INFECTION AND IMMUNITY, Feb. 2003, p. 647–655
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Vol. 71, No. 2
The msbB Mutant of Neisseria meningitidis Strain NMB Has a Defect
in Lipooligosaccharide Assembly and Transport to
the Outer Membrane
Deborah M. B. Post,1Margaret R. Ketterer,1Nancy J. Phillips,2
Bradford W. Gibson,2and Michael A. Apicella1*
Department of Microbiology, University of Iowa, Iowa City, Iowa 52242,1and Department of
Pharmaceutical Chemistry, School of Pharmacy, University of California,
San Francisco, California 941432
Received 25 July 2002/Returned for modification 30 August 2002/Accepted 13 November 2002
A deletion-insertion mutation in msbB, a gene that encodes a lipid A acyltransferase, was introduced into
encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of the same strain.
These mutants were designated NMBA11K3 and NMBA11K3cap-, respectively. Neither lipooligosaccharide
(LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both
were easily extracted from NMBA11K3cap-. Immunoelectron microscopy using monoclonal antibody (MAb)
6B4, which recognizes the terminal Gal?1-4GlcNAc of LOS, demonstrated that NMB, NMBcap-, and
NMBA11K3cap- expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3.
However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Mass-spectrometric
analyses demonstrated that the relative amounts of the lipid A-specific C12:0 3-OH and C14:0 3-OH present
in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25- and 23-fold, respec-
tively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from
NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substan-
tially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences
the assembly of the lipid A and consequently the incorporation of porin in the outer membrane.
Neisseria meningitidis is one of the leading causes of bacterial
meningitis worldwide (1). Meningococcal disease affects
mainly children and young adults. The rapid progression of
meningococcal disease makes proper diagnosis and subsequent
treatment often vital to the survival of infected individuals. If
not properly diagnosed and treated, meningococcal infections
can lead to shock and death within a matter of hours (35). Due
to the detrimental effects caused by these bacteria, a better
understanding of meningococcal pathogenesis may prove valu-
able in the management of systemic meningococcal disease.
One of the major virulence factors of N. meningitidis is the
capsular polysaccharide. N. meningitidis serogroups are based
on the capsular polysaccharide. Five serogroups, A, B, C, Y,
and W-135, are most often associated with invasive meningo-
coccal strains. Polysaccharide vaccines have been developed
for serogroups A, C, Y, and W-135. Additionally, recent work
on polysaccharide conjugate vaccines has shown the improved
efficacy of these vaccines in infants and young adults (21, 37).
Currently a vaccine for serogroup B is not available. The cap-
sular polysaccharide for this serogroup is poorly immunogenic,
due to its similarity to human neural adhesion molecules (35).
An additional virulence factor present in N. meningitidis is
lipooligosaccharide (LOS). LOS is the principal glycolipid
present in the outer membrane of N. meningitidis and is com-
posed of the oligosaccharide chain extensions, the core, and
the lipid A. The oligosaccharide chain extensions have been
shown to play a role in molecular mimicry (8, 19, 34). The lipid
A of N. meningitidis is similar in structure to lipid A from other
gram-negative bacteria (11, 12, 28). The lipid A portion of
these bacteria is known to be responsible for many of the
adverse effects seen with gram-negative bacterial infections
HtrB and MsbB are the acyltransferases responsible for the
addition of the secondary acyl substitutions onto the lipid A (3,
5, 16). Unlike Escherichia coli, N. meningitidis does not require
the presence of the two 2-keto-3-deoxyoctulosonic acid (Kdo)
groups for full lipid A acylation (32). Previous work with htrB
and msbB mutants demonstrated that the lipid A portion of
their LOS and lipopolysaccharide (LPS) structures were mod-
ified (12, 18, 28). These modified forms of LOS and LPS were
reduced in their toxicity (9, 16) and in their ability to stimulate
cytokine secretion (7, 24). Additionally, the Haemophilus in-
fluenzae and Salmonella enterica serovar Typhimurium htrB
mutants were reduced in their virulence (9, 16).
Due to the importance of the lipid A structure in pathogen-
esis we wished to explore the possibility that there is an msbB
homologue in N. meningitidis. A gene which showed high sim-
ilarity to the htrB and msbB genes from E. coli was identified.
This gene was cloned, and deletion-insertion mutants were
made in N. meningitidis encapsulated strain NMB and N. men-
ingitidis acapsular mutant strain NMBcap-. These mutants were
subsequently designated NMBA11K3 and NMBA11K3cap-, re-
spectively. In this study we report our chemical and immuno-
chemical analyses of these mutants.
* Corresponding author. Mailing address: The University of Iowa,
Department of Microbiology, 51 Newton Rd., Iowa City, IA 52242.
Phone: (319) 335-7807. Fax: (319) 335-9006. E-mail: michael-apicella
MATERIALS AND METHODS
Bacterial strains, plasmids, and culture conditions. All bacterial strains and
plasmids used in this study are described in Table 1. E. coli was grown in
Luria-Bertani medium at 37°C and supplemented with appropriate antibiotics.
Encapsulated N. meningitidis serogroup B strain NMB was isolated from the
bloodstream of a patient with meningococcal sepsis. An acapsular NMB mutant
(PBCC7232-NMB ?siaA-D) was a gift from Wyeth-Lederle Vaccines and Pedi-
atrics (West Henrietta, N.Y.). This strain was designated NMBcap- for the
studies presented here. N. meningitidis organisms were grown on gonococcal agar
(Becton Dickinson, Sparks, Md.) supplemented with 1% IsoVitaleX or on brain
heart infusion (BHI) agar (Becton Dickinson) supplemented with 2.5% heat-
inactivated fetal calf serum (FCS) at 37°C in 5% CO2. Liquid cultures of N.
meningitidis were grown in BHI broth supplemented with 2.5% FCS or in gono-
coccal broth supplemented with 1% IsoVitaleX at 37°C. Kanamycin-resistant N.
meningitidis was grown on supplemented BHI agar plates or in supplemented
BHI broth with 50 ?g of kanamycin/ml. N. meningitidis organisms grown in the
presence of kanamycin were grown without CO2. For growth curve cultures, an
overnight bacterial culture was used to inoculate a 3-ml culture to an optical
density at 600 nm (OD600) of 0.05. Cultures were grown at 37°C with agitation.
Readings were taken once every hour.
Recombinant DNA and transformation methods. Restriction and modifying
enzymes were purchased from New England Biolabs (Beverly, Mass.) and Pro-
mega (Madison, Wis.). Standard recombinant DNA protocols were performed as
previously described (22). Transformation of E. coli with plasmid DNA was done
by the CaCl2method (6). Transformation of N. meningitidis was performed as
previously described (27).
DNA isolation. Plasmid DNA was prepared with the QIAprep Spin Miniprep
kit or the QIAprep Midiprep kit, according to manufacturer’s instructions (Qia-
gen Inc., Valencia, Calif.). Chromosomal DNA was isolated with the Puregene
DNA isolation kit (Gentra Systems, Minneapolis, Minn.).
DNA sequencing and analysis. DNA sequencing reactions were performed by
using dye terminator cycle sequencing chemistry with AmpliTaq DNA polymer-
ase and the FS enzyme (PE Applied Biosystems, Foster City, Calif.). The reac-
tions were run on and analyzed with an Applied Biosystems model 373A stretch
fluorescence automated sequencer at the University of Iowa DNA Facility. All
primers were either commercially available or were purchased from either Ge-
nosys Corporation (Aldrich, Milwaukee, Wis.) or IDT Technologies (Coralville,
Iowa). Sequence analysis was performed using Assembly LIGN, version 1.0
(Oxford Molecular Group Inc., Oxford, United Kingdom), MacVector (Oxford
Molecular Group Inc.), and Wisconsin Package, version 10.0 (Genetics Com-
puter Group, Madison, Wis.).
Cloning and mutagenesis of the N. meningitidis msbB gene. Cloning and mu-
tagenesis of the msbB gene was performed as previously described (18). Briefly,
the E. coli htrB gene was used to search the Neisseria gonorrhoeae strain FA1090
sequence at the University of Oklahoma website. The sequence that showed
highest homology to the E. coli gene was used to design PCR primers. Since the
genomes of N. gonorrhoeae and N. meningitidis are highly homologous, primers
gchtrB3 (5?-CAACAGGCGGCGGTGGAACAG-3?) and gchtrB4 (5?-TTCGG
CATCCACTCCCCTTTG-3?) were used for amplification of the N. meningitidis
strain NMB msbB gene. The 1,443-bp PCR product was cloned with the TA
cloning vector pCR2.1 (Invitrogen, Carlsbad, Calif.) and was subsequently sub-
cloned into pUC19. This construct was transformed into E. coli DH5? cells
(Invitrogen) and was subsequently designated pNMBA11pUC19. Restriction
enzymes BclI and BssHII deleted 138 bp from the msbB gene. A kanamycin
resistance cassette was inserted into the modified msbB gene, and the resulting
construct was designated pNMBA11K3. The proper construct was confirmed by
using PCR and restriction enzyme digests. Plasmid DNA from pNMBA11K3 was
used for transformation with N. meningitidis strains NMB and NMBcap-. Trans-
formants were selected for on BHI plates containing kanamycin.
Southern blot and PCR analyses. Hybridization experiments were carried out
according to the manufacturer’s protocols. All probes were labeled by either
PCR labeling or random labeling with digoxigenin-labeled deoxynucleoside
triphosphates (Boehringer Mannheim Corp., Indianapolis, Ind.). Primers
gchtrB3 and -4 were used to perform PCRs.
SDS-PAGE of LOS. LOS was isolated from 6 liters of BHI broth supple-
mented with 2.5% FCS for strains NMB and NMBcap- and 6 liters of BHI broth
supplemented with 2.5% FCS and 50 ?g of kanamycin/ml for strain
NMBA11K3cap- by using a modified hot-phenol-water preparation (18). Sam-
ples were run on a Tris-Tricine sodium dodecyl sulfate-polyacrylamide gel elec-
trophoresis (SDS-PAGE) gel as previously described by Lesse et al. (13). Silver
staining was done according to a previously described protocol (30).
Isolation and SDS-PAGE of MPs. Overnight 10-ml broth cultures were har-
vested by centrifugation at ?3,800 ? g for 10 min. The cell pellet was resus-
pended in membrane preparation (MP) buffer (50 mM Tris-HCl, 150 mM NaCl,
10 mM EDTA, pH 7.4) and warmed at 56°C for 30 min. Then, cultures were
cooled to room temperature. Cultures were passed through a syringe by using
needles of different gauges to shear the cells. The process was repeated 10 times
for each gauge of needle. This procedure was first performed with a 20-gauge
needle, then with a 22-gauge needle, and last with a 25-gauge needle. Sheared
cells were centrifuged at 16,000 ? g for 15 min. The supernatant from this spin
was centrifuged at 25,000 ? g for 20 min. The supernatant from this spin was
centrifuged at 30,000 ? g for 20 min. Finally, the supernatant from the previous
spin was centrifuged at 100,000 ? g for 2 h 15 min. All spins were performed at
20°C. The pellet obtained from the last spin was glass-like and was used as the
sample for SDS-PAGE. Samples were separated on a 4 to 20% Tris-Tricine
gradient gel (13). Equal quantities of proteins were loaded onto each gel, as
determined by spectrophotometric readings. Coomassie blue staining and silver
staining were performed according to previously described protocols (17, 30).
Western blot analyses of LOS and MPs. Western blot analyses were per-
formed as previously described by Towbin et al. (29). MAb 6B4, which recognizes
the terminal Gal?1-4GlcNAc moiety of the oligosaccharide chain extension (2),
was utilized to detect LOS. The porin antibody 3H1 was a gift from Milan Blake
(Baxter Hyland Immuno, Columbia, Md.). The blots were developed by using the
Super Signal West Pico chemiluminescent substrate according to the manufac-
turer’s instructions (Pierce, Rockford, Ill.).
SEM and TEM analyses. For microscopy studies, all bacterial strains were
grown in BHI broth to an OD600of 0.8 to 1.0. Samples for transmission electron
microscopy (TEM) analysis were placed in an equal volume of 4% paraformal-
dehyde (final concentration, 2%). Samples for scanning electron microscopy
(SEM) analysis were settled onto silicon wafers and fixed in 2% paraformalde-
hyde. Cells for immuno-TEM were dehydrated by using a standard graded-
ethanol series, followed by embedment in London Resin White (Ted Pella Inc.,
Redding, Calif.). For resolution of cell membrane morphology by TEM, bacterial
cells were treated with osmium according to standard protocols and then dehy-
drated through graded ethanols and embedded in Epon acrylic resin. Thin
sections of embedded cells were mounted on nickel grids. The Epon-embedded
TABLE 1. Strains and plasmids used in this study
Strain or plasmidGenotype, relevant phenotype, or selection marker Source or reference
E. coli DH5?
F??80dlacZ?M15 ?(lacZYA-argF)U169 deoR recA1 endA1
Wild type, serogroup B
?siaA-siaD, serogroup B
Kanamycin resistant, msbB mutant
Kanamycin resistant, msbB mutant, ?siaA-siaD
?) phoA supE44 ??thi-1 gyrA96 relA1
N. meningitidis NMB
N. meningitidis NMBcap-
N. meningitidis NMBA11K3
N. meningitidis NMBA11K3cap-
Ampicillin, cloning vector
Ampicillin, cloning vector
Ampicillin, msbB PCR product in pUC19 vector
Ampicillin, kanamycin, insertion-deletion msbB mutant in pUC19
New England Biolabs
648 POST ET AL.INFECT. IMMUN.
sections were stained with 5% uranyl acetate and lead citrate for contrast.
Samples for SEM and immuno-TEM were treated with neuraminidase (1 U/ml;
Oxford GlycoScience, Novato, Calif.) for 2 h at 37°C prior to labeling with MAb
6B4. Following overnight incubation with the primary antibody, specimens were
incubated with goat anti-mouse IgM conjugated to gold beads, either 12-nm gold
bead–conjugate (Jackson ImmunoResearch, West Grove, Pa.) for TEM or
25-nm gold bead–conjugate for SEM (EMS, Ft. Washington, Pa.). The TEM
samples were finally counter-stained with 5% uranyl acetate. Immunolabeled
samples for SEM were incubated in 2.5% glutaraldehyde to cross-link the anti-
bodies and then processed by using a standard graded-ethanol series. These
samples were carbon coated before being viewed on an S-4000 scanning electron
microscope (Hitachi, Mountain View, Calif.). TEM samples were viewed with a
Hitachi H-7000 transmission electron microscope. All samples were viewed with
microscopes located in the Central Microscopy Research Facility at the Univer-
sity of Iowa.
GC/MS analysis of membrane fatty acids. The MPs from strains NMB,
NMBA11K3, NMBcap-, and NMBA11K3cap- were treated with 0.5 ml of 10%
(wt/wt) BF3-methanol (Supelco, Bellefonte, Pa.) and heated at 100°C for 6 h.
Samples were allowed to cool to room temperature and then were treated with
0.5 ml of saturated NaCl solution, followed by 0.5 ml of high-performance liquid
chromatography grade hexanes (Aldrich, St. Louis, Mo.). After the samples were
vortexed and centrifuged, the organic layers were removed and transferred to
clean vessels. The aqueous layers were then extracted a second time with 0.5 ml
of hexanes. The combined organic layers were evaporated to dryness under a
stream of nitrogen and later redissolved in hexanes for gas chromatography/mass
spectrometry (GC/MS) analysis. Samples were analyzed with a Hewlett-Packard
5890 gas chromatograph interfaced with a VG70SE mass spectrometer. The gas
chromatograph was equipped with an on-column injector (J & W Scientific,
Folsom, Calif.), and samples were separated on a 30-m by 0.25-mm BPX70
column with a 0.25-?m film thickness (SGE, Inc., Austin, Tex.). The initial oven
temperature, 100°C, was held for 5 min, and then data acquisition was started
and the samples were eluted by using a temperature gradient from 100 to 220°C
at 4°C/min. The carrier gas was helium at ?6 lb/in2. Relative peak areas were
measured from the total ion chromatograms for each run and normalized to the
Cloning and mutagenesis of the N. meningitidis msbB gene.
The N. meningitidis msbB gene was amplified by PCR and
cloned. This gene has been previously shown to be able to
complement for both the temperature sensitivity and the LPS
phenotype of an E. coli htrB mutant (18). A deletion-insertion
mutation was generated in the N. meningitidis msbB gene and
the resulting construct was designated pNMBA11K3.
Transformations of pNMBA11K3 into N. meningitidis
strains NMB and NMBcap- were performed as previously de-
scribed (27). Selection for transformants was done on plates
containing kanamycin. Southern blot analyses and PCR dem-
onstrated that the proper mutations had been incorporated
into the N. meningitidis NMB and NMBcap- genomic DNA
(data not shown). The resulting transformants were designated
NMBA11K3 and NMBA11K3cap-, respectively. These muta-
tions should not have a polar effect on downstream genes.
First, a kanamycin cassette, which has previously been shown
to produce nonpolar mutations (15), was utilized to construct
the pNMBA11K3 mutant. Second, the msbB gene is not part of
an operon. We sequenced over 200 bp of DNA downstream
from the msbB gene, and no open reading frames were found.
In addition, the annotated sequence from N. meningitidis strain
MC58 indicates that the closest gene is almost 300 bp down-
stream from the msbB gene, and it is transcribed in the oppo-
site orientation. Proper capsule expression phenotypes for the
four different strains were confirmed by using MAb 2-2-B, a
serogroup B-specific capsular MAb, which was a gift from
Wendell Zollinger (Walter Reed Army Institute of Research,
Silver Spring, Md.) (data not shown).
Comparison of growth rates of N. meningitidis strains NMB,
NMBcap-, NMBA11K3, and NMBA11K3cap-. To determine
whether the mutation in the msbB gene had any effect on the
growth rate of the bacteria, growth curves were determined.
These growth curves demonstrated that there was no differ-
ence in the growth rates of strains NMB, NMBcap-, and
NMBA11K3cap- (data not shown). However, there was a re-
duction in the growth rate of strain NMBA11K3 compared
with those of the other strains. In the first 8 h of growth,
NMBA11K3 reached 50% of the density of the other strains,
and after 24 h growth was reduced by approximately one-third.
The growth curves were performed three times, and the results
of all three experiments were consistent.
Characterization of the LOS by SDS-PAGE and Western
blot analysis. Silver staining showed that the NMBA11K3cap-
LOS migrated through the gel slightly faster than the NMB
and NMBcap- LOS (Fig. 1A) and that the NMBA11K3cap-
LOS stained brown instead of black. This staining pattern was
FIG. 1. Characterization of NMBA11K3cap- LOS by SDS-PAGE
and Western blot analyses. (A) Silver staining analysis of an SDS-
PAGE gel. Lane 1, NMB LOS; lane 2, NMBcap- LOS; lane 3,
NMBA11K3cap- LOS. The sialylated LOS (top band) is absent from
the NMBcap- LOS because the sialylation genes were deleted in this
strain. A different glycoform of LOS is visible in the NMBcap- LOS
sample where the sialylated LOS band would normally migrate.
(B) Western blot analysis with MAb 6B4. Lane 1, NMB LOS; lane 2,
NMBcap- LOS; lane 3, NMBA11K3cap- LOS.
VOL. 71, 2003N. MENINGITIDIS AND LIPID A 649
consistent with previous reports of LPS and LOS isolated from
htrB and msbB mutants (12, 18, 23). Western blot analysis
NMBA11K3cap- was performed. The blot showed that MAb
6B4 bound to all of the LOS samples tested (Fig. 1B). These
results indicated that the oligosaccharide portion of the
NMBA11K3cap- LOS was intact. LOS could not be purified
from NMBA11K3 by using phenol-water, proteinase K, and
petroleum ether-phenol extraction methods. Subsequent MPs
from NMBA11K3 failed to reveal the presence of LOS bands.
TEM analyses of MAb 6B4-immunolabeled NMB, NMBA
11K3, NMBcap-, and NMBA11K3cap-. Figure 2 shows micro-
graphs representative of each of the samples. Immunoelectron
micrographs of NMB, NMBcap-, and NMBA11K3cap- (Fig.
2A, C, and D, respectively) show the typical diplococcus struc-
ture of N. meningitidis. These micrographs also show structur-
ally intact membranes and an electron-dense cytoplasm. The
NMBA11K3 sample (Fig. 2B) shows bacteria that still have the
coccoid shape but that are somewhat larger than the bacteria
from the NMB, NMB cap-, and NMBA11K3cap- samples.
FIG. 2. Examination of MAb 6B4-immunolabeled N. meningitidis using TEM analyses. (A) NMB; (B) NMBA11K3; (C) NMBcap-;
(D) NMBA11K3cap-; (E and F) Epon-embedded sections showing the structure of the bacterial cell membranes of NMB (E) and NMBA11K3
(F). Scale bars, 1 ?m (A to D) and 100 nm (E and F).
650POST ET AL.INFECT. IMMUN.
Additionally, there appear to be patches, instead of an even
distribution, of electron-dense material in the cytoplasm. Im-
NMBA11K3cap- (Fig. 2A, C, and D, respectively) show the
meningococci were labeled circumferentially with MAb 6B4. The
immunoelectron micrograph of NMBA11K3 (Fig. 2B) shows
the meningococci were labeled with MAb 6B4 predominately
in the cytoplasm of the bacteria, with essentially no MAb 6B4
label present on the outer membrane. Higher-power electron
microscopy revealed that both NMB and NMBA11K3 had
evidence of bilamellar outer membranes (Fig. 2E and F, re-
SEM analyses of MAb 6B4-immunolabeled NMB, NMBA
11K3, NMBcap-, and NMBA11K3cap-. Figure 3 shows micro-
graphs representative of data collected from each of the sam-
ples. All strains showed the typical diplococcus shape when
viewed by SEM. NMB, NMBcap-, and NMBA11K3cap- (Fig.
3A, C, and D, respectively) showed surface labeling with MAb
6B4. MAb 6B4 did not label the surface of NMBA11K3 (Fig.
SDS-PAGE and Western blot analyses of MPs from NMB,
NMBcap-, NMBA11K3, and NMBA11K3cap-. Both silver
staining and Coomassie blue staining demonstrated that there
were differences in the components of the MP from NMBA
11K3 compared with those from NMB, NMBcap-, and
NMBA11K3cap- (Fig. 4). Silver-staining analysis showed that
there was no detectable LOS present in the NMBA11K3 sam-
ple (Fig. 4A). Western blot analysis utilizing MAb 6B4 con-
firmed the absence of a full-length LOS structure in the MP
from NMBA11K3 (Fig. 5B). Examination of the MP also sug-
gested that there were decreases in the levels of at least two
other components of the outer membrane, those with molec-
ular masses of ?42 and ?35 kDa, in NMBA11K3 compared
with levels in its parent strain, NMB. Western blot analyses
determined that the proteins with altered levels were PorA and
PorB, respectively (Fig. 5A). The expected molecular masses
of these proteins are approximately 42 kDa for PorA and 35.7
kDa for PorB.
MS analyses of MP fatty acids from NMB, NMBcap-,
NMBA11K3, and NMBA11K3cap-. Fatty acid methyl esters
were prepared from the MPs and were subsequently analyzed
by GC/MS (Fig. 6 and Table 2). All of the expected fatty acids
were present in each sample; however, the relative abundances
of the fatty acids detected from the samples varied. For both
the encapsulated and acapsular strains, the hydroxylated fatty
acids derived exclusively from lipid A (C12:0 3-OH and C14:0
3-OH) had lower relative abundances in the msbB mutants
than in the parental strains. This phenomenon was most dra-
matic for the encapsulated strains, where the levels of C12:0
3-OH and C14:0 3-OH were 25- and 23-fold higher, respec-
tively, in the NMB sample than in the NMBA11K3 sample.
The NMBcap- sample had C12:0 3-OH and C14:0 3-OH levels
that were both approximately twofold higher than those of the
NMBA11K3cap- sample. Additionally, the C12:0 fatty acid,
which is also found in lipid A, was recovered in lower relative
abundance in the msbB mutants than in their respective pa-
FIG. 3. SEM analyses of MAb 6B4-immunolabeled N. meningitidis. (A) NMB; (B) NMBA11K3; (C) NMBcap-; (D) NMBA11K3cap-. Scale
bars, 100 nm.
VOL. 71, 2003N. MENINGITIDIS AND LIPID A651
rental strains (Table 2). Compared to the lipid A fatty acid
ratios in the NMBcap- strain, the relative amounts of C12:0,
C12:0 3-OH, and C14:0 3-OH detected in the NMBA11K3cap-
strain suggest the loss of a single C12:0 fatty acid from the lipid
A structure in the msbB mutant. This observation is consistent
with molecular mass measurements of the intact lipid A from
NMB, NMBcap-, and NMBA11K3cap- obtained by matrix-
assisted laser desorption ionization–time of flight (MALDI-
TOF) MS, which showed a shift to lower mass (?182 Da,
corresponding to the loss of a lauric acid residue) for the msbB
mutant (data not shown). No LOS could be isolated from the
NMBA11K3 mutant strain; therefore, MALDI-TOF analysis
of the intact lipid A was not possible. These data demonstrate
that the relative abundance of the lipid A fatty acids present in
the MPs from the msbB mutants is dramatically reduced com-
pared with their relative abundance in the parental strains.
However, this reduction is significantly more pronounced in
the encapsulated msbB mutant (NMBA11K3) than in the
acapsular msbB mutant (NMBA11K3cap-). These data suggest
that the amounts of lipid A, and hence LOS, expressed in the
outer membranes of NMBA11K3 and NMBA11K3cap- are
significantly reduced compared to the amounts expressed in
the outer membranes of their parent strains. Additionally, the
relative amounts of C16:0 in the MPs from the msbB mutants
were higher than those in the MPs from the wild-type strains.
Consistent with these findings, a previous study by Steeghs et
al. also found that the relative amounts of short-chain fatty
acids increased in a LOS-deficient N. meningitidis lpxA mutant
Previous studies involving htrB mutants from E. coli, H.
influenzae, and S. enterica serovar Typhimurium have demon-
strated that they exhibit a number of phenotypes (10, 12, 16,
28). E. coli, H. influenzae, and S. enterica serovar Typhimurium
htrB mutants were all shown to be initially sensitive to temper-
atures above 32°C (10, 12, 28). However, work from our lab-
oratory and others has demonstrated that N. gonorrhoeae and
E. coli msbB mutants are not temperature sensitive (18, 24). In
agreement with these studies,
NMBA11K3cap- were able to grow on solid medium at 37°C.
Growth curves demonstrated that NMBA11K3cap- was able to
grow at the same rate as NMB and NMBcap-. However,
NMBA11K3 had a slower growth rate and was unable to reach
the same OD, after 56 h of growth, as the other three strains.
Since the msbB mutants were able to grow at 37°C and since
NMBA11K3cap- was able to grow at the same rate as NMB
and NMBcap-, it seems unlikely that the lag in the growth of
NMBA11K3 is due to temperature sensitivity.
An additional characteristic of htrB and msbB mutants is
modification of the LPS and LOS structures. Studies per-
formed with S. enterica serovar Typhimurium and H. influenzae
FIG. 4. Silver staining (A) and Coomassie blue staining (B) analy-
ses of MPs from N. meningitidis strains NMB (lane 1), NMBcap- (lane
2), NMBA11K3 (lane 3), and NMBA11K3cap- (lane 4).
FIG. 5. Western blot analyses of MPs from N. meningitidis strains
NMB (lanes 1), NMBcap- (lanes 2), NMBA11K3 (lanes 3), and
NMBA11K3cap- (lanes 4). (A) MAb 3H1 for porin; (B) MAb 6B4 for
LOS. Expected molecular masses: PorA, ?42 kDa; PorB, ?35 kDa;
LOS, ?5 kDa.
652POST ET AL.INFECT. IMMUN.
htrB mutants demonstrated that the lipid A structures of both
were modified (12, 28). The lipid A from the H. influenzae htrB
mutant was determined to be approximately 90% tetraacyl and
10% pentaacyl instead of the normal hexaacyl structure. Ad-
ditionally, studies of an N. gonorrhoeae msbB mutant demon-
strated that the lipid A was pentaacyl instead of hexaacyl (18).
In the study presented here, we were able to isolate and begin
to characterize LOS from NMBA11K3cap-. The increase in
migration rate and the change in the staining pattern of the
NMBA11K3cap- LOS were consistent with data that have been
previously reported for msbB and htrB mutants (12, 18, 23).
Additionally, the binding of MAb 6B4, which recognizes the
terminal two sugars of the oligosaccharide, to NMBA11K3cap-
LOS indicates that the oligosaccharide region of the LOS is
intact. Similar to findings with the N. gonorrhoeae msbB mutant
(18), MS analysis of the NMBA11K3cap- lipid A demonstrated
that it is missing one lauric acid (C12:0) substitution and thus
may have a pentaacyl rather than a hexaacyl structure. These
results are consistent with a previous study of an N. meningi-
tidis strain H44/76 msbB mutant (33).
We were unable to isolate LOS from NMBA11K3 despite
using a variety of standard methods. Immuno-SEM using MAb
6B4 demonstrated a lack of surface labeling of NMBA11K3,
while NMB, NMBcap-, and NMBA11K3cap- all showed sur-
face binding of the antibody. Immuno-TEM micrographs
showed that, unlike what was found for NMB, NMBcap-, and
NMBA11K3cap-, there was no MAb 6B4 binding to the outer
membrane of NMBA11K3. However, binding of MAb 6B4 was
visible in the cytoplasm of NMBA11K3. Since we could not
isolate LOS from this mutant, we speculate that this labeling
represents MAb 6B4 binding to the Gal?1-4GlcNAc of the
oligosaccharide chain extensions still attached to its carrier,
undecaprenol phosphate. MAb 6B4 has been previously shown
to bind to lacto-N-neotetraose-ceramide in human erythro-
cytes (14). That study, by Mandrell et al., demonstrated that
MAb 6B4 is able to recognize its epitope on a lipid carrier
FIG. 6. GC/MS chromatograms of fatty acid methyl esters derived from membrane preparations of the designated N. meningitidis strains.
Relevant peaks are labeled in each chromatogram. The y axes show the relative peak intensities of the summed ion abundances.
VOL. 71, 2003N. MENINGITIDIS AND LIPID A653
other than lipid A. Recent work in our laboratory with a
meningococcal bacA mutant suggests that N. meningitidis LOS
is assembled similarly to LPS (D. Post, A. Zaleski, E. Johan-
sen, B. Gibson, and M. Apicella, unpublished data). Based on
these studies and the current model for LPS assembly (36), it
appears that the oligosaccharide chain extensions are assem-
bled on the carrier lipid undecaprenol phosphate. Then, the
oligosaccharide chain extensions and the lipid A-core region
are transported to the periplasm by separate mechanisms,
where they are subsequently ligated and transported to the
outer membrane. The failure to isolate the lipid A-core region
alone suggests that there is a defect in the assembly of the lipid
A-core complex in NMBA11K3. Interestingly, recent findings
by Tzeng et al. demonstrated that two N. meningitidis strain
NMB mutants, defective in Kdo biosynthesis and Kdo transfer
to the lipid A, expressed an LOS that consisted only of lipid A
(31, 32). These results demonstrate that the lipid A can be
assembled and transported to the bacterial surface in the ab-
sence of Kdo.
MS analyses of outer membranes isolated from NMBA11K3
demonstrated that the amounts of the lipid A-specific C12:0
3-OH and C14:0 3-OH present in these samples were 25- and
23-fold, respectively, less than the amount present in the MP
from NMB. These data taken together with the microscopy
data suggest that the amount of LOS expressed in the outer
membranes of NMBA11K3 is significantly reduced compared
to the amount expressed in the outer membranes of its parent
strain. MP from NMBA11K3cap- showed 2- and 1.7-fold de-
creases in the amounts of C12:0 3-OH and C14:0 3-OH, re-
spectively, present in these samples compared to the amounts
present in MP from NMBcap-. Additionally, the relative
amounts of C16:0 in the MPs from the msbB mutants were
higher than those in the MPs from their respective wild-type
strains. These data suggest that both NMBA11K3 and
NMBA11K3cap- are defective in their abilities to assemble
their LOS and that the presence of the capsular polysaccharide
makes the defect in LOS assembly and subsequent transport
more pronounced. These results also suggest that NMBA11K3
may place other lipids, most likely C16:0, the lipid that anchors
the capsule to the outer membrane, in their outer membranes
to compensate for the loss of the lipid A portion of the LOS.
These mutants may preferentially express the lipidated capsule
on their surfaces when the lipid A is altered. However, if the
capsule components are absent in the msbB mutant, the me-
ningococcus may be able to incorporate the modified LOS
structure into the outer membrane. Interestingly, Steeghs et al.
determined that the presence of the capsular polysaccharide is
essential for the viability of the LOS-deficient N. meningitidis
lpxA mutant H44/76(pHBK30) (25), further suggesting that the
C16:0 lipid may have a role in stabilizing the outer membranes
of LOS-depleted mutants.
Western blot analyses demonstrated that the levels of porin
expressed in the outer membrane of NMBA11K3 were also
altered. Since porin is known to closely associate with LOS (4),
it was not surprising to see a decrease in the levels of PorA and
PorB in the outer membranes. SDS-PAGE analyses of MP
from NMBA11K3 demonstrated that a number of other pro-
teins expressed in the outer membrane were present at levels
similar to those in the NMB MP. An explanation for the
decrease in the levels of porin and LOS could be that they are
transported to the outer membrane as a complex. The modi-
fication in the lipid A structure may have decreased the effi-
ciency of LOS assembly; therefore, the transport and subse-
quent surface expression of the whole complex are altered.
A decrease in LOS and LPS expression on the bacterial
surface has not been previously reported for any htrB or msbB
mutant. Previous studies of an N. meningitidis msbB mutant by
van der Ley et al. did not report any changes in the amount of
LOS or porin expressed on the bacterial surface (33). One
explanation for this difference may be the use of different
strains of N. meningitidis in our respective studies.
Further study is required to more clearly determine the
components involved in the maintenance of the outer mem-
brane structure of NMBA11K3. This strain may prove to be an
important tool for further elucidating the mechanisms of LOS
assembly and transport in pathogenic Neisseria. Additionally,
since NMBA11K3 is not greatly impaired in its growth rate and
since the surface expression of LOS is markedly reduced, this
strain may prove useful in the future development of menin-
The University of Iowa DNA facility is supported in part by the
Diabetes Endocrinology Research Center with National Institutes of
Health grant DK25295 and by the College of Medicine. Research in
M. A. Apicella’s laboratory was supported by AI45728 and AI44642.
D. M. B. Post’s work was in part supported by NIH training grant
T32A107511. Research in B. W. Gibson’s laboratory was supported by
AI44642 and by the UCSF mass spectrometry facility, which is partially
supported by NCRR 06164.
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Editor: J. T. Barbieri
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