Fax: + 41 1 733 4659, Tel.: + 41 1 733 4642, E-mail: jennings@cytos.com Abbreviations: GNA, genome-derived Neisseria antigen; LPSS, lipo-polypeptide signal sequence; MipA, MltA-interact
Trang 1GNA33 from Neisseria meningitidis serogroup B encodes
a membrane-bound lytic transglycosylase (MltA)
Gary T Jennings1*, Silvana Savino1*, Elisa Marchetti1, Beatrice Arico`1, Thomas Kast2, Lucia Baldi1, Astrid Ursinus2, Joachim-Volker Ho¨ltje2, Robert A Nicholas3, Rino Rappuoli1and Guido Grandi1
1
I.R.I.S., Chiron S.p.A., Siena, Italy;2Max Planck Institute fur Entwicklungsbiologie, Abteilung Biochemie, Tubingen, Germany;
3
Department of Pharmacology, University of North Carolina at Chapel Hill, NC, USA
In a previous study, we used the genome of serogroup B
Meningococcusto identify novel vaccine candidates One of
these molecules, GNA33, is well conserved among
Men-ingococcusB strains, other Meningococcus serogroups and
Gonococcusand induces bactericidal antibodies as a result of
being a mimetic antigen of the PorA epitope P1.2 GNA33
encodes a 48-kDa lipoprotein that is 34.5% identical with
membrane-bound lytic transglycosylase A (MltA) from
Escherichia coli In this study, we expressed GNA33, i.e
MeningococcusMltA, as a lipoprotein in E coli The
lipo-protein nature of recombinant MltA was demonstrated by
incorporation of [3H]palmitate MltA lipoprotein was
purified to homogeneity from E coli membranes by
cation-exchange chromatography Muramidase activity was
con-firmed when MltA was shown to degrade insoluble murein sacculi and unsubstituted glycan strands HPLC analysis demonstrated the formation of 1,6-anhydrodisaccharide tripeptide and tetrapeptide reaction products, confirming that the protein is a lytic transglycosylase Optimal muramidase activity was observed at pH 5.5 and 37C and enhanced by Mg2+, Mn2+and Ca2+ The addition of Ni2+ and EDTA had no significant effect on activity, whereas
Zn2+ inhibited activity Triton X-100 stimulated activity 5.1-fold Affinity chromatography indicated that MltA interacts with penicillin-binding protein 2 from Meningo-coccusB, and, like MltA from E coli, may form part of a multienzyme complex
Neisseria meningitidis is a Gram-negative, capsulated
b-proteobacterium capable of causing severe meningitis
and septicemia with a fatality rate of 10% [2] The
complete 2 272 351-bp genomic sequence of Meningococcus
serogroup B (strain MC58) has been determined and used
by us to identify novel vaccine candidates against this
pathogenic organism [1,2] We amplified, cloned and
expressed in Escherichia coli selected ORFs encoding
teins with predicted surface exposure Recombinant
pro-teins were purified, immunized in mice, and the resultant
sera analysed by FACS, ELISA, and bactericidal assay
GNA33 was positive in all three analyses and highly
conserved (99.2± 0.7%) among 22 strains of
Meningococ-cusB, nine strains from Meningococcus serogroups A, C, Y,
X, Z, W135, and 95.8% conserved in Neisseria gonorrhoeae
[1] Further study revealed that GNA33 elicits protective
antibodies to meningococci by mimicking a surface-exposed
epitope on loop 4 of porin A in strains with serosubtype
P1.2 [3]
The ORF of GNA33 encodes a protein 441 amino acids
in length with an N-terminal 20-amino-acid lipopolypep-tide signal sequence (LPSS) with a consensus lipoprotein-processing site, LAAC [4] Sequence comparison showed that GNA33 is 34.5% identical and 41.3% homologous with the 38-kDa membrane-bound lytic transglycosylase A (MltA) from E coli (Fig 1) In E coli, four additional exo-specific lytic transglycoylases (MltB, MltC, MltD, and Slt70) have been identified and/or characterized [5–8] These lytic transglycosylases exhibit no significant sequence homology with each other With the exception
of Slt70 (soluble lytic transglycosylase), they are all lipoproteins that attach to the outer membrane [7–10] Homologues of all these lytic transglycosylases have been identified in Meningococcus B [2], which, like their E coli counterparts, also exhibit little sequence conservation with each other
Lytic transglycosylases are a unique class of lysozyme-like enzymes that catalyze cleavage of the b-1,4-glycosidic bond between N-acetylmuramic acid (MurNAc) and N-acetyl glucosamine (GlcNAc) However, unlike lysozyme where the glycosyl moiety is transferred to H2O, cleavage by lytic transglycosylases is followed by an intramolecular transgly-cosylation [10] In this reaction, the glycosidic linkage between the muramyl and glucosaminyl residues is trans-ferred to the C6 position of the muramyl residue to form terminal 1,6-anhydromuramic acid-containing products [10] By virtue of their ability to cleave the polysaccharide backbone of the peptidoglycan layer, lytic transglycosylases are thought to play a role in synthesis and degradation of the murein sacculus It has been proposed that lytic transglycosylases play important roles in cellular elongation,
Correspondence to G T Jennings, Cytos Biotechnology AG,
Wagistrasse 25, CH-8952 Zu¨rich-Schlieren, Switzerland.
Fax: + 41 1 733 4659, Tel.: + 41 1 733 4642,
E-mail: jennings@cytos.com
Abbreviations: GNA, genome-derived Neisseria antigen; LPSS,
lipo-polypeptide signal sequence; MipA, MltA-interacting protein; MltA,
membrane-bound lytic transglycosylase A; PBP, penicillin-binding
protein.
*Note: these authors contributed equally to this work.
(Received 29 March 2002, revised 14 June 2002,
accepted 20 June 2002)
Trang 2septation, recycling of muropeptides, and pore formation
[7,10,11]
Current models of cell wall synthesis in Gram-negative
bacteria predict the necessity for murein synthases and
lytic enzymes to interact in a co-ordinated and controlled
manner [10] Indeed, interactions between lytic
cosylases (MltA, MltB and Slt70), bifunctional
transgly-cosylase-transpeptidases (PBP1A, PBP1B, PBP1C),
transpeptidases (PBP2, PBP3), and endopeptidases
(PBP4 and PBP7) of E coli have been reported [12,13]
In particular, affinity chromatography and/or surface
plasmon resonance have shown interactions between
MltA, PBP1B, PBP1C, PBP2, PBP3 and a newly
identi-fied scaffolding protein, MipA [14] It is thought that,
through such interactions, the enzymes required for cell
wall metabolism associate and form a multienzyme
complex [10,14] An enzyme complex would not only
provide a means for regulating peptidoglycan synthesis but
would also provide a way to control the potentially
autolytic activity of proteins such as MltA To date, no
evidence of these associations in Neisseria species has been
reported
In this study we cloned GNA33 (MltA) from
Meningo-coccus serogroup B The recombinant lipoprotein was
expressed in E coli, purified, and assayed for its
muram-idase and lytic transglycosylase activity In addition, we used
affinity chromatography to investigate the hypothesis that
MltA associates with other enzymes involved in
peptido-glycan metabolism and thus may be part of a multienzyme
complex
E X P E R I M E N T A L P R O C E D U R E S
Vector construction Three versions of meningococcal mltA were amplified by PCR and cloned into the expression vector pET21b+ (Novagen) via 5¢ NdeI and 3¢ XhoI restriction sites These included a full-length form incorporating its endogenous 20-amino-acid LPSS, a form containing a 19-amino-acid LPSS from an unrelated Meningococcus B lipoprotein, GNA1946 [1], and a truncated form lacking any leader sequence (Fig 1)
Full-length mltA was amplified using a forward primer containing an NdeI restriction site (5¢-CGCGGATCCCA TATGAAAAAATACCTATTCCGC-3¢) incorporating the ATG start codon The reverse primer (5¢-CCCGCTC GAGTTACGGGCGGTATTCGG-3¢) contained a XhoI restriction site and was used for all three constructs The construct containing the GNA1946 LPSS was made using a forward primer (5¢-GGGAATTCCATATGAAAACCTT CTTCAAAACCCTTTCCGCCGCCGCGCTAGCGCT CATCCTCGCCGCCTGCCAAAGCAAGAGCATC-3¢) spanning the entire leader of GNA1946 and containing 18 nucleotides overlapping the mltA sequence A conservative double nucleotide substitution (underlined) was made in a region of the primer encoding the GNA1946 LPSS This substitution introduced an NheI restriction and was designed to allow the GNA1946 LPSS to be ligated into any of the meningococcus genes that we have previously expressed in pET-21b[1] This restriction site was not used
Fig 1 Amino acid sequence of MltA from
N meningitidis serogroup B: comparison with
MltA from E coli The amino-acid sequence
of MltA from Meningococcus B (strain 2996)
(NmMltA) was compared with MltA from
E coli (EcMltA) using the GAP program
included in the Genetics Computer Group
(GCG) Wisconsin Package version 10.0 The
20-amino-acid LPSS is underlined The LPSS
was identified using the program PSORT
avail-able at http://psort.nibb.ac.jb The
19-amino-acid LPSS from the Meningococcus B gene
GNA1946 (GNA1946L), was used to replace
the MltA leader peptide and is shown above
the meningococcal sequence Amino acids are
identified by the standard single letter code.
Trang 3in this study The truncated gene lacking the 20-amino-acid
leader peptide was amplifed using the forward primer,
5¢-CGCGGATCCCATATGCAAAGCAAGAGCATCC
AAA-3¢
PCR was performed in a reaction volume of 100 lL
comprising 10 mM Tris/HCl (pH 8.3), 50 mM NaCl,
1.5 mMMgCl2, 0.8 mMdNTPs, 40 lMeach oligonucleotide
primer, and 2.5 U TaqI DNA polymerase (PerkinElmer,
Boston, MA, USA) Template DNA for the reaction was
200 ng genomic DNA from Neisseria meningitidis B 2996
The primary denaturation step was performed at 94C for
3 min and the remainder of the first five cycles with
denaturation, annealing and polymerization conditions of
94C for 40 s, 52 C for 40 s and 72 C for 1 min,
respectively The annealing temperature was increased to
65C for the next 30 cycles, and a final 7 min extension at
72C completed the reaction PCR products were purified
using the Qiagen Gel Extraction Kit Ligations and
transformations into E coli DH5 were performed as
described by Sambrook et al [15] After selection,
amplifi-cation and purifiamplifi-cation, the plasmids were used to transform
E coli BL21(DE3) (Novagen, Madison, WI, USA) The
genomic sequence of Meningococcus B is known for the
strain MC58 [2] The nucleotide sequence of mltA from
strain 2996 has 17 nucleotide substitutions (of which 16 are
silent) with respect to mltA from strain MC58 Only one of
these base changes results in an amino-acid substitution,
Ser312 to Ala
Expression and purification of recombinant MltA
E coli BL21(DE3) cells harboring the three versions of
pET21b-MltA (see above) were grown at 30C in Luria–
Bertani medium containing 100 lgÆmL)1 ampicillin until
the D550 reached 0.6–0.8 Isopropyl thio-b-D-galactoside
was added to a final concentration of 1.0 mM, and the
culture shaken for an additional 3 h Cells were collected by
centrifugation at 8000 g for 15 min at 4C All subsequent
procedures were performed at 4C
For purification of lipidated MltA, cells were
resuspend-ed in 25 mL 50 mM phosphate/300 mM NaCl, pH 8.0,
containing complete protease inhibitor (Roche, Basel,
Switzerland) Bacteria were disrupted by osmotic shock
with two or three passages through a French Press (SLM
Aminco) Unbroken cells were removed by centrifugation
at 5000 g for 15 min, and membranes sedimented by
centrifugation at 100 000 g for 45 min The pellet was
resuspended in 20 mM Tris/HCl (pH 8.0)/1.0M NaCl
containing complete protease inhibitor, and the suspension
mixed for 2 h After centrifugation at 100 000 g for
45 min, the pellet was resuspended in 20 mM Tris/HCl
(pH 8.0) containing 1.0MNaCl, 5.0 mgÆmL)1Chaps, 10%
(v/v) glycerol and complete protease inhibitor The solution
was stirred overnight, centrifuged at 100 000 g for 45 min,
and the supernatant dialysed for 6 h against 20 mMBicine
(pH 8.5)/120 mM NaCl/5.0 mgÆmL)1 Chaps/10% (v/v)
glycerol The dialysate was cleared by centrifugation at
13 000 g for 20 min and applied to a Mono S FPLC
ion-exchange column (Pharmacia, Uppsala, Sweden) at a flow
rate of 0.5 mLÆmin)1 Elution was performed using a
stepwise NaCl gradient
The protein was also expressed and purified in a form
lacking the LPSS After expression and harvesting, cells
were resuspended in 20 mMBicine (pH 8.5)/20 mMNaCl/ 10% (v/v) glycerol containing complete protease inhibitor and disrupted with a Branson Sonifier 450 The sonicate was centrifuged at 8000 g for 30 min to remove unbroken cells, and MltA was precipitated from the supernatant by the addition of saturated (NH4)2SO4 solution MltA was precipitated between 35% and 70% saturation and was collected by centrifugation at 8000 g for 30 min The pellet was dissolved in 20 mMBicine (pH 8.5)/20 mMNaCl/10% (v/v) glycerol and dialysed against this buffer overnight The dialysate was centrifuged at 13 000 g for 20 min, and the supernatant was applied to an FPLC Mono S ion-exchange column at a flow rate of 0.5 mLÆmin)1 The protein was eluted from the column with a stepwise NaCl gradient Purifications were analysed by SDS/PAGE [16], and protein concentration determined by the Bradford method West-ern-blot analysis was performed using polyclonal antisera as described previously [1]
Palmitate labelling Palmitate incorporation by recombinant MltA was con-firmed as described by Kraft et al [17] Briefly, E coli BL21(DE3) harbouring one of the three pET21b-MltA constructs were grown at 30C in Luria–Bertani medium containing 100 lgÆmL)1 ampicillin and 5 lCiÆmL)1 [3H]palmitate (Amersham) until the D550nm reached 0.4–0.8 Expression of recombinant protein was induced for 1 h by the addition of isopropyl b-D-thiogalactoside (final concentration 1 mM), and the bacteria harvested by centrifugation at 3000 g for 15 min Cells were washed twice with cold NaCl/Pi, suspended in 20 mMTris/HCl (pH 8.0)/
1 mMEDTA/1.0% (w/v) SDS, lysed by boiling for 10 min, and centrifuged for 10 min at 13 000 g Cold acetone was added to the supernatant, and, after 1 h at)20 C, protein was collected at 13 000 g for 10 min Protein was resus-pended in 1.0% (w/v) SDS, boiled with SDS/PAGE sample buffer, and subjected to SDS/PAGE using a 12.5% separating gel Gels were fixed for 1 h in 10% (v/v) acetic acid, and soaked for 30 min in Amplify solution (Amer-sham) The gel was vacuum-dried under heat and exposed
to Hyperfilm (Kodak) overnight at)80 C
Assay for muramidase activity Purified, recombinant MltAs expressed with the GNA1946 LPSS or without an LPSS were assessed for their ability to degrade insoluble murein sacculi into soluble muropeptides
by the method of Ursinus & Holtje [18] Murein lysis activity was determined using peptidoglycan radiolabelled with meso-2,6-diamino-3,4,5-[3H]pimelic acid as substrate Enzyme (3–10 lg total) was incubated for 45 min at 37C
in a total volume of 100 lL comprising 10 mMTris/maleate (pH 5.5), 10 mM MgCl2, 0.2% (v/v) Triton X-100 and [3H]diaminopimelic acid-labelled murein sacculi ( 10 000 c.p.m.) The assay mixture was placed on ice for 15 min with 100 lL 1.0% (w/v) N-cetyl-N,N,N-trime-thylammonium bromide, and the precipitated material separated by centrifugation at 10 000 g The radioactivity
in the supernatant was measured by liquid-scintillation counting The E coli lytic transglycosylase Slt70 was used as
a positive control for the assay, and the negative control comprised the above assay solution without enzyme
Trang 4Assay for lysis of poly(MurNAc-GlcNAc) glycan strands
The ability of MltA to utilize purified glycan strands as
substrate was determined by the method described by
Ursinus & Holtje [18] Poly(MurNAc-GlcNAc)n>30,
labelled with N-acetyl-D-1-[3H]glucosamine, was incubated
with 3 lg MltA in 10 mM Tris/maleate (pH 5.5)/10 mM
MgCl2/0.2% (v/v) Triton X-100 for 30 min at 37C The
reaction was stopped by boiling for 5 min, and the pH of
the sample adjusted to 3.5 by addition of 10 lL 20% (v/v)
phosphoric acid The components of the assay were then
separated by RP-HPLC on a Nucleosil 300 C18column as
described by Harz et al [19] The E coli lytic
transglycos-ylase MltA was used as a positive control in the assay A
negative control was performed in the absence of enzyme
Analysis of reaction products
The nature of the reaction products resulting from the
digestion of unlabelled E coli murein sacculus were
deter-mined by RP-HPLC as described by Glauner [20] Murein
sacculi digested with the muramidase Cellosyl were used to
calibrate and standardize the Hypersil ODS column
Gel filtration
The molecular masses of the recombinant proteins were
estimated using either FPLC Superose 12 (H/R 10/30) or
Superdex 75 gel-filtration columns (Pharmacia) The
buf-fers were 20 mM Bicine (pH 8.5) with and without
5.0 mgÆmL)1Chaps, respectively In addition, each buffer
contained 150–200 mM NaCl and 10% (v/v) glycerol
Proteins were dialysed against the appropriate buffer and
applied in a volume of 200 lL Gel filtration was performed
with a flow rate of 0.5–2.0 mLÆmin)1 and the eluate
monitored at 280 nm Fractions were collected and
analysed by SDS/PAGE Blue Dextran 2000 and the
molecular-mass standards ribonuclease A,
chymotryp-sin A, ovalbumin A, and BSA (Pharmacia) were used to
calibrate the columns The molecular mass of the sample
was estimated from a calibration curve of Kav vs log
(molecular mass) of the standards
Preparation of membrane extracts for affinity
chromatography
A detergent-solubilized membrane extract was prepared
from an acapsulated N meningitidis strain, M7 An
over-night culture of strain M7 was inoculated into 2 L
Muller-Hinton broth containing 0.25% (w/v) glucose, and grown at
37C in an atmosphere of 5.0% CO2 When the D550
reached 0.6, the culture was cooled on ice and harvested by
centrifugation at 8000 g; all the following steps were
performed at 4C The pellet was resuspended in 10 mM
Tris/HCl (pH 8.0) containing complete protease inhibitor
and DNase (10 lgÆmL)1), and the cells were disrupted with
a French Press Membranes were spun down at 100 000 g
for 45 min and resuspended in 10 mM Tris/maleate
(pH 6.8) containing 2.0% (v/v) Triton X-100, 10 mM
MgCl2, 150 mMNaCl and EDTA-free complete protease
(buffer I) After stirring overnight, membrane debris was
removed by centrifugation (100 000 g for 45 min), and the
supernatant containing solubilized protein stored at)20 C
Affinity chromatography Purified leaderless MltA (10 mgÆmL)1gel) was coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer’s protocol CNBr-activated Sepharose 4B prepared without protein and where the functional groups were neutralized with Tris was used as a control for nonspecific binding to the resin Disposable columns containing either control or MltA-coupled resin were prepared and equilibrated with 20 col vol buffer I Solu-bilized membrane extract was applied to both columns at a flow rate of 0.25 mLÆmin)1, then washed with 5· 1.0 mL buffer I Retained proteins were eluted by increasing the NaCl concentration in a stepwise fashion Salt concentra-tions of 300 mM, 600 mMand 1.0Min buffer I were applied
in 5· 1.0 mL aliquots, and the eluates retained for analysis
by SDS/PAGE, penicillin-binding assay, and Western blot Penicillin-binding assay
Penicillin-binding proteins (PBPs) were identified using the
125I-labelled Bolton–Hunter derivative of ampicillin pre-pared as described previously [21] Briefly, 4 lL (2.4 lg total) of the labelled ampicillin derivative was incubated for
30 min at 37C with 40 lL of the fractions eluted from control and MltA-coupled affinity columns The reaction was stopped by the addition of 4 lL penicillin G (60 mgÆmL)1), and the reaction complexes separated by SDS/PAGE and visualized by autoradiography
Preparation of antisera to PBP2 Recombinant PBP2 from N gonorrhoeae was purified as a soluble, active form PBP2 was expressed in the cytoplasm
of E coli as a fusion protein to maltose-binding protein (MBP) with a His6tag at its N-terminus Codons 44–581, which encode the entire periplasmic domain of PBP2, were fused in-frame to the C-terminus of MBP via an interven-ing tobacco etch virus (TEV) protease site The fusion protein was overexpressed in E coli, purified on a Ni2+/ nitrilotriacetate column, and cleaved with His6–TEV protease (fusion protein/TEV protease, 20 : 1, w/w) in
50 mM Tris/HCl (pH 8.0)/500 mM NaCl/10% glycerol After digestion, PBP2 was again purified by metal chelate affinity chromatography to remove uncut fusion protein, His6–MBP and the protease PBP2 was not eluted in the flow through, which contained unrelated contaminant proteins, but was eluted from the column with 10 mM imidazole Purified PBP2 was judged to be at least 95% pure by SDS/PAGE The protein was concentrated to
6 mgÆmL)1and stored at)80 C Purified PBP2 was used
to immunize mice, and antisera were collected as described
b y Pizza et al [1]
Western blot Fractions eluted from the MltA-coupled affinity column were separated by discontinuous SDSPAGE using a 12.5% separating gel [15] Proteins were electroblotted onto a nitrocellulose membrane and probed with antisera to PBP2 diluted 1 : 1000 Immunoreactive proteins were detected using the enhanced chemiluminescent method (Amersham, Chicago, IL, USA) and fluorography
Trang 5R E S U L T S
Cloning and expression inE coli
Expression of MltA in E coli was observed when the
gene was cloned with either its own 20-amino-acid LPSS
or the 19-amino-acid LPSS from an unrelated
Meningo-coccus lipoprotein, GNA1946 However, the level of
expression was much lower when the native leader
peptide was used (result not shown) Hence, for purposes
of purification and characterization, we used the clone
incorporating the LPSS from GNA1946 MltA cloned
without a leader peptide was expressed very efficiently
and represented about 20% of total cellular protein as
judged by densitometry This truncated, soluble form of
the protein was used for affinity chromatography (see
below)
MltA incorporating the LPSS from GNA1946 was
routinely expressed at 30C because expression of the
recombinant protein at 37C resulted in lysis of host
cells Lysis at 37C was observed within 60 min of
induction of expression and could be prevented by the
addition of 12% (w/v) sucrose and 10 mM MgSO4
Overexpression of E coli MltA also results in formation
of spheroplasts and cell lysis [9] However, in contrast
with our results, lysis due to overexpression of E coli
MltA occurs at 30C, but not at 37 C With E coli
MltA, this effect is due to the temperature sensitivity of
its muramidase activity, which exhibits maximum activity
at 30C and a 93% reduction in activity at 37 C It also
has been reported that a 55-fold overexpression of E.coli
lytic transglycosylase MltB resulted in rapid cell lysis at
37C [8] Similar to our observation with Meningococcus
MltA, autolysis induced by overexpression of E coli
MltB was also prevented by osmotic protection during
growth
Purification of recombinant proteins
Recombinant MltA lipoprotein was purified from the
membrane fraction of E coli as described in
Experimen-tal Procedures Analysis of the purification by SDS/
PAGE showed that MltA lipoprotein was localized in
the membrane fraction (Fig 2, lane 2) Western-blot
analysis with polyclonal sera raised against MltA failed
to detect MltA in any of the soluble fractions obtained
before Chaps extraction, demonstrating exclusive
local-ization of the lipoprotein to the membrane fraction
(result not shown) After solubilization of MltA with
Chaps, it was necessary to maintain NaCl at a minimum
concentration of 120 mM to prevent the lipoprotein from
precipitating The predicted pI for MltA is 10.5 The
basic nature of the protein enabled FPLC
cation-exchange chromatography to be performed under
condi-tions that allowed almost complete removal of
contam-inating proteins in a single step (Fig 2, lane 4) Similarly,
this property was exploited to perform a simple two-step
procedure for the purification of the truncated version of
MltA, which involved salting out and cation exchange
The leaderless form is found exclusively in the cytosolic
fraction of E coli and was purified to homogeneity as
judged by SDS/PAGE with Coomassie blue staining
(Fig 2, lane 5)
Molecular mass The molecular masses of the lipoprotein and truncated forms of MltA were determined under denaturing condi-tions by SDS/PAGE (Fig 2) The two forms of the protein migrate to the same position in the gel (Fig 2), and, from a calibration plot of log mass vs relative mobility of protein standards, the masses of both forms of MltA were calculated to be 44.5 kDa This is in agreement with the molecular mass of 45 869 Da predicted from the amino-acid composition of the protein excluding the first 19-amino-acids of the leader peptide As the lipoprotein expressed with its 2138-Da leader sequence migrates to the same position as leaderless MltA, it is reasonable to conclude that the signal peptide is cleaved when this clone
is expressed The presence of detergent in the purification prevented an accurate estimation of molecular mass for MltA lipoprotein using molecular exclusion chromatogra-phy As truncated MltA lacking its LPSS was purified in the absence of detergent, we determined the native molecular mass using this form of the protein (see Experimental
Fig 2 SDS/polyacrylamide gel showing the purification and molecular mass of recombinant forms of MltA Proteins were separated by SDS/ PAGE on a 12.5% separating gel and stained with Coomassie Brilliant Blue Lane M, molecular-mass standards; lane 1, bacterial lysate after expression; lane 2, membrane fraction after 100 000 g centrifugation; lane 3, soluble fraction after extraction of membrane fraction with 0.5% CHAPS; lane 4, an aliquot from the peak fraction from Mono S FPLC ion-exchange chromatography; lane 5, truncated MltA (expressed without the LPSS) after Mono S FPLC ion-exchange chromatography.
Trang 6procedures) Truncated MltA was eluted with a Kav
corresponding to a molecular mass of 31 600 Da This
value is low compared with that of the denatured protein
suggesting either an interaction with the column or a smaller
than expected Stokes’ radius Nevertheless the native
molecular mass of the truncated form of MltA is more
indicative of a monomer than a dimer
Confirmation that MltA is a lipoprotein
To test if recombinant MltA expressed with either its
endogenous LPSS or GNA1946 LPSS was lipidated, the
ability of the proteins to incorporate [3H]palmitate was
examined Proteins extracted from cells grown in the
presence of the radiolabel were examined by SDS/PAGE
and autoradiography (Fig 3) A labelled band with a
molecular mass of 44 kDa was observed for MltA cloned
with either its own leader or the leader from GNA1946 The
radiolabel was not incorporated when MltA lacking an
LPSS was expressed
MltA is a muramidase Both the purified lipoprotein and truncated form of MltA showed muramidase activity when assayed for their ability
to degrade murein sacculi to soluble muropeptides How-ever, the activity observed with the lipoprotein form was 21.6-fold higher than the activity of the truncated form For this reason, the lipoprotein was chosen for further kinetic analyses The activity of MltA lipoprotein was enhanced 5.1-fold by the addition of 0.2% (v/v) Triton X-100 to the assay, whereas Triton X-100 had no measurable effect on the activity of the truncated soluble form of the protein Biochemical and kinetic properties of the enzyme The effect of pH on muramidase activity was determined in Tris/maleate buffer over the pH range 5.0–8.0 The optimal
pH for the reaction was determined to be 5.5 (data not shown) The optimum pH for lytic transglycosylase activity
b y MltA from E coli is 4.5 [18] Enzyme activity was measured over the temperature range 18–42C Maximum activity was observed at 37C (data not shown) As we observed that MltA has 77% of the activity at 30C as it does at 37C, the stability of cells expressing Meningococcus MltA at 30C is unlikely to be due solely to a temperature-dependent decrease in murein lytic activity as previously described for E coli MltA (see above)
The effect of ions on muramidase activity was determined
by performing the reaction with a variety of bivalent cations, at a final concentration of 10 mM Maximum activity was found with Mg2+, which stimulated activity 2.1-fold Mn2+and Ca2+stimulated enzyme activity to a similar extent, whereas Ni2+and EDTA had no significant effect on activity In contrast, Zn2+significantly inhibited enzyme activity (data not shown)
Initial-rate kinetic analyses were performed with sub-strate concentrations ranging from 2.6 to 52.0 mgÆL)1 An analysis of the Michaelis–Menten curve (data not shown) showed that the enzyme exhibits typical first-order and zero-order kinetics As the substrate for the reaction is insoluble,
it is not possible to determine the Km for the reaction in molar terms [18] However, the apparent Kmof 8.2 mgÆL)1 determined from a double-reciprocal Lineweaver–Burk plot
is slightly lower than the value (52.6 mgÆL)1) ob tained previously for MltA from E coli [18]
Substrate specificity and reaction product The ability of MltA to lyse isolated glycan strands comprising poly(MurNAc-GlcNAc)n>30was demonstrated when we separated 1,6-anhydrodisaccharide subunit reac-tion products from the oligosaccharide substrate by HPLC (Fig 4) The same elution profile was observed when we assayed E coli MltA in a control experiment (result not shown) The use of isolated glycan strands as a substrate further demonstrates homology with E coli MltA, which is also capable of utilizing both murein sacculi and isolated glycan strands [18,22]
HPLC analysis of the digestion products after incubation
of MltA with murein sacculi showed two major peaks eluted with retention times of 52.4 and 68.9 min (Fig 5) By comparing the elution profile of the calibration standard,
it was determined that these major reaction products
Fig 3 Demonstration that MltA is a lipoprotein E coli BL21(DE3)
harbouring pET21b-MltA cloned with its own LPSS, with the LPSS
from GNA1946, or without a leader sequence were grown in the
presence of [3H]palmitate Expression of recombinant protein was
induced for 1 h at 30 C by the addition of 1 m M isopropyl b- D
-thiogalactoside Cells were then washed, lysed and protein precipitated
as described in Experimental Procedures Proteins were separated by
SDS/PAGE using a 12.5% separating gel, and the labelled proteins
were visualized by autoradiography Lane 1, MltA cloned without an
LPSS; lane 2, MltA cloned with its own LPSS; lane 3, MltA cloned
with the LPSS from GNA1946 Molecular masses of marker proteins
are indicated on the left and the position of MltA is indicated by an
arrow.
Trang 7corresponded to 1,6-anhydrodisaccharide tripeptide and
tetrapeptide, respectively The formation of the 1,6-anhydro
intramolecular bond within the muramic acid moiety
confirms that the enzyme is indeed a lytic transglycosylase
(Fig 5)
MltA–Sepharose affinity chromatography of membrane
proteins
The leaderless form of MltA was expressed, purified and
covalently bound to CNBr-activated Sepharose This
col-umn was used to isolate MltA-interacting proteins from a
membrane fraction of Meningococcus B Proteins were
eluted with a stepwise NaCl gradient and assayed for
penicillin-binding activity by incubation with 125I-labelled
ampicillin PBPs were visualized by SDS/PAGE and
auto-radiography (Fig 6) A control column prepared without
MltA was used to assess the specificity of binding The most
intensely labelled band at 62 kDa observed in the starting
material was retained by MltA–Sepharose during loading
and washing, but was completely eluted with 300 mMNaCl
In contrast, the intensely labelled 46-kDa band observed in
the starting material was not retained by the column and was
eluted in the flow through Vollmer et al [14] reported that
400 mMNaCl was sufficient to completely disrupt binding of
PBPs to E coli MltA When the autoradiograph and
Coomassie blue-stained gel were overlaid, it was not possible
to see a protein band corresponding to the 62-kDa
radioactive band This is characteristic of PBPs, which are
typically of low abundance; for example, E coli PBP2 is present at only 50 copies per cell [23,24] To date, four PBPs have been identified in Meningococcus B: PBP1, PBP2, PBP3 and PBP4 These proteins have predicted molecular masses of 88.9 kDa, 63.6 kDa 50.5 kDa and 34.1 kDa, respectively [25,26] Hence we reasoned that the 62-kDa PBP specifically retained during affinity chromatography is PBP2 To confirm this hypothesis, we analysed affinity-chromatography fractions by Western-blot analysis using polyclonal antisera raised against PBP2 from Gonococcus (Fig 7) Gonococcal PBP2 has 98% sequence identity with PBP2 from Meningococcus serogroup B Immunoblots showed an immunoreactive band with a molecular mass of
62 kDa in the starting material and in the fraction obtained after elution with 300 mM NaCl Moreover, this band migrated to the same position as purified gonococcal PBP2 The 88-kDa immunoreactive band observed in the starting material was not retained by the MltA affinity column Taken together these results demonstrate an interaction involving MltA and PBP2
D I S C U S S I O N
A genomics-based approach to vaccine discovery previ-ously identified GNA33 as a potential vaccine candidate
Fig 5 HPLC analysis of muropeptides after digestion of murein sacculi withMltA Isolated murein sacculi were digested with purified MltA and reduced with sodium borohydride The resulting muropeptides were separated by RP-HPLC on a Hypersil ODS column Elution was performed with a linear gradient from 50 m M sodium phosphate (pH 4.32) to 50% methanol in 50 m M sodium phosphate (pH 4.95) The column was calibrated and standardized with murein sacculi digested with the muramidase Cellosyl.
Fig 4 HPLC analysis demonstrating hydrolysis of isolated glycan
strands Poly(MurNAc-GlcNAc) n>30 was incubated without (A) or
with (B) MltA as described in Experimental procedures At the
com-pletion of the incubation, the sample was passed over a Nucleosil 300
C 18 column to which was applied 0.1 m M sodium phosphate buffer
(pH 2), 5% acetonitrile for 5 min, 100% methanol for 5 min, and
again starting buffer The radioactivity of the eluate was monitored.
The peak eluted between 20.2 and 22.2 min corresponds to intact
glycan strands In a control assay in which MltA was replaced with
E coli MltA, the same elution profile as seen in (B) was observed (data
not shown).
Trang 8against meningococcal infection Sequence comparison
predicted that GNA33 encodes a lipoprotein homologous
to the lytic transglycosylase MltA from E coli To
definitively identify and characterize GNA33, we cloned
and expressed the ORF of GNA33 in E coli with and
without an LPSS Although the level of expression of the
truncated form was 20-fold higher than of the lipoprotein
form, incorporation of an LPSS in MltA increased
specific activity by 22-fold Incorporation of3[H]palmitate,
cleavage of the leader peptide, and localization of the
protein to the membrane fraction all suggest that
recom-binant MltA is correctly processed as a lipoprotein in
E coli Moreover, purification of enzymatically active
protein and lysis of host cells during expression confirmed
the fidelity of the heterologous expression system When
MltA was expressed with its own LPSS, the level of
expression was low The level of expression was increased
significantly by fusing codons 21–441 of MltA to an LPSS
from an unrelated Meningococcus B lipoprotein,
GNA1946 This LPSS in combination with MltA is
obviously efficiently processed by the
lipoprotein-process-ing machinery of E coli
We demonstrated that recombinant MltA is capable of
lysing murein sacculi, confirming that the protein is a
muramidase The lipoprotein produced two major
reac-tion products, 1,6-anhydrodisaccharide tripeptide and tetrapeptide, confirming that the protein is indeed a lytic transglycosylase Of the four exo-specific lytic transglycos-ylases in E coli studied to date, only MltA is capable of utilizing unsubstituted murein glycan strands as substrate [18] The ability of meningococcal MltA to also utilize the unsubstituted substrate shows a functional similarity between the two homologues Furthermore, in many of the biochemical parameters assessed, such as pH opti-mum, Km and requirement for bivalent cations, the
N meningitidis and E.coli enzymes are similar [9,18] These results confirm the sequence-based prediction that GNA33 is a homologue of E coli lytic transglycosylase MltA For these reasons, we assigned the name MltA to GNA33
In this study, we used affinity chromatography to demonstrate an association between meningococcal MltA and PBP2 The ability to interact with a PBP is a characteristic common to MltA from N meningitidis and
E coli and is the first description of such an association beyond that reported for E coli E coli MltA is thought
to form part of an enzyme complex composed of murein synthases and muramidases This association is believed
to facilitate the co-ordinated action of different enzymes involved in enlargement and septation of the murein sacculus [10] Reconstitution experiments with E coli
Fig 6 PBP assay of proteins fractionated by affinity chromatography
on MltA-sepharose Aliquots of fractions obtained from the elution of
the meningococcal membrane extract from either a MltA–Sepharose
or control column were assayed for the presence of PBPs with
125
I-labelled ampicillin as detailed in Experimental Procedures The
labelled fractions were subjected to SDS/PAGE on a 10% separating
gel and visualized by autoradiography after 100 h exposure SM is
membrane extract before addition to the column C indicates eluates
obtained from the control column, and T represents eluates from the
MltA–Sepharose column Shown are the first two fractions from the
wash with buffer I (150 m M NaCl) and each of the elution steps in
300 m M NaCl, 600 m M NaCl and 1 M NaCl The position of
molec-ular-mass markers is indicated.
Fig 7 Western blot of proteins fractionated by affinity chromatography
on MltA–Sepharose Aliquots of fractions obtained after elution of the meningococcal membrane extract from the MltA–Sepharose column were analysed by immunoblotting with anti-PBP2 sera Immunore-active bands were detected by enhanced chemiluminesence as described in Experimental procedures Lane 1, purified gonococcal PBP2; lane 2, membrane extract from meningococcus before addition
to the column; lane 3, fraction obtained after elution with 300 m M
NaCl The positions of molecular-mass markers are shown.
Trang 9MltA and PBP1B demonstrated the necessity for the
structural protein MipA, and it has been proposed that
this enzyme serves as a scaffold for assembly of the
multienzyme complex [14] We performed an extended
homology search of the Meningococcus B genome but
failed to identify a homologue of MipA A similar
situation exists for Haemophilus influenzae, which contains
a homologue of MltA but not MipA [14] A BLAST
search showed that N meningitidis PBP2 and E coli
PBP3 have 39% identity and 59% homology over a
541-amino-acid overlap and revealed that meningococcal
PBP2 is more homologous to E coli PBP3 than PBP2
In fact, Meningococcus does not have a homologue of
E coli PBP2, which is involved in maintaining the
characteristic rod shape of the bacterium Interestingly,
it is the presence of either PBP2 or PBP3 in the enzyme
complex of E coli that confers a specific function to the
complex [14] In E coli, PBP2 is known to be responsible
for cell elongation, whereas PBP3 is involved in septum
formation [27,28] It will be interesting to determine if
such an enzyme complex exists in Meningococcus, the
nature and composition of the protein components, and
in particular the function of the association between
MltA and PBP2
We initially reported that antibodies raised against
GNA33 are bactericidal, a property known to correlate
with protective effects in humans [1] It was subsequently
discovered that antibodies elicited by vaccination with
GNA33 are bactericidal because MltA is an effective
mimetic antigen of the PorA epitope P1.2 [3] In its own
right, MltA may be a useful vaccine for the prevention of
disease caused by P1.2 strains Furthermore, it has been
suggested that substituting strain specific PorA loops into
MltA or its subdomains may generate immunogenic
mimetics of other serotype PorA epitopes [3] The ease of
expression and purification demonstrated in this work
further suggests the great potential that MltA offers as a
recombinant vaccine candidate against meningococcal
infection A direct role for lytic transglycosylases in
meningococcal disease is suggested by an investigation of
genes required for bacteraemic disease In an infant rat
model of N meningitidis infection, Sun and co-workers
[29] used insertional mutagenesis to identify genes
essen-tial for pathogenesis, one of which was the gene encoding
MltB A further role for lytic transglycosylases in disease
may be associated with their reaction products The
1,6-anhydrodisaccharide-containing metabolites, such as
those shown here to be produced by meningococcal
MltA, have been shown to have diverse biological
activities For instance, the cytopathology of respiratory
epithelium that is characteristic of Bordetella pertussis
infection is caused by 1,6-anhydromuramic
acid-contain-ing products [30] The same compounds are also capable
of inducing sleep and arthritis [31,32] Perhaps most
importantly is the potential of 1,6-anhydromuramyl
peptides to induce meningeal inflammation [33] Hence
lytic transglycosylases such as MltA may be directly
involved in the pathogenesis associated with
meningoc-cocal infection The potential that lytic transglycosylases
offer as targets for disease intervention combined with
their importance in growth, septation, recycling of
peptidoglycan, and pore formation makes them worthy
of further investigation
A C K N O W L E D G E M E N T S
We would like to thank Vega Masignani and Maria Scarselli for sequence comparisons and database searches, and Mariagrazia Pizza for many helpful discussions We are also grateful to Giorgio Corsi for preparing the figures and to Catherine Mallia for formatting and submitting the manuscript.
R E F E R E N C E S
1 Pizza, M., Scarlato, V., Masignani, V., Giuliani, M.M., Arico, B., Comanducci, M., Jennings, G.T., Baldi, L., Bartolini, E., Capecchi, B et al (2000) Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing Science 287, 1816–1820.
2 Tettelin, H., Saunders, N.J., Heidelberg, J., Jeffries, A.C., Nelson, K.E., Eisen, J.A et al (2000) Complete genome sequence of Neisseria meningitidis serogroup B strain MC58 Science 287, 1809–1815.
3 Granoff, D.M., Moe, G.R., Giuliani, M.M., Adu-Bobie, J., Santini, L., Brunelli, B., Piccinetti, F., Zuno-Mitchell, P., Lee, S.S., Neri, P et al (2001) A novel mimetic antigen eliciting protective antibody to Neisseria meningitidis J Immunol 167, 6487–6496.
4 Hayashi, S & Wu, H.C (1990) Lipoproteins in bacteria.
J Bioenerg Biomemb 22, 451–469.
5 Betzner, A.S & Keck, W (1989) Molecular cloning, over-expression and mapping of the slt gene encoding the soluble lytic transglycosylase of Escherichia coli Mol Gen Genet 219, 489–491.
6 Dijkstra, A.J., Hermann, F & Keck, W (1995) Cloning and con-trolled overexpression of the gene encoding the 35 kDa soluble lytic transglycosylase from Escherichia coli FEBS Lett 366, 115–118.
7 Dijkstra, A.J & Keck, W (1996) Identification of new members of the lytic transglycosylase family in Haemophilus influenzae and Escherichia coli Microb Drug Resist 2, 141–145.
8 Ehlert, K., Holtje, J.-V & Templin, M.F (1995) Cloning and expression of a murein hydrolase lipoprotein from Escherichia coli Mol Microbiol 16, 761–768.
9 Lommatzsch, J., Templin, M.F., Kraft, A.R., Vollmer, W & Holtje, J.-V (1997) Outer membrane localization of murein hydrolases: MltA, a third lipoprotein lytic transglycosylase in Escherichia coli J Bacteriol 179, 5465–5470.
10 Holtje, J.-V (1998) Growth of the stress-bearing and shape-maintaining murein sacculus of Escherichia coli Microbiol Mol Biol Rev 62, 181–203.
11 Holtje, J.-V & Tuomanen, E.I (1991) The murein hydrolases of Escherichia coli: properties, functions and impact on the course of infections in vivo J Gen Microbiol 137, 441–454.
12 vonRechenberg, M., Ursinus, A & Holtje, J.-V., (1996) Affinity chromatography as a means to study multienzyme complexes involved in murein synthesis Microb Drug Resist 2, 155–157.
13 Romeis, T & Holtje, J.-V (1994) Specific interaction of penicillin-binding proteins 3 and 7/8 with soluble lytic transglycosylase in Escherichia coli J Biol Chem 269, 21603–21607.
14 Vollmer, W., von Rechenberg, M & Holtje, J.-V (1999) Demonstration of molecular interactions between the murein polymerase PBP1B, the lytic transglycosylase MltA, and the scaffolding protein MipA of Escherichia coli J Biol Chem 274, 6726–6734.
15 Sambrook, J., Fritsch, E.F & Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
16 Laemmli, U.K & Favre, M (1973) Maturation of the head of bacteriophage T4 I DNA packaging events J Mol Biol 80, 575– 599.
17 Kraft, A.R., Templin, M.F & Holtje, J.-V (1998) Membrane-bound lytic endotransglycosylase in Escherichia coli J Bacteriol.
180, 3441–3447.
Trang 1018 Ursinus, A & Holtje, J.-V (1994) Purification and properties of a
membrane-bound lytic transglycosylase from Escherichia coli.
J Bacteriol 176, 338–343.
19 Harz, H., Burgdorf, K & Holtje, J.-V (1990) Isolation and
separation of the glycan strands from murein of Escherichia coli by
reversed-phase high-performance liquid chromatography Anal.
Biochem 190, 120–128.
20 Glauner, B (1988) Separation and quantification of muropeptides
with high-performance liquid chromatography Anal Biochem 17,
451–464.
21 Schwarz, U., Seeger, K., Wengenmayer, F & Strecker, H (1981)
Penicillin-binding proteins of Eschericia coli identified with a
125 I-labelled derivative of ampicillin FEMS Microbiol Lett 10,
107–109.
22 Romeis, T., Vollmer, W & Holtje, J.V (1993) Characterization of
three different lytic transglycosylases in Escherichia coli FEMS
Microbiol Lett 111, 141–146.
23 Dougherty, T.J., Kennedy, K., Kessler, R.E & Pucci, M.E (1996)
Direct quantitation of the number of individual
penicillin-binding proteins per cell in Escherichia coli J Bacteriol 178, 6110–
6115.
24 Spratt, B.G (1977) Properties of the penicillin-binding proteins of
Escherichia coli K12 Eur J Biochem 72, 341–352.
25 Ropp, P.A & Nicholas, R.A (1997) Cloning and characterization
of the ponA gene encoding penicillin-binding protein 1 from
Neisseria gonorrhoeae and Neisseria meningitidis J Bacteriol 179,
2783–2787.
26 Zhang, Q.Y & Spratt, B.G (1989) Nucleotide sequence of the penicillin-binding protein 2 gene of Neisseria meningitides Nucleic Acids Res 17, 5383.
27 Park, J.T & Burman, L (1973) FL-1060: a new penicillin with a unique mode of action Biochem Biophys Res Commun 51, 863–868.
28 Schmidt, L.S., Botta, G & Park, J.T (1981) Effects of furazlocillin, a beta-lactam antibiotic which binds selectively
to penicillin-binding protein 3, on Escherichia coli mutants deficient in other penicillin-binding proteins J Bacteriol 145, 632–637.
29 Sun, Y.H., Bakshi, S., Chalmers, R & Tang, C.M (2000) Func-tional genomics of Neisseria meningitidis pathogenesis Nat Med.
6, 1269–1273.
30 Cookson, B.T., Tyler, A.N & Goldman, W.E (1989) Primary structure of the peptidoglycan-derived tracheal cytotoxin of Bor-detella pertussis Biochemistry 28, 1744–1749.
31 Krueger, J.M., Pappenheimer, J.R & Karnovsky, M.L (1982) Sleep-promoting effects of muramyl peptides Proc Natl Acad Sci USA 79, 6102–6106.
32 Fleming, T.J., Wallsmith, D.E & Rosenthal, R.S (1986) Arthropathic properties of gonococcal peptidoglycan fragments: implications for the pathogenesis of disseminated gonococcal disease Infect Immun 52, 600–608.
33 Tuomanen, E., Hengstler, B., Zak, O & Tomasz, A (1986) Induction of meningeal inflammation by diverse bacterial cell walls Eur J Clin Microbiol 5, 682–684.