(Received for publication, February 5, 1997, and in revised form, May 7, 1997)
From the Department of Microbiology & Immunology and
Molecular Biology Institute and the § Department of
Psychiatry & Biobehavioral Sciences, UCLA School of Medicine,
Los Angeles, California 90095
Surface proteins of Staphylococcus
aureus are anchored to the cell wall by a mechanism requiring a
COOH-terminal sorting signal. Previous work demonstrated that the
sorting signal is cleaved at the conserved LPXTG motif and
that the carboxyl of threonine (T) is linked to the staphylococcal cell
wall. By employing different cell wall lytic enzymes, surface proteins
were released from the staphylococcal peptidoglycan and their
COOH-terminal anchor structure was revealed by a combination of mass
spectrometry and chemical analysis. The results demonstrate that
surface proteins are linked to a branched peptide
(NH2-Ala--Gln-Lys-(NH2-Gly5)-Ala-COOH) by an amide bond between the carboxyl of threonine and the amino of the
pentaglycine cross-bridge that is attached to the
-amino of lysyl.
This branched anchor peptide is amide-linked to the carboxyl of
N-acetylmuramic acid, thereby tethering the COOH-terminal end of surface proteins to the staphylococcal peptidoglycan.
Gram-positive pathogens cause a variety of different human diseases, which often present therapeutic difficulty because of bacterial antibiotic resistance (1). The treatment of hospital-acquired infections of Staphylococcus aureus and Enterococcus faecium is particularly challenging since many of these strains are multiply resistant to all classes of antibiotics (2). In search for a novel target of antibiotic therapy, we have begun to characterize the anchoring of surface proteins to the cell wall of S. aureus. Staphylococcal surface proteins are thought to be instrumental during the establishment and maintenance of human infections by either promoting the attachment to specific host tissues or preventing the phagocytic clearance of the invading bacteria (3, 4).
Cell wall anchoring of surface proteins in S. aureus
requires both an NH2-terminal signal (leader) peptide and a
COOH-terminal cell wall sorting signal (5). The 35-residue sorting
signal harbors an LPXTG sequence motif that has been found
conserved within the sorting signals of more than 100 surface proteins
of Gram-positive bacteria (6, 7) and serves as the recognition sequence
for the proteolytic cleavage between its threonine (T) and glycine (G)
residues (8). Previous work reported the purification of a surface
protein that had been released from the staphylococcal peptidoglycan by
lysostaphin digestion (9). The COOH-terminal peptide of this molecule
was cut at engineered trypsin cleavage sites and characterized with
ESI-MS1 and Edman
degradation, revealing the addition of two and three glycine residues
to the threonine of the LPXTG motif (9). Because lysostaphin, a glycyl-glycine endopeptidase (10), cleaves the pentaglycine cross-bridges of the staphylococcal peptidoglycan predominantly at their central glycine residue (11), it was suggested
that surface proteins could be anchored via an amide linkage between
the carboxyl of threonine and the amino of the pentaglycine
cross-bridge (9). Nevertheless, previous work left unresolved the
chemical structure of the cell wall anchor of surface proteins. This
question was addressed here, and we demonstrate that surface proteins
are linked to a branched peptide (NH2-Ala--Gln-Lys-(NH2-Gly5)-Ala-COOH).
This branched anchor peptide is amide-linked to
N-acetylmuramic acid, thereby tethering the peptide backbone
to the glycan chains of the cell wall. Unlike the highly cross-linked
wall peptides within the peptidoglycan, these branched anchor peptides
are not substituted at their terminal D-alanine.
To express the hybrid
protein Seb-MH6-Cws, the coding sequence for the protein A
cell wall sorting signal harboring the engineered methionine-histidine
affinity tag was amplified from the chromosomal DNA of S. aureus strain 8325-4 (12) with the primers Seb-6His (AAGGTACCATGCATCACCATCACCATCACGCTCAAGCATTACCAGAAACT) and spa-2 (AAGGATCCTTATCATTTCAAATAAGAATGTGTT). The PCR product was digested with
KpnI and BamHI and cloned into the corresponding
sites of pSEB-FnBP (7) to generate pHTT4, which was transformed into S. aureus OS2 (5). The coding sequence of the 11 amidase
was amplified from purified phage DNA (12) with the primers
(AACATATGCAAGCAAAATTAACTAAAAAT and
AAGGATCCCTAGTGATGGTGATGGTGATGACTGATTTCTCCCCATAAGTC), digested with
NdeI and BamHI and cloned into the corresponding
sites of the T7 expression vector pET-9a (13) to yield pHTT2. After
transformation of Escherichia coli BL1 (DE3) pLysS (13), the
resulting strain was employed for the induced expression of the
11
amidase.
S. aureus OS2 harboring pHTT4 was grown overnight in tryptic soy broth supplemented with chloramphenicol (10 µg/ml) and diluted 1:40 into the same medium. Generally 4 liters of culture were grown with shaking at 250 rpm and 37 °C for 5 h. Cells were harvested by centrifugation at 8000 × g for 15 min. Pellets were suspended in 100 ml of water, and the cell wall carbohydrates were extracted by the addition of 100 ml of an ethanol-acetone (1:1) mixture (14) and incubation for 30 min on ice. The cells were collected by centrifugation and washed with 300 ml of ice-cold water. Cell pellets were suspended in 30 ml of 0.1 M Tris-HCl buffer (pH 7.5 for lysostaphin and amidase digests, pH 6.8 for mutanolysin), and the peptidoglycan was digested with either lysostaphin (33 µg/ml, Ambi), amidase (67 µg/ml), or mutanolysin (333 units/ml, Sigma) and incubated for either 2 (lysostaphin) or 16 h (amidase and mutanolysin) at 37 °C. Peptidoglycan-released surface proteins were separated from the acetone-protoplasts (14) by centrifugation at 17,000 × g for 15 min, and the collected supernatant was filtered through a 0.2-µm pore size membrane. Solubilized Seb-MH6-Cws surface protein was purified by affinity chromatography on Ni-NTA-Sepharose (Qiagen). Briefly, a column with 2 ml of bed volume was washed with 15 ml of equilibration buffer (50 mM Tris, 150 mM NaCl, pH 7.5), loaded with 30 ml of cell wall extract, washed first with 30 ml of wash buffer (50 mM Tris, 150 mM NaCl, 10% glycerol, pH 7.5) and then with 30 ml of equilibration buffer. Bound protein was eluted with 5 ml of elution buffer (0.5 M imidazole, 50 mM Tris, 150 mM NaCl, 10% glycerol, pH 7.5).
Amidase and lysostaphin-solubilized Seb-MH6-Cws were precipitated with 7% trifluoroacetic acid (v/v), solutions were incubated on ice for 30 min, centrifuged for 15 min at 17,000 × g, and the collected pellets were washed in acetone and dried. After affinity chromatography, the mutanolysin-solubilized Seb-MH6-Cws protein was dialyzed against 4 liters each of 50 mM of NH4HCO3 for 1, 3, and 24 h and then dried under vacuum. The dried pellets of the affinity-purified Seb-MH6-Cws proteins were suspended into 600 µl of 70% formic acid. A crystal of CnBr was added, and the reaction was incubated for 16 h at room temperature. The cleaved peptides were dried under vacuum, washed twice with 100 µl of water, and suspended in 1 ml of buffer A (6 M guanidine hydrochloride, 0.1 M NaH2PO4, 0.01 M Tris, pH 8.0). A 1-ml column of Ni-NTA-Sepharose was pre-equilibrated with 10 ml of buffer A and loaded with the CnBr-cleaved peptides. The column was washed successively with 10 ml of buffer A, 10 ml of buffer B (8 M urea, 0.1 M NaH2PO4, 0.01 M Tris, pH 8.0), and 10 ml of buffer C (same as buffer B, but pH 6.3). The anchor peptides were finally eluted with 2 ml of 0.5 M acetic acid. The eluate was divided into equal volumes, one of which was desalted on C-18 matrix prior to MALDI-MS, whereas the other was subjected to RP-HPLC purification. Preparation of anchor peptides from 4 liter of staphylococcal culture (5 × 1013 colony forming units) typically yielded 1 nM (2 µg) of purified compound. Because of the relative small yield of anchor peptides, we chose to analyze our compounds by the more sensitive MALDI-MS and ESI-MS rather than fast atom bombardment mass spectrometry. The latter method has been used extensively in the past for the study of bacterial peptidoglycans (11, 15-17).
MALDI-MSA C-18 matrix cartridge (Analtech) was prewashed
with 10 ml of CH3CN, followed by 10 ml of 0.1%
trifluoroacetic acid in water. One ml of the affinity chromatography
eluate was loaded on the cartridge, washed with 10 ml of 0.1%
trifluoroacetic acid, and eluted with 3 ml of 60% CH3CN.
The desalted anchor peptides were dried under vacuum and suspended in
20 µl of CH3CN:water:formic acid (50:50:0.1). 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
(-cyano-4-hydroxycinnamic acid, 2 mg/200 µl
CH3CN:water:trifluoroacetic acid (70:30:0.1)) and mass
measured using an external calibration.
For further purification of anchor peptides, the
affinity chromatography eluate was subjected to RP-HPLC on a C-18
column (2 × 250-mm, C18 Hypersil, Keystone Scientific). The
separation was carried out at 40 °C with a linear gradient from 99%
H2O (0.1% trifluoroacetic acid) to 99% CH3CN
(0.1% trifluoroacetic acid) in 90 min at a flow rate of 0.2 ml/min.
The elution of anchor peptides was monitored at 215 nm, and 1-min
fractions were collected. Vacuum dried fractions were generally
re-dissolved in 50 µl of water:CH3CN:formic acid
(50:50:0.1). A Perkin-Elmer Sciex API III triple quadrupole mass
spectrometer was tuned and calibrated by flow injection (10 µl/min)
of a mixture of PPG 425, 1000, and 2000 (3.3 × 105,
1 × 10
4, and 2 × 10
4
M, respectively) in water:methanol (1:1) containing 2 mM ammonium formate and 0.1% CH3CN.
Calibration across the m/z range 10-2400 was effected by
multiple ion monitoring of eight PPG solution signals (typically the
singly charged ions at m/z 58.99, 326.25, 906.67, 1254.92, 1545.13, 1863.34, and 2010.47 and the doubly charged ion at
m/z 520.4). The ion spray voltage operated at 4.5 kV using
hydrocarbon-depleted air for the spray nebulization ("zero" grade
air, 40 p.s.i., 0.6 liter/min), and spectra were generated with a
curtain gas produced from the vapors of liquid nitrogen. Samples (10 µl) were introduced into the ionization source by flow injection.
ESI-MS spectra were obtained at instrument conditions sufficient to
resolve the isotopes of the PPG/NH4+
singly charged ion at m/z 906 with 40% valley, an orifice
voltage of 60, and step size during data collection of 0.3 Da.
Deconvolution of the series of multiply charged ions and
calculation of peptide or protein molecular weight was achieved with
the HypermassTM computer program. Daughter ion spectra were obtained
using degraded mass resolution to improve the sensitivity of detection,
and a step size of 1 Da was used for data collection. Under these
conditions, the isotopes of the PPG/NH4+
singly charged ion at m/z 906 were not resolved from one
another.
E. coli
BL1(DE3) pLysS, pHTT2 was grown overnight in LB medium containing
chloramphenicol (30 µg/ml) and kanamycin (50 µg/ml). The culture
was diluted 1:50 into 500 ml of the same medium and incubated with 250 rpm shaking at 37 °C. When the A600 reached 0.2, T7 polymerase was induced by the addition of 1 mM
isopropyl-1-thio--D-galactopyranoside and the culture
was incubated for another 4 h. Cells were harvested by
centrifugation at 8000 × g for 15 min and suspended in
20 ml of F
buffer (50 mM Tris-HCl, 10 mM EDTA,
20% sucrose, 1 mM dithiothreitol, pH 8.0). Lysozyme
(Sigma) was added to a final concentration of 0.1 mg/ml, and the
suspension was sonicated on ice with 30 10-s pulses over 30 min. The
lysate was centrifuged for 10 min at 14,000 × g to
collect inclusion bodies harboring staphylococcal amidase. The pellets
were extracted with 30 ml of a solution containing CH3CN:isopropanol:water (3:3:14) and centrifuged for 10 min
at 14,000 × g, and the supernatant was discarded. The
pellets were suspended in 20 ml of buffer B, and insoluble material was
removed by centrifugation for 10 min at 14,000 × g.
Proteins soluble in the supernatant were filtered through a 0.2-µm
pore size membrane prior to affinity chromatography. A 2.5-ml
Ni-NTA-Sepharose column was pre-equilibrated with 15 ml of buffer B,
loaded with the filtrate, washed first with 30 ml of buffer B, and then
with 30 ml of buffer C. Amidase was eluted with 5 ml of buffer E (same
as buffer B, but pH 4.5). The purity and molecular mass of
staphylococcal amidase were determined by 12% SDS-PAGE analysis, which
was in agreement with the predicted mass. As judged on Coomassie or
silver-stained SDS-PAGE, the amidase preparation was 100% pure. The
eluate was dialyzed against 1 liter of 50 mM Tris-HCl, 1 M urea, 0.005% Tween 80, pH 7.5, for 24 h at 4 °C
without stirring, followed by a second dialysis against the same buffer
without urea for 16 h at 4 °C and slow stirring.
An experimental scheme was
developed that allowed for the selective purification of COOH-terminal
peptides harboring the cell wall anchor structure of surface proteins
(Fig. 1). Seb-MH6-Cws is a
recombinant protein consisting of the NH2-terminal signal (leader) peptide and mature region of staphylococcal enterotoxin B (18)
with a fused COOH-terminal cell wall sorting signal of protein A (5).
At the fusion joint between these two sequences, a methionine (M)
residue followed by six histidines (H6) was inserted. This
engineered insertion serves as an affinity tag for the rapid purification of solubilized surface protein by chromatography on
nickel-Sepharose. Purified proteins were cleaved with CnBr at their
methionine residues, thereby separating the COOH-terminal anchor
peptides from the remainder of the polypeptide chains. The anchor
peptides were further purified by a second round of affinity
chromatography on nickel-Sepharose and analyzed by MALDI-MS.
Structure of Lysostaphin-released Anchor Peptides
Digestion
of the staphylococcal cell wall with lysostaphin resulted in the
solubilization of Seb-MH6-Cws, and most of these polypeptides migrated uniformly on SDS-PAGE with a mass of
approximately 30,000 Da (Fig. 1). Some of the lysostaphin-released
material appeared as distinct species with higher molecular mass, which is likely caused by an incomplete digestion of the peptidoglycan. After
CnBr cleavage of Seb-MH6-Cws, three major anchor peptides were identified by MALDI-MS with m/z 1726, 3836, and 3867 in
addition to ions with lesser intensity (m/z 1668, 1759, and
3777; Fig. 2A). To purify
individual anchor peptides, we subjected the affinity-purified sample
to RP-HPLC on C-18 resin (Fig. 2B). Two major absorption peaks eluted at 24-29% and at 34-37% CH3CN,
respectively. The material that eluted at 24-29% CH3CN
consisted of two different peptides with an observed average compound
mass of 1665.42 and 1722.58, as measured by ESI-MS (Fig.
2C). Amino acid analysis and Edman degradation confirmed the
structure of these two peptides to be
NH2-HHHHHHAQALPET-Gly-Gly-(Gly)-COOH (calculated mass
1665.76 and 1722.81), indicating that they differed by the addition
of a COOH-terminal glycine residue (mass 57) (Table
I). (To facilitate identification of
residues within anchor peptides, all amino acids specified by the
mRNA coding sequence are printed in the single-letter code, whereas
linked cell wall residues are indicated in the three-letter code.) This
result confirmed our previous observation that COOH-terminal anchor
peptides can be liberated from the staphylococcal cell wall by the
cleavage with lysostaphin between the second and the third glycyl
residue of the pentaglycine cross-bridge (9).
|
The anchor peptides that eluted from the RP-HPLC at 34-37% CH3CN were ESI-MS measured to be peptides with masses of 3777, 3830, and 3858 (data not shown). Edman degradation and amino acid analysis revealed that these compounds consisted of COOH-terminal anchor peptides with additional 16 upstream residues of Seb sequence (NH2-VDSKDVKIEVYLTTKKGTMHHHHHHAQALPET-Gly-Gly-(Gly)-COOH). The observed compound masses can thus be explained as anchor peptides with two and three glycyl residues linked to the threonyl of the LPXTG motif, similar to the peptides described above. This result indicated that the CnBr cleavage at the engineered methionine-histidine site occurred with reduced efficiency (50-60%) as compared with the cleavage at other methionyl residues (data not shown).
Structure of Amidase-released Anchor PeptidesMuralytic
amidases cleave the bacterial wall peptides at their amide linkage
between the amino of L-alanyl and the carboxyl of
N-acetylmuramic acid (19, 20). Since no such enzyme was available to us, we cloned, overexpressed, and purified the amidase gene of staphylococcal phage 11 (21) in E. coli.2 Amidase cleavage
of the staphylococcal peptidoglycan solubilized Seb-MH6-Cws
as two distinct species, both of which migrated more slowly on SDS-PAGE
than the lysostaphin-released counterpart (Fig. 1). Edman degradation
of amidase-solubilized Seb-MH6-Cws (without prior CnBr
cleavage) revealed the expected NH2-terminal sequence (ESQPDP). In contrast to the sequencing data obtained for the lysostaphin-released species, we observed a half-molar amount of
phenylthiohydantoin-coupled alanine in addition to the
NH2-terminal glutamic acid after the first cleavage cycle
(data not shown). This result indicated that some of the
amidase-released species harbored two amino-terminal residues, glutamic
acid and alanine, consistent with a branched peptide structure and with
the cleavage of the amide bond between L-alanyl and
N-acetylmuramic acid by the added amidase (22).
After CnBr cleavage and affinity purification, the amidase-released
anchor peptides were subjected to MALDI-MS (Fig.
3A). Five major ions with
m/z 2235, 2717, 2758, 4335, and 4857 were identified and
these compounds were further purified by RP-HPLC (Fig. 3B).
An average ESI-MS mass of 2235.05 was observed for the anchor peptide
eluting at 26% CH3CN (Fig. 3C). When subjected to Edman degradation, this compound yielded the sequence
NH2-A/HHHHHHAQALPET-Gly-Gly-Gly-Gly-Gly-COOH and two
phenylthiohydantoin-coupled residues, histidine and alanine, were
identified at equimolar concentrations during cycle 1 (Table I). This
result is consistent with the structure of a branched peptide
consisting of surface protein linked via the pentaglycine cross-bridge
to the -amino of lysine in the wall peptide and a calculated average
mass of 2235.35 as shown in Fig. 4. An
NH2-terminal alanyl of the wall peptide, liberated via
amidase cleavage, is likely linked to D-isoglutamine and
presumably no further amino acid was released from the wall
peptide due to the nature of its iso-peptide bond (23) (see Fig. 4 for
a structural model). The amino acid analysis was consistent with the
presented structure (Table II).
|
To characterize the entire structure of the compound with mass 2235, we
performed CID of the triply charged parent ion (m/z 746) in
an MS/MS experiment. The resulting daughter ion spectrum was examined
for the presence of breakdown products (15, 24, 25) and compared with
our structural model (Fig. 4). The structure of the branched peptide
was revealed by the presence of the daughter ions at m/z
201, 258, 342, 399, and 982. The ion at m/z 201 was interpreted to be Ala--Gln. The observation that no glutamine was
released by Edman degradation during cycle two suggests that the two
amino acids are linked by the characteristic isopeptide bond. The ion
at m/z 399 comprises the entire stem peptide
(NH2-Ala-
-Gln-Lys-Ala-COOH), whereas the ion at
m/z 342 represented the loss of Ala from this structure
(NH2-Ala-
-Gln-Lys-COOH). Evidence for the bond between the fifth glycyl and the
-amino of lysyl was provided by the fact
that Edman degradation during cycles 19 and higher (i.e. after the fifth glycyl) did not release phenylthiohydantoin-coupled amino acids (Table I) and by the presence of the ions at m/z 258 (NH2-Lys-(NH2-Gly)-Ala-COOH) and 982 (NH2-Lys(NH2-H6AQALPET-Gly5)-COOH), which comprise this bond. A summary of the observed daughter ions and
their interpretation is presented in Table
III.
|
The anchor peptide with an observed ESI-MS average mass of 2713.48 eluted at 28% CH3CN and upon Edman degradation also
yielded a sequence of
NH2-HHHHHHAQALPET-Gly-Gly-Gly-Gly-Gly-COOH. However, in
contrast to the compound with a mass of 2235, no release of phenylthiohydantoin-coupled alanine was observed during the first cycle. These observations are consistent with the linkage of surface protein to a cell wall tetrapeptide that is amide-linked to
N-acetylmuramyl-(1-4)-N-acetylglucosamine (19, 26) (calculated average compound mass 2713.83). This was a
surprising finding because no N-acetylglucosaminidase or N-acetylmuramidase had been added that would have released
surface proteins with linked disaccharide. One explanation for this
result may be that the well known endogenous
N-acetylglucosaminidase of S. aureus (20, 27) had
released surface proteins during the prolonged incubation with amidase.
Nevertheless, the availability of the compound with mass 2713 provided
us with an opportunity to study the linkage of anchor peptides to the
glycan strands of the staphylococcal cell wall.
The structure of the 2713-Da anchor fragment was characterized by MS/MS
using the parent ion at m/z 905. The resulting daughter ion
spectrum was compared with our model (Fig.
5). The presence of
N-acetylhexosamines in this structure was revealed by the
characteristic ions at m/z 187, 168, and 138 (25).
N-Acetylhexosamine was also indicated as the compound with
mass 203, and the resulting branched peptide with linked
N-acetylmuramic acid generated doubly and triply charged
ions at m/z 1246 and 832, respectively
(MurNAc-L-Ala-D--Gln-L-Lys(NH2-H6AQALPET-Gly5)-D-Ala-COOH). The linkage of L-alanine in the stem peptide to the lactyl
moiety of N-acetylmuramic acid could be demonstrated with
the ions at m/z 1154 and 770 (lactyl-Ala-
-Gln-Lys(NH2-H6AQALPET-Gly5)-Ala-COOH). Several other ions confirmed the sequence of the stem peptide to be
identical to that of the amidase-released anchor peptide (Fig. 5).
Although the data identify the presence of
N-acetylhexosamine and N-acetylmuramic acid, they
cannot determine the sequence of these two sugars in the
disaccharide-linked anchor peptide. If this sample resulted from the
solubilization by a glucosaminidase, its structure would
likely be
MurNAc-(Ala-
-Gln-Lys(NH2-H6AQALPET-Gly5)-Ala-COOH)-(
1-4)-GlcNAc. All daughter ions observed in this MS/MS experiment are listed in Table
IV for a comparison of the observed and
calculated masses with the proposed structures.
|
Average compound masses of 4342, 4821, and 4849 were measured by ESI-MS for amidase-released peptides eluting at 36-38% CH3CN (Fig. 3). Similar to the observations made with the partial CnBr cleavage of lysostaphin-solubilized Seb-MH6-Cws, these mass measurements are consistent with peptides harboring an additional 16 upstream residues of Seb fused to the amidase-released anchor structures as a result of an uncleaved methionine adjacent to the engineered histidine tag.
Muramidase Release of Surface ProteinsMuramidase cleaves the
staphylococcal cell wall at the glycan chains (20), i.e. the
1-4 glycosidic bond between N-acetylmuramic acid and
N-acetylglucosamine, and released Seb-MH6-Cws as
a spectrum of species with increasing mass due to linked peptidoglycan
(Fig. 1). NH2-terminal sequencing of the
muramidase-solubilized Seb-MH6-Cws also yielded two
phenylthiohydantoin-coupled residues after the first cycle: glutamic
acid and alanine. The presence of phenylthiohydantoin-alanine can be
explained by the alkali release of ester-linked D-alanine from cell wall teichoic acid (28, 29) during the sequencing cycle.
Because D-alanyl-decorated polyribitol teichoic acids are linked to N-acetylmuramic acid within the glycan chains,
muramidase digestion of the staphylococcal peptidoglycan can solubilize
surface proteins with attached teichoic acids (30, 31). Although
several attempts were made to purify large quantities of the
muramidase-released, CnBr-cleaved anchor species, we failed to obtain
sufficient material for a more detailed chemical, ESI-MS, or MS/MS
analysis.
The cell wall anchor structure of surface proteins in S. aureus has been revealed by a combination of mass spectrometry and molecular biology techniques. Surface proteins are tethered to the cell
wall via an amide bond between the carboxyl of their COOH-terminal
threonine and the amino of the pentaglycine cross-bridge, which is
attached to the -amino of lysyl within a peptidoglycan tetrapeptide.
This branched anchor peptide is linked to N-acetylmuramic acid, thereby attaching surface proteins to the glycan chains of the
staphylococcal cell wall. We think that surface proteins may be linked
to a precursor molecule of peptidoglycan synthesis rather than to the
mature, assembled cell wall, because the staphylococcal peptidoglycan
is highly cross-linked and has very few free pentaglycine amino groups
(11, 23, 32-34). During the biosynthesis of bacterial peptidoglycans,
a membrane-bound disaccharide-precursor molecule (undecaprenyl-PO4-PO4-MurNAc-(L-Ala-D-
-Gln-L-Lys-(NH2-Gly5)-D-Ala-D-Ala-COOH)-(
1-4)-GlcNAc) is assembled in the cytoplasm (35, 36) and translocated across the
membrane to serve as a substrate for transglycosylation and transpeptidation reactions (37, 38) (Fig.
6). Such a precursor molecule could also
serve as the peptidoglycan substrate for the cell wall sorting reaction
of surface proteins. To characterize a cell wall sorting intermediate
consisting of surface protein with linked peptidoglycan precursor, we
have begun to purify and characterize such a compound from
staphylococcal protoplasts.3
The structural elucidation of such an intermediate may thus resolve the
nature of the peptidoglycan substrate for the cell wall sorting reaction.
Digestion of the staphylococcal peptidoglycan with either lysostaphin or amidase solubilized surface proteins as species with uniform mass. This can be explained by our model of the cell wall anchor structure (Fig. 6). Both lysostaphin and amidase cleaved the branched anchor peptide at sites that are proximal to its linkage with the glycan strands. Hence, a single enzymatic cut completely solubilized surface proteins from the otherwise tightly cross-linked cell wall of S. aureus. In contrast, muramidase digestion released surface proteins as a large spectrum of fragments with increasing mass due to linked peptidoglycan fragments. If the anchor peptides were randomly linked to N-acetylmuramyl, i.e. anywhere along the glycan chains of S. aureus, the release of surface proteins would require no more than two muramidase cuts at neighboring sites. In such a scenario, one would expect surface proteins also to be solubilized as a uniform species rather than the observed spectrum of fragments. An alternative possibility, which would be consistent with the data presented here, is that surface proteins may be tethered to distinct sites of the glycan chains that cannot be directly cleaved by muramidase. Although an N-acetylmuramic acid-linked anchor peptide has been characterized in this report, its flanking sugar moieties and relative position within the glycan chains have not yet been identified.
The disaccharide containing compound with mass 2713 was found in the amidase but not in the muramidase-digested sample; however, the source of this molecule could not be determined. One explanation for its occurrence is that the 2713-Da compound may have been solubilized from the staphylococcal peptidoglycan by a specific enzymatic activity, for example N-acetylglucosaminidase (20, 27). Alternatively, this compound may represent a peptidoglycan (disaccharide) precursor molecule that is linked to surface protein. Such precursors are generally attached to lipid; however, the trifluoroacetic acid treatment of the amidase-digested sample could have removed a phosphodiester-linked undecaprenyl decoration.
Because all cell wall anchor peptides of surface proteins identified here consist of unsubstituted tetrapeptides, we propose that surface proteins linked to a peptidoglycan precursor molecule could be incorporated into the cell wall by a transglycosylation reaction (39) (Fig. 6). The linked anchor structure may subsequently be cleaved at its terminal D-alanyl-D-alanine by a carboxypeptidase activity (38) to generate the mature anchor peptide unit. This model takes into account that all cell wall peptides are thought to first be synthesized as a pentapeptide precursor by the addition of D-alanyl-D-alanine to the muramyl-linked tripeptide in the bacterial cytoplasm (40). After assembly into the cell wall, the pentapeptides may be either cross-linked to generate substituted tetrapeptides or cleaved by carboxypeptidases, thereby yielding free (unsubstituted) tetrapeptides (38). Nevertheless, it also seems possible that pentapeptide-peptidoglycan precursor molecules may be cleaved by carboxypeptidases and that the resulting tetrapeptide precursors serve as substrates for the sorting reaction of surface proteins. Our current data cannot distinguish between these possibilities, but they may be addressed in the future by combining our mass spectrometric analysis of anchor structures with the antibiotic inhibition of peptidoglycan precursor synthesis.
A notable feature of the cell wall anchor structure of surface proteins in S. aureus is its lack of cross-linking with neighboring wall peptides. The regulated release of proteins from the bacterial surface has been proposed for several different Gram-positive organisms (41, 42). Such release of surface proteins is thought to be important during the pathogenesis of bacterial infections as a mechanism for either the escape from the host's immune surveillance or the selective alteration of bacterial surface properties. An extensive cross-linking between anchor peptides and the neighboring cell wall would require a multitude of muralytic enzymes to release surface proteins from the peptidoglycan. The demonstrated anchor structure, however, can easily be released by an enzymatic activity that selectively cleaves the glycan strands of the cell wall without an extensive solubilization of the bacterial peptidoglycan. We thus predict that surface proteins released into bacterial culture supernatants harbor COOH-terminally linked cell wall tetrapeptides with attached glycan fragments.
Murein (Braun's) lipoprotein is a structural element of the envelope
of Gram-negative bacteria (43, 44). In contrast to Gram-positive
surface proteins, which display their free NH2-terminal domains on the bacterial surface, an NH2-terminal lipid
modification inserts Braun's lipoprotein into the inner leaflet of the
outer membrane (the N-terminal cysteine contains thioether-linked,
2,3-palmitoylated glycerol as well as N-acyl palmitoyl)
(45). The short polypeptides are thought to be assembled into a
trimeric structure (46) and about one third of all murein lipoprotein
is linked to the peptidoglycan (47, 48). This is accomplished via an
amide linkage between the -amino of the lipoprotein's COOH-terminal
lysine and the carboxyl of m-diaminopimelic acid in the wall
peptide (17, 48). When engineered to harbor a COOH-terminal sorting
signal, lipoproteins can also be linked to the cell wall of
Gram-positive organisms; however, such polypeptides have not yet been
identified to occur naturally in bacterial envelopes (49).
An important conclusion that can be drawn from the cell wall anchor
structure is that the amide bond exchange reaction of surface protein
sorting is not sensitive to the -lactam inhibition of the classical
transpeptidation reaction catalyzed by penicillin-binding proteins
(50). We tested this prediction and found that neither methicillin nor
penicillin G could inhibit surface protein anchoring to the
staphylococcal cell wall (data not shown). The sorting reaction may
thus represent a novel target for antibiotic therapy if its amide bond
exchange reaction proves to be essential for either viability or
pathogenesis.
We are indebted to Nghia Truong for the purification of staphylococcal amidase. We also thank Simon Daefler (Rockefeller University), Richard Stevens (UCLA), and members of the Schneewind laboratory for critical review of this work. We also thank four reviewers for their comments on this manuscript. Protein sequencing was performed by Dr. Audree Fowler at the UCLA Microsequencing Facility, which is partially supported by Grant CA 16042-20 from the National Cancer Institute to the Jonsson Comprehensive Cancer Center.