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J. Biol. Chem., Vol. 278, Issue 34, 31521-31528, August 22, 2003
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From the
Unité des Neisseria and Centre
National de Référence des Méningocoques,
Plate-forme de Protéomique, and
¶Unité de Pathogénie
Bactérienne des Muqueuses, Institut Pasteur, 28 rue du Dr Roux, 75724
Paris Cedex 15, France
Received for publication, May 7, 2003 , and in revised form, June 6, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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-lactam
antibiotics. Indeed, few N. meningitidis strains carry
plasmid-encoded -lactamases. However, meningococcal strains with reduced
susceptibility to penicillin due to a decreased affinity of the
penicillin-binding protein 2
(PBP2)1 associated
with mosaic penA genes
(2,
3) have been reported worldwide
(4). The penA gene
codes for the PBP2, which is homologous to high molecular weight class B PBPs
and is closely related to Escherichia coli PBP3 and Streptococcus
pneumoniae PBP2x. Therefore, reduced susceptibility to penicillin G in
N. meningitidis is expected to be associated with changes in the
peptidoglycan structure, as previously seen in S. pneumoniae
(5), Haemophilus
influenzae (6), and the
related human pathogen Neisseria gonorrhoeae
(7).
Several virulence factors are involved in bacteria-host interactions and
are responsible for preventing bacterial clearance and allowing colonization.
However, little is known about the role of peptidoglycan in meningococcal
pathogenesis, partly because the structure of the N. meningitidis
peptidoglycan is not known. Indeed, besides the central roles of peptidoglycan
in bacterial growth, shape maintenance and cell division, there is an
increasing amount of evidence suggesting that its degradation products play a
major biological role in the innate immune response of the host. The tracheal
cytotoxin from Bordetella pertussis is a peptidoglycan degradation
product, GlcNAc-(-1,4)-1,6-anhydromuramyl-L
-alanyl-D-glutamyl-meso-diaminopimelate-D-alanine
(8). The same product from
N. gonorrhoeae is cytotoxic for human fallopian tube epithelial cells
(9). Furthermore, several
different muropeptides induce somnogenic, pyrogenic, and hematological effects
(10,
11), which are directly
relevant to the pathogenesis of N. meningitidis. Accordingly, N.
gonorrhoeae has been used as a model organism to study the peptidoglycan
of Neisseria spp., with particular emphasis on its structure and the
biological activity of its degradation products
(9,
12–14).
However, the structural analysis of muropeptide species was only partial.
Interestingly, N. meningitidis is cytopathic to human nasopharyngeal
epithelial cells, which constitute the first line of host defense
(15).
The importance of penicillin resistance in meningococcal infection treatment failure and the potential biological properties of its peptidoglycan led us to perform a detailed structural analysis of the muropeptide composition of N. meningitidis using reverse-phase high pressure liquid chromatography (HPLC), matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and post source decay analysis (MALDI-PSD). In addition, we compared the peptidoglycan composition of a series of clinical meningococcal strains with different levels of reduced susceptibility to penicillin G.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Peptidoglycan Preparation—Meningococci were inoculated in 500 ml of GCB liquid and incubated at 37 °C in a 5% CO2 atmosphere until the optical density at 600 nm reached 0.6–0.7. Peptidoglycan was isolated by an adapted version of the method developed for E. coli (19). Cultures were swirled in ice-alcohol baths and then centrifuged at 8,000 x g for 30 min at 4 °C. The cells were washed once with ice-cold 20 mM sodium acetate (pH 5.0) and resuspended in 10 ml of the same buffer. The cells were added dropwise to 10 ml of boiling 4% SDS buffered with 20 mM sodium acetate (pH 5.0) and boiled for a further 30 min. After cooling to room temperature overnight, the SDS-insoluble material was collected by centrifugation at 100,000 x g for 30 min. The pellet was washed 5 times with warm water and resuspended in 5 ml of 100 mM Tris-HCl (pH 7.5) and 10 mM NaCl. DNase and RNase (Sigma) were added at a concentration of 10 and 50 µg/ml, respectively, for 2 h at 37 °C. The peptidoglycan-associated proteins were removed by incubation with 50 µg/ml of proteinase K (Invitrogen) overnight at 37 °C. The SDS-insoluble material was reextracted with boiling 1% SDS for 15 min. The material was collected and washed by centrifugation four times as described above. The peptidoglycan pellet was resuspended in distilled water, lyophilized, and stored at –80 °C. The lyophilized peptidoglycan was resuspended in 20 mM sodium phosphate buffer (pH 4.8) and completely digested with 25 µg/200 µl of muramidase from Streptomyces globisporus (Sigma) for 18 h at 37 °C. The enzyme reaction was stopped by boiling the sample for 3 min, and insoluble contaminations were removed by centrifugation. The digested peptidoglycan was mixed with an equal volume of 0.5 M sodium borate buffer (pH 8.0) in which sodium borohydride (10 mg/ml) had been freshly dissolved for 20 min exactly at room temperature. Excess borohydride was destroyed with 20% phosphoric acid, the pH of the samples was adjusted to 4.0, and the samples were filtered (0.2-µm pore size).
Reverse-phase HPLC Analysis and Desalting of Muropeptides—For HPLC analysis, we used a linear gradient from 50 mM sodium phosphate buffer (pH 4.33), with 1 µg/ml sodium azide to compensate for base-line drift, to 50 mM sodium phosphate buffer (pH 5.1) containing 15% methanol for 120 min on a Hypersil ODS column (4.6 x 250 nm; 5-µm particles; ThermoHypersil-Keystone) at room temperature using a flow rate of 0.5 ml/min. UV detection was carried out at 205 nm. Isolated muropeptides were desalted by HPLC on the same column using a 0–50% acetonitrile gradient in 0.05% trifluoroacetic acid, water. During the desalting step, some HPLC peaks were further separated in two muropeptide species that were designated a and b. The desalted muropeptides were lyophilized and stored at –20 °C.
Mass Spectrometry—Desalted muropeptides were analyzed by
MALDI-MS using 5-chloro-2-mercaptobenzothiazole as the MALDI matrix as
previously described (20).
Alternatively, samples were desalted a second time directly on the sample
plate and analyzed using 
-cyano-4-hydroxycinnamic acid as the MALDI
matrix. Briefly, muropeptides were dissolved in 1 µl of
acetonitrile/H2O/trifluoroacetic acid (70:30: 0.1%). Subsequently,
0.5 µl of the sample and 0.5 µl of a 6-fold dilution of
-cyano-4-hydroxycinnamic acid were loaded onto the MALDI sample plate
(96 x 2 Teflon plate, Applied Biosystems Inc., Framingham, MA). Samples
were air-dried at room temperature and washed for 30 s with 0.7 µl of cold
water (0.1% trifluoroacetic acid). After removing the liquid with a pipette,
samples were recrystallized with 0.5 µl of
acetonitrile/H2O/trifluoroacetic acid (70:30:0.1%). MALDI-MS
measurements were taken on a Voyager-DE STR spectrometer (Applied Biosystems
Inc., Framingham, MA) equipped with a nitrogen laser (337 nm). The instrument
was operated in the delayed extraction mode and in the reflector mode.
Measurements were taken in the reflector mode with an acceleration voltage of
20 kV, 76% grid voltage, and a delay extraction of 150 ns. Each mass spectrum
was an average of 250–500 laser shots. Masses were measured using close
external calibration with a mixture of four peptides (des-Arg 1-bradykinin,
[M+H]+: 904.4681; angiotensin 1, [M+H]+: 1296.6853;
neurotensin, [M+H]+: 1672.9175; ACTH 1839, [M+H]+:
2465.1989). For PSD experiments, the timed ion selector was used to select the
[M+Na]+ value of the precursor ion.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The elution conditions used to separate the meningococcal muropeptides
differed from those described by Glauner
(19). Better separation was
achieved if the elution step was carried out at room temperature (25 °C)
rather than at 55 °C. Consequently, it was not possible to assign
structures unambiguously by directly comparing the HPLC muropeptide profiles
of E. coli and N. meningitidis. Therefore, several major
muropeptide peaks were further analyzed by MALDI-PSD.
Fig.
2,Fig. 2 shows the
MALDI-PSD spectra of muropeptides 8a (Fig.
2A), 12a (Fig.
2B), and 16 (Fig.
2C). The molecular masses of these muropeptides
([M+Na]+ of 1887.5331 m/z, 1929.8141
m/z, and 2853.2243 m/z, respectively) are
consistent with several alternative structures. Indeed, muropeptide 8a may be
either GlcNAc-MurNAc-tetra-tetra-GlcNAc-MurNAc or
GlcNAc-Mur-NAc-tetra-(N(
)-Ala)-tri-GlcNAc-MurNAc, and
muropeptide 12a corresponded to the O-acetylated derivative of
muropeptide 8a. The MALDI-PSD spectra of both of these muropeptides generated
a 964.3584 m/z fragment that corresponds to
GlcNAc-MurNAc-tetrapeptide, suggesting that both molecules are completely
symmetrical. This is also suggested by the absence of a
GlcNAc-MurNAc-tripeptide (893.3604 m/z) or a
GlcNAc-MurNAc-pentapeptide (1035.4346 m/z) in the MALDI-PSD
spectra of either muropeptide. Therefore, the muropeptide 8a and its
O-acetylated derivative (muropeptide 12a) correspond to
GlcNAc-MurNAc-tetra-tetra-GlcNAc-MurNAc and not to
GlcNAc-MurNAc-tetra-(N()-Ala)-tri-GlcNAc-MurNAc resulting from
L,D-endopeptidase activity. Furthermore, the MALDI-PSD
spectra of muropeptide 12a (Fig.
2B) revealed a small 1859.1779 m/z
fragment, corresponding to the parent ion minus an alanine residue. In the
positive ion mode, muropeptides fragment preferentially from the C-terminal
end as b ions (20). Therefore,
the alanine residue is located at the C terminus, which is consistent with the
structure of GlcNAc-MurNAc-tetra-tetra-GlcNAc-MurNAc. Finally, the MALDI-PSD
spectra of muropeptide 16 (Fig.
2C) also indicated that this muropeptide is completely
symmetrical (absence of 893.3604 m/z and 1958.8322
m/z ion fragments). Additionally, the presence of two ion
fragments of 1636.7225 m/z and 1841.3890
m/z, corresponding to fragments 1725.6344
m/z and 1929.7173 m/z, respectively, minus
an alanine residue (Fig.
2C), again suggest that this molecule contains an alanine
residue at its C terminus. We concluded that muropeptide 16 corresponds to the
trimeric muropeptide [GlcNAc-MurNAc-tetra]3 carrying an
O-acetyl group.
Given the structural assignment of these three major muropeptides, the molecular masses of the other muropeptides and their relative retention times compared with these major muropeptides, and the temperature-dependent nature of the elution profile according to structure (19), we were able to assign structures to all muropeptides species unambiguously (Table II).
Interestingly, some differences were observed between the N. meningitidis and the related pathogen N. gonorrhoeae chromatogram profiles (7, 12). We found no evidence of muropeptides carrying glycine residues in N. meningitidis as observed in gonococci (12, 13). Furthermore, the extent of O-acetylation of dimers and trimers differed in meningococci and in gonococci (21). We were unable to estimate the percentage of anhydromuropeptides and, therefore, to calculate the average length of meningococcal glycan chains. In fact, most anhydro-muropeptides co-eluted with other muropeptides that constituted the major fraction of the HPLC peaks (for example, muropeptides 8b and 12b) (Fig. 1 and Table II). Finally, contrary to the report by Dougherty (12), indicating the presence of tetramers in gonococcal peptidoglycan similarly to in E. coli (22), we found no evidence for such structures in meningococci, with trimers being the most polymerized muropeptide species identified.
Degree of Cross-linking and O-Acetylation—This analysis permitted us to assess the degree of cross-linking and of disaccharide O-acetylation. Our calculations took into account the presence of muropeptide mixtures in some peaks. Two scenarios were encountered, (i) one single muropeptide was found to be the major species in a given peak, and for the purpose of our calculations, we decided to consider it to be the only structure, or (ii) several of the different muropeptides in a single peak were present in significant amounts. In such cases, the peak area was allocated proportionally to each structure. We calculated the degree of cross-linking according to Glauner (19) and found a percentage of around 38.5% for strain LNP8013, consistent with that determined for other Gram-negative bacteria (6, 22–24).
The degree of disaccharide O-acetylation was calculated as
follows: 
(OAc monomers + di-OAc dimers + tri-OAc trimers + 
OAc dimers + 
di-OAc trimers + 
OAc trimers)/
muropeptides. Note that we used the peak area and not the molar percentage of
each muropeptide for our calculation, meaning that we took the molar
proportion of disaccharides directly into account. We estimated that one in
every three disaccharides is O-acetylated, which is similar to
previous estimates for gonococci
(25).
Our data also enabled us to estimate an empirical formula to calculate the shift in retention time of a muropeptide modified by O-acetylation. Each O-acetylation increases the retention time by 25.18 min divided by the number of disaccharide units of a given muropeptide. For example, the retention time of a dimer with one O-acetylated disaccharide will be 13 min longer than that of the non-O-acetylated dimer, whereas if both disaccharides are O-acetylated it will be 26 min longer. Based on this empirical formula, we were able to compare observed and calculated retention times, resulting in a S.D. of ± 0.89 min (Table III). The calculated retention times were close to the observed ones in all cases except for the major monomer GlcNAc-MurNAc tetrapeptide.
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Comparison of the Muropeptide Composition of Different Meningococcal Strains—Table IV summarizes the results obtained for two additional penicillin-susceptible and seven penicillin-intermediate meningococcal strains harboring mosaic penA genes (Table I). Penicillin G MICs ranged from 0.006 to 1 µg/ml (Table I). Comparison of the HPLC profiles of the different strains did not reveal any major differences. No new peaks were observed in the penicillin-intermediate strains compared with the three susceptible strains, indicating that the resistance probably did not arise due to a shift from D,D-transpeptidation to L,D-transpeptidation as observed in enterococci (26, 27). Variations in the degree of cross-linking and O-acetylation appeared to be associated with the genetic background rather than with the resistance phenotype. The percentage of cross-linking was between 37.2 and 42.5%, whereas the percentage of O-acetylation per disaccharide was between 31.6 and 40.3%. Furthermore, the amount of muropeptides carrying dipeptide or tripeptide chains appeared to differ according to the genetic background of each strain, and no correlation was observed with resistance. These findings might reflect differences in the amount of dipeptides and tripeptides that accumulate in the peptidoglycan cytoplasmic precursor pool of each strain. However, given the percentage of muropeptides carrying a pentapeptide chain, we observed a gross correlation between penicillin G MIC of the strain and the accumulation of pentapeptide chains in the meningococcal peptidoglycan (Table IV and Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Most of what was known about the structure of Neisseria spp. peptidoglycans was derived from data obtained for the closely related pathogen N. gonorrhoeae (7, 12, 13). However, these studies were partial, restricted to the major muropeptide components and failed to determine the extent of muropeptide O-acetylation. Our MALDI-MS analysis could detect fentomolar concentrations of the different muropeptides. This allowed us to identify 14 different O-acetylated muropeptides. Furthermore, besides the GlcNAc-MurNAc tetrapeptide (and some traces of the disaccharide pentapeptide), we found that mainly dimers and trimers were O-acetylated (Table II). We found approximately the same diversity of monomeric structures as Martin et al. (13), with the exception of muropeptides carrying glycine residues replacing the D-alanine residues on the peptide backbone. These muropeptides were also found by Garcia-Bustos and Dougherty (21). The presence of glycine has also been reported for other bacteria (22). However, this might just reflect different culture conditions as observed for Staphylococcus aureus (20).
The partial characterization of the muropeptide composition of the
gonococcal peptidoglycan, in particular of dimers and trimers, could not
exclude the presence of the A2pm-A2pm type of cross-bridges. Because these
cross-bridges are due to penicillin-insensitive
L,D-transpeptidation, we wanted to determine whether
meningococci and potentially other Neisseria spp. are able to
synthesize direct A2pm-A2pm bridges. These muropeptides were first described
in the peptidoglycan of E. coli
(19,
22) and have since been shown
to be associated with -lactam resistance in enterococci
(26). Meningococci cannot
contain major muropeptides with A2pm-A2pm type cross-bridges because they are
highly susceptible to penicillin and other -lactam antibiotics.
Furthermore, MALDI-PSD analysis of the major muropeptides 8a, 12a, and 16
indicated that these muropeptides consist of
[GlcNAc-MurNAc-tetra]n, where n = 2 or 3, which
excludes the presence of major L,D-endopeptidase
activity. Based on the relative retention times of other minor muropeptide
components (for example, muropeptides 7 and 17, a tetra-tri dimer and its
anhydro derivative, which had retention times of 64.8 and 94.7 min,
respectively), we can conclude that these muropeptides also have a
D-Ala-A2pm bridge. In fact, muropeptides 7 and 17 would have
displayed lower retention times than those observed
(19), in particular
muropeptide 17, which would have been expected to elute at about 85 min rather
than at 94.7 min. Taken together, we found no evidence for the presence of
A2pm-A2pm bridges in N. meningitidis.
Another particular feature of the N. meningitidis peptidoglycan is the absence of higher oligomers than trimers (22) and of muropeptides derived from covalently linked lipoproteins as observed in E. coli (32). Dougherty described the presence of tetramers in gonococci (12) after using coupled Sephadex G50-G25 columns to fractionate muropeptides. However, subsequent studies only detected trimers (21), in agreement with our analysis. Thus, Neisseria peptidoglycan consists of a maximum of two layers. In fact, despite the apparent complexity of the HPLC profile due to the extent of O-acetylation, the peptidoglycan of Neisseria has less muropeptide variants than that of E. coli. Our results also suggest that N. meningitidis does not require a covalent linkage of the outer membrane via lipoproteins for cell envelope integrity.
Finally, we compared the muropeptide profiles of several meningococcal
strains with different levels of susceptibility to penicillin G. Reduced
susceptibility to penicillin in N. meningitidis did not appear to be
correlated with any new muropeptides, thus excluding a switch in the nature of
the cross-bridge as a mechanistic basis for resistance, as observed in
pneumococci, staphylococci, and enterococci
(5,
26,
27,
33). Furthermore, we did not
observe any significant differences in the degree of cross-linking between the
strains, as is the case in -lactam-resistant S. aureus strains
(33–35).
In H. influenzae, non--lactamase-mediated antibiotic resistance
has been shown to be associated with an increase in tripeptides. This may
involve an increase in the activity of a
D,D-carboxypeptidase, which would create more
tripeptides in the final cell wall
(6). When we compared the
percentage of pentapeptide chains incorporated into the meningococcal
peptidoglycan layer, we observed a correlation between penicillin G MIC values
and the amount of muropeptides carrying an intact pentapeptide (monomers,
dimers, or trimers) (Table IV
and Fig. 3). However, the
variations in monomers, dimers, or trimers were heterogeneous. These results
might simply reflect the different genetic backgrounds of the studied strains.
This increase in pentapeptide chains suggests that the peptidoglycan of
meningococcal penicillin-intermediate strains is more inclined to de
novo peptidoglycan incorporation. This is a net advantage over the
susceptible strains, as our data suggest that meningococci do not possess
enzymes capable of performing L,D-transpeptidation. In
the presence of penicillin, penicillin-intermediate strains would have more
available sites to incorporate new peptidoglycan and to regenerate their
existing layer. Alternatively, increasing the accessibility of anchoring sites
in the existing peptidoglycan might make it possible for the existing
synthetic machinery to cope with the presence of penicillin by competing more
efficiently for the active site of PBPs.
Reduced susceptibility to penicillin in N. meningitidis has been shown to be associated with mosaic penA genes encoding altered PBP2s (2, 3). All penicillin-intermediate strains tested harbored an altered penA allele different from that of the penicillin-susceptible strains, as defined by restriction fragment length polymorphism analysis (Table I). This suggests that the changes observed in the peptidoglycan of penicillinintermediate strains are associated with alterations in PBP2s. The presence of pentapeptide chains is a hallmark of newly synthesized peptidoglycan that has not been processed by D,D-carboxypeptidases. This suggests a decrease in D,D-carboxypeptidase activity in penicillin-intermediate strains that has not been yet identified in N. meningitidis. In general, D,D-carboxypeptidases are encoded by low molecular weight PBPs (36). Indeed, the genome analysis of two meningococcal strains predict the existence of three homologs of these low molecular weight PBPs, including PBP3 (37, 38). PBP2 belongs to the high molecular weight class B PBPs and is, therefore, predicted to catalyze the D,D-transpeptidation of peptidoglycan lipid-linked precursors rather than to function as a D,D-carboxypeptidase. However, our results suggest that the meningocccal PBP2 may have D,D-carboxypeptidase activity. The increased number of pentapeptide chains as monomers could also reflect less efficient D,D-transpeptidase activity of altered PBP2s. Alternatively, it is possible that both D,D-transpeptidase and D,D-carboxypeptidase activities are reduced. However, we did not detect a marked decrease in the degree of cross-linking that may be due to the fact that we examined the steady state of peptidoglycan biosynthesis or that the decreased D,D-transpeptidase activity of altered PBP2s is compensated by the D,D-transpeptidase activity of the meningococcal PBP1, which belongs to the high molecular weight class A PBPs. Constructions of isogenic strains carrying wild-type and altered penA genes should make it possible to determine the role of PBP2 in the N. meningitidis peptidoglycan modifications (40).
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FOOTNOTES |
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|| Recipient of a postdoctoral fellowship from the Fundação para a Ciência e a Tecnologia, Portugal (SFRH/BPD/1567/2000). To whom correspondence should be addressed. Tel.: 33-1-40-61-32-73, Fax: 33-1-40-61-36-40; E-mail: bonecai{at}pasteur.fr.
1 The abbreviations used are: PBP, penicillin-binding protein; HPLC, high
pressure liquid chromatography; MALDI-MS, matrix-assisted laser desorption
ionization mass spectrometry; PSD, post source decay; GlcNAc,
N-acetylglucosamine; MurNAc, N-acetylmuramic acid; A2pm,
diaminopimelic acid; MIC, minimum inhibitory concentration.
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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