2080 was explained as
L-Ala-D-iGln-L-Lys(Gly1)-D-Ala-D-Ala linked to the COOH-terminal threonine of surface proteins. Compounds with m/z 2486 and 2557 represented the disaccharide-linked
species of the aforementioned anchor peptides, whereas the observation of ions at m/z 2528 and 2599 suggested N,O-6
diacetylation of muramoyl in similar structures. Thus, the data
revealed that all anchor peptides were linked to the peptidoglycan of
the femA mutant strain UK17 via monoglycyl
cross-bridges.
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Fig. 4.
Cell wall anchor structure of surface
proteins in femA mutant staphylococci. A,
11 hydrolase-solubilized Seb-MH6-Cws was purified on
nickel-NTA Sepharose and cleaved with CnBr, and the COOH-terminal
anchor peptides were isolated by a second affinity chromatography step.
The samples were desalted over C18 column and analyzed by MALDI-MS. The
numbers indicate the m/z values of the identified
ions. B, the calculated mass of predicted anchor peptide
structures (Calc m/z) in the peptidoglycan of
femA mutant strain UK17 was compared with the observed ion
signals (Obs m/z).
11 hydrolase-digested peptidoglycan of
strain BB308 yielded four main ion clusters. For better analysis of the
signals at m/z 2000-3000, the spectrum was drawn on an
expanded scale in Fig. 5B.
Observation of ions with m/z 2266 and 2745 was consistent
with the calculated mass of anchor peptides harboring
L-Ala-D-iGln-L-Lys(Gly4Ser1)-D-Ala and
MurNAc-[L-Ala-D-iGln-L-Lys(Gly4Ser1)-D-Ala]-(1-4)-GlcNAc linked to the carboxyl of threonine. Anchor peptides linked to pentapeptide species via similar Gly4Ser1
cross-bridges were also observed: m/z 2337 (L-Ala-D-iGln-L-Lys(Gly4Ser1)-D-Ala-D-Ala), 2815 (MurNAc-(L-Ala-D-iGln-L-Lys(Gly4Ser1)-D-Ala-D-Ala)-(1-4)-GlcNAc), and 2859 (6-OAc-MurNAc-(L-Ala-D-iGln-L-Lys(Gly4Ser1)-D-Ala-D-Ala)-(1-4)-GlcNAc). In addition, signals of anchor peptides were identified that harbored either wild-type pentaglycyl (m/z 2237, 2309, 2715, 2757, 2787, 2829) or monoglycyl cross-bridges (m/z 2007, 2080, 2529, 2601). Compounds with m/z 4370 and 4887 were due to
incomplete CnBr cleavage of Seb-MH6-Cws, and their anchor
structures were similar to those described for ions with m/z
2266 and 2787.
View larger version (42K):
[in a new window]
Fig. 5.
Cell wall anchor structure of surface
proteins in femAB mutant staphylococci. A,
11 hydrolase-solubilized Seb-MH6-Cws was purified on
nickel-NTA Sepharose and cleaved with CnBr, and the COOH-terminal
anchor peptides were isolated by a second affinity chromatography step.
The samples were desalted over C18 column and analyzed by MALDI-MS. The
numbers indicate the m/z values of the identified
ions. B, same mass spectrum as shown in A but
drawn to an expanded scale to reveal all ion signals at m/z
2000-3000. C, the calculated mass of predicted anchor
peptide structures (Calc m/z) in the peptidoglycan of
femAB mutant strain BB308 was compared with the observed ion
signals (Obs m/z).
-amino of lysyl is directly linked to the
D-alanyl at position four of a neighboring cell wall
subunit. The remaining 50% is composed of monoglycyl cross-bridges,
similar to the UK17 parent (29). MALDI-MS analysis of anchor peptides
released from the peptidoglycan of UK31 with 11 hydrolase treatment
revealed prominent ions with m/z 1454, 2010, 2081, 2488, 2530, 2559, 3557, and 4184 (Fig. 6). The
compound with m/z 1454 is likely a COOH-terminal degradation
product of surface protein; its mass is consistent with the peptide
sequence NH2-HHHHHHAQALPE (calculated mass of 1450 Da). The
ion at m/z 3557 can be explained as a COOH-terminal peptide
with a structure similar to that of m/z 1454 and an
additional 16 upstream residues due to incomplete CnBr cleavage. The
observed compound with m/z 2010 represented anchor peptide
linked to the monoglycyl cross-bridge of a cell wall tetrapeptide
(L-Ala-D-iGln-L-Lys(Gly1)-D-Ala). Ions with m/z 2081, 2488, 2530, 2559, and 2601 were
explained as anchor peptide linked to monoglycyl cross-bridges with
additional peptidoglycan structures: 2081 (L-Ala-D-iGln-L-Lys(Gly1)-D-Ala-D-Ala), 2488 (MurNAc-(L-Ala-D-iGln-L-Lys(Gly1)-D-Ala-D)-(1-4)-GlcNAc), 2530 (6-OAc-MurNAc-(L-Ala-D-iGln-L-Lys(Gly1)-D-Ala)-(1-4)-GlcNAc), 2559 (MurNAc-(L-Ala-D-iGln-L-Lys(Gly1)-D-Ala-D-Ala)-(1-4)-GlcNAc), 2601 (6-OAc-MurNAc-(L-Ala-D-iGln-L-Lys(Gly1)-DAla-D-Ala)-(1-4)-GlcNAc). None of the observed signals were consistent with anchor peptide structures in which the carboxyl of threonine was linked directly to
the -amino of lysine.
View larger version (29K):
[in a new window]
Fig. 6.
Cell wall anchor structure of surface
proteins in femAX mutant staphylococci. A,
11 hydrolase-solubilized Seb-MH6-Cws was purified on
nickel-NTA Sepharose and cleaved with CnBr, and the COOH-terminal
anchor peptides were isolated by a second affinity chromatography step.
The samples were desalted over C18 column and analyzed by MALDI-MS. The
numbers indicate the m/z values of the identified
ions. B, the calculated mass of predicted anchor peptide
structures (Calc m/z) in the peptidoglycan of
femAX mutant strain UK31 was compared with the observed ion
signals (Obs m/z).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-amino of lysyl within the cell wall of the femAX
strain UK31, indicating that not all cross-bridges serve as a substrate
for the sorting reaction. S. aureus BB308 carries a
transposon insertion in the promoter of the femAB operon
(18). Peptidoglycan analysis of this strain revealed the presence of
pentaglycyl, tetraglycyl-monoseryl, and monoglycyl as well as small
amounts of triglycyl cross-bridges. Analysis of anchor peptides
purified from the peptidoglycan of S. aureus BB308 showed
that surface proteins were mostly linked to tetraglycyl-monoseryl as
well as pentaglycyl. Although monoglycyl containing murein is known to
be the most abundant species in strain BB308, we observed little
surface protein anchoring to this cross-bridge, suggesting that the
sortase activity of S. aureus preferred cross-bridges
containing five residues. BB308 also contains small amounts of
peptidoglycan with triglycyl cross-bridges; however, we could not
identify surface protein linked to this species. This is likely due to
the low abundance of triglycyl murein subunits in the peptidoglycan
BB308 since the results from strain UT34-2 (femB) indicate
that surface proteins can be linked to triglycyl amino donors.
-lactam resistance in fem mutants of strain
BB270 suggests that the PBP2' enzyme cannot efficiently recognize the
altered peptidoglycan cross-bridges (20). This is corroborated by
electron microscopic studies of fem strains that revealed
gross defects in morphology as well as cell lysis (29). Furthermore, all fem strains contain reduced amounts of cross-linked
peptidoglycan (29). Introduction of the femA or
femB mutation into strains that do not express the PBP2'
enzyme also caused significant changes in the amount of peptidoglycan
cross-linking as well as cell wall physiology. Thus, similar to the
sorting reaction, staphylococcal PBPs can employ different amino group
donors for transpeptidation, although these cross-bridges cannot fully
substitute for the wild-type pentaglycyl substrate.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom reprint requests should be addressed: Dept. of
Microbiology and Immunology, UCLA School of Medicine, 10833 Le Conte Ave., Los Angeles, CA 90095. Tel.: 310-206-0997; Fax: 310-267-0173; E-mail: olafs{at}ucla.edu.
-lactamase; MALDI-MS, matrix-assisted laser desorption ionization
mass spectrometry; MurNAc, N-acetylmuramic acid; PAGE, polyacrylamide gel electrophoresis; PBP, penicillin binding
protein.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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