Lipooligosaccharide structure contributes to multiple steps in the virulence of Neisseria meningitidis.

Abstract

Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with host epithelial cells and is a major factor contributing to the human proinflammatory response to meningococci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The wild-type strain [NeuNAc-Galbeta-GlcNAc-Galbeta-Glcbeta-Hep2 (GlcNAc, Glcalpha) 3-deoxy-D-manno-2-octulosonic acid (KDO2)-lipid A; 1,4' bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the mouse model. In contrast, the Hep2 (GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB) mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induction of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of this study further define the importance of the LOS structure as a virulence component involved in multiple steps in the pathogenesis of N. meningitidis.

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INFECTION AND IMMUNITY, Feb. 2006, p. 1360–1367
0019-9567/06/$08.00?0 doi:10.1128/IAI.74.2.1360–1367.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 2
Lipooligosaccharide Structure Contributes to Multiple Steps
in the Virulence of Neisseria meningitidis
Laura Plant,1* Johanna Sundqvist,1Susu Zughaier,2Lena Lo ¨vkvist,1
David S. Stephens,2,3and Ann-Beth Jonsson1
Department of Medical Biochemistry and Microbiology, Biomedical Centrum, Uppsala University, Uppsala, Sweden1;
Division of Infectious Diseases, Department of Medicine, Emory University, Atlanta, Georgia2;
and Laboratories of Microbial Pathogenesis, VA Medical Center, Atlanta, Georgia3
Received 19 September 2005/Returned for modification 7 November 2005/Accepted 22 November 2005
Lipooligosaccharide (LOS) of Neisseria meningitidis has been implicated in meningococcal interaction with
host epithelial cells and is a major factor contributing to the human proinflammatory response to meningo-
cocci. LOS mutants of the encapsulated N. meningitidis serogroup B strain NMB were used to further determine
the importance of the LOS structure in in vitro adherence and invasion of human pharyngeal epithelial cells
by meningococci and to study pathogenicity in a mouse (CD46 transgenic) model of meningococcal disease. The
wild-type strain [NeuNAc-Gal?-GlcNAc-Gal?-Glc?-Hep2(GlcNAc, Glc?) 3-deoxy-D-manno-2-octulosonic acid
(KDO2)-lipid A; 1,4? bisphosphorylated], although poorly adherent, rapidly invaded an epithelial cell layer in
vitro, survived and multiplied early in blood, reached the cerebrospinal fluid, and caused lethal disease in the
mouse model. In contrast, the Hep2(GlcNAc) KDO2-lipid A (pgm) mutant, which was highly adherent to
cultured epithelial cells, caused significantly less bacteremia and mortality in the mouse model. The Hep2-
KDO2-lipid A (rfaK) mutant was shown to be moderately adherent and to cause levels of bacteremia and
mortality similar to those caused by the wild-type strain in the mouse model. The KDO2-lipid A (gmhB)
mutant, which lacks the heptose disaccharide in the inner core of LOS, avidly attached to epithelial cells but
was otherwise avirulent. Disease development correlated with expression of specific LOS structures and was
associated with lower adherence but rapid meningococcal passage to and survival in the bloodstream, induc-
tion of proinflammatory cytokines, and the crossing of the blood-brain barrier. Taken together, the results of
this study further define the importance of the LOS structure as a virulence component involved in multiple
steps in the pathogenesis of N. meningitidis.
Neisseria meningitidis is a frequent inhabitant of the upper res-
piratory tract of humans and is a major cause of meningitis and
fulminant septicemia in otherwise healthy individuals. Among the
important virulence factors involved in meningococcal pathogen-
esis, endotoxin, or lipooligosaccharide (LOS), is a major compo-
nent inducing proinflammatory responses in meningococcal sep-
sis and meningitis (15, 36). Furthermore, the morbidity and
mortality of meningococcal sepsis are directly correlated with
levels of circulating meningococcal LOS (3, 4).
Meningococcal LOS interacts with human cells, resulting in
the production of proinflammatory cytokines and chemokines,
including interleukin 1 (IL-1), IL-6, and tumor necrosis factor
(TNF), that are important in the pathogenesis of meningococ-
cal disease (3, 42). While pili and Opa and Opc outer mem-
brane proteins are also critical, LOS is one of the structures
important in mediating meningococcal attachment to (23, 24)
and invasion into (30) epithelial cells. The role of LOS in these
events is further substantiated by the findings that LOS-defi-
cient meningococcal mutants show impaired adherence (2)
and reduced induction of serum cytokines (35) compared to
the wild-type strain.
The structure of N. meningitidis LOS has been characterized
both immunologically (33) and biochemically (6, 8, 19, 20, 40).
Meningococcal LOS lacks the repeating O antigens of enteric
lipopolysaccharide but maintains a conserved inner core com-
posed of heptose and 3-deoxy-D-manno-2-octulosonic acid bound
to lipid A, to which variable ? and ? chain saccharides are at-
tached (15). Lipid A is the active moiety through its ability to
upregulate the inflammatory response. Changes in the configura-
tion or the conformational structure of lipid A affect the biolog-
icalresponse(27).Variationinthecompositionofmeningococcal
LOS is also postulated to mediate changes in host immune re-
sponses and bacterial virulence (28, 37).
In order to further define the role of LOS in meningococcal
pathogenesis, each of a series of genetically and structurally
defined LOS mutants of the serogroup B meningococcal strain
NMB was examined for its capacity to adhere to and invade
epithelial cells in comparison to the wild-type parent strain.
Also, the pathogenicities of the parent and of each of the
mutants were examined by use of a mouse model of meningo-
coccal disease (9), and survival in the blood of these mice was
assessed. Our results show that structural variations of LOS
affect attachment and invasion of epithelial cells, access to and
survival in the bloodstream, and the subsequent innate im-
mune response and morbidity in vivo.
MATERIALS AND METHODS
Bacterial strains. Neisseria meningitidis serogroup B strain NMB (encapsu-
lated; L2/4 immunotype) and the genetically defined mutants of this strain (pgm,
rfaK, and gmhB mutants) are shown in Fig. 1 and described in references 12, 15,
25, 29, 34, and 41. The meningococcal strain NMB and the pgm, rfaK, and gmhB
* Corresponding author. Mailing address: Department of Medical
Biochemistry and Microbiology, Biomedical Centrum, Uppsala Uni-
versity, PO Box 582, Uppsala, Sweden. Phone: 46 18 471 46 05. Fax: 46
18 50 98 76. E-mail: Laura.Plant@imbim.uu.se.
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mutants were grown on GC agar with Kellogg’s supplement (16) at 37°C in 5%
CO2. The antibiotics for LOS mutants were used at the following concentrations:
for the pgm and gmhB mutants, tetracycline at 5 ?g/ml; and for the rfaK mutant,
spectinomycin at 60 ?g/ml. The M7 strain used in the whole-cell enzyme-linked
immunosorbent assay (ELISA) is an unencapsulated mutant (NMB synA::Tn916
mutant) (32).
Cells. Detroit 562 human pharyngeal cells (ATCC CCL 138) were maintained
in modified Eagle medium (Sigma) supplemented with 10% inactivated fetal
bovine serum (Sigma), 2 mM L-glutamine (Sigma), 1 mM sodium pyruvate
(Gibco), and 1? nonessential amino acids (Gibco) at 37°C in 5% CO2.
Electron microscopy. Bacteria were resuspended to an optical density at 600
nm (OD600) of 0.3 in Tris-Mg buffer (10 mM Tris-HCl [pH 7.4] and 10 mM
MgCl2) and mounted on copper grids. The grids were stained with 1% ammo-
nium molybdate and analyzed with a JEOL 1230 electron microscope.
Western blotting. Bacterial protein extracts (750 ng) were separated by 10%
(PilC expression) and 15% (Opa expression) sodium dodecyl sulfate-polyacryl-
amide gel electrophoresis (SDS-PAGE) by standard methods and transferred to
polyvinylidene difluoride membranes. The membranes were blocked overnight
with 5% nonfat dried milk in Tris-buffered saline (TBS; 0.05 M Tris and 2 M
NaCl, pH 7.4) containing 0.05% Tween 20. PilC was identified by incubation with
the K3 antibody that recognizes both PilC1 and PilC2 (polyclonal rabbit antibody
diluted 1:5,000). The Opa proteins were detected with the 4B12/C11 antibody,
which recognizes all Opa proteins (1) (monoclonal mouse antibody diluted
1:5,000). Incubation with primary antibodies was followed by incubation with
horseradish peroxidase-conjugated goat anti-rabbit antibody (Bio-Rad; diluted
1:10,000) or horseradish peroxidase-conjugated goat anti-mouse antibody (Santa
Cruz Biotechnologies; diluted 1:10,000). A chemiluminescence kit from Perkin
Elmer Life Sciences was used for detection.
Examination of LOS expression. Tricine SDS-PAGE was used to examine
the LOS expression of all bacterial mutants. Whole-cell lysates of bacterial
strains were prepared by incubation of bacterial suspensions (8 mg) with
proteinase K (2.5 ?g/ml), Tris-HCl (50 mM), CaCl2(1 mM), and glycerol
(2%) at 60°C for 18 h. Following digestion, loading buffer (1 M Tris-HCl, 10%
glycerol, 2% SDS, 0.5% 2-?-mercaptoethanol, and 0.0125% bromophenol
blue) was added, 200 ng of sample was separated on a 16.5% gel, and LOS
was detected by silver staining (39).
Capsule quantification by whole-cell ELISA. The expression of capsule by
various mutants used in this study was quantified by a whole-cell ELISA method
as described previously (31). Briefly, cells were harvested from overnight growth
on GC plates and suspended in phosphate-buffered saline. A 50-?l aliquot of a
1:3 dilution of cell suspensions at an OD650of 0.1 was applied and dried at 37°C
overnight. Antibodies specific for serogroup B (2-2-B) and serogroup A (14-1-A)
were used at 1:2,000 and 1:30,000 dilutions, respectively. The M7 strain does not
express capsule due to the synA mutation and was used as a negative control for
the whole-cell ELISA.
Adhesion and invasion assay. Detroit 562 cells were grown in 24-well tissue
culture plates to full confluence. Suspensions of meningococci were added to
triplicate wells at a multiplicity of infection of 100. Infected cells were incubated
at 37°C in 5% CO2for 2 h and washed five times with media to remove unbound
bacteria. Adhered bacteria were quantified after 2 h by lysing the cells for 5 min
with 1% saponin and serially diluting the samples and spreading them on GC
agar plates. The number of invaded bacteria was determined after 3, 4, 5, and 6 h
by incubating cells in medium containing 100 ?g/ml gentamicin for 1 h to kill
extracellular bacteria before the saponin treatment. Both assays were conducted
in at least three independent experiments, and data were normalized to results
for the original inocula. The sensitivity of all bacterial strains to gentamicin was
determined in cell media by recovery of viable bacteria following a 1-h incubation
in gentamicin at 0, 0.1, 1, 10, and 100 ?g/ml.
Growth curve. To monitor the growth of bacteria in cell culture medium,
100-?l suspensions of bacteria (OD600? 0.1) were inoculated into 2 ml of cell
culture medium and incubated at 37°C in 5% CO2. The optical density at 600 nm
was measured at various time points after inoculation.
Animal infection assay. A mouse model of meningococcal infection (9, 10) was
used to examine the pathogenicity of the LOS mutants. Briefly, 5- to 8-week-old
CD46 transgenic mice (n ? 17) were infected intraperitoneally (i.p.) with 3 ? 108
bacteria in two independent experiments. Blood samples were taken from the tail
1, 4, 8, and 24 h after infection and spread on GC agar plates to determine the
bacterial load in blood. At 4, 8, and 24 h postinfection, blood smears were made
for analysis of neutrophil infiltration. Serum was collected 24 h postchallenge for
analysis of proinflammatory cytokine levels in blood. The cisterna magna was
punctured 24 h postinfection, and the cerebrospinal fluid (CSF) was checked for
absence of red blood cells and spread on GC plates for a viable count.
FIG. 1. Genotypes and structures of the serogroup B N. menin-
gitidis LOS mutants used in this study. The wild-type NMB strain
and the pgm::Tn916 (enzyme deficiency: phosphoglucomutase),
rfaK::?(sp) (enzyme deficiency: ?1-2-N-acetyl-glucosamine trans-
ferase), and gmhB::Tn916 (enzyme deficiency: 2-D-manno-heptose
phosphatase) LOS mutants were assessed for virulence.
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Survival assay. Blood from CD46 transgenic mice was collected by retro-
orbital bleeding and heparin treated. Whole blood was diluted 1:1 with bacterial
suspension in Detroit cell media (1 ? 107bacteria) in a final volume of 300 ?l.
Bacterial survival was determined by serial dilutions and plating of the blood-
bacteria mixture at 3, 6, 9, and 12 h postinfection. Survival was monitored in
freshly isolated blood in two independent experiments.
Immunoassays for cytokines in serum. Concentrations of murine IL-6 and
TNF were measured in the sera of N. meningitidis-challenged mice at 24 h
postinjection by using sandwich ELISAs according to the manufacturer’s recom-
mendations (Diaclone).
Blood smears. Samples (approximately 5 ?l) of blood were obtained from the
tail vein of infected mice, and smears were made on glass slides. The slides were
fixed with methanol and left to dry. The smear samples were stained with
Wright’s stain according to the manufacturer’s recommendations (Sigma). Sam-
ples were analyzed by light microscopy.
Statistical analysis. Experiments were evaluated with Microsoft Excel. A
two-tailed Student’s t test was used to assess significance in the adhesion and
invasion assays and in the cytokine assays. Lethality experiments were assessed
using Fisher’s exact test, and bacteremia and in vitro blood survival was moni-
tored with a nonparametric Mann-Whitney test. All experiments were performed
in sets of at least two independent experiments.
RESULTS
Expression of virulence factors by LOS mutants. N. menin-
gitidis strain NMB and the LOS mutants have been previously
described and are shown in Fig. 1 (12, 15, 25, 29, 34, 41). The
mutant strains showed growth rates similar to that of the wild-
type strain, with the rfaK and gmhB mutants having lower final
bacterial numbers in the stationary phase (Fig. 2A); however,
growth rates were not significantly different (P ? 0.05). N.
meningitidis NMB and each mutant expressed the SDS-PAGE
LOS profiles expected (Fig. 2B) on the basis of their nuclear
magnetic resonance-defined structures (Fig. 1), and Western
blotting of whole-cell preparations revealed that the parent
and the mutants expressed PilC (Fig. 2B). The wild-type strain
expressed a two-band Opa repertoire and the pgm and rfaK
mutants expressed a third Opa of higher molecular weight,
while the gmhB mutant expressed one Opa in common with the
parent and a unique Opa of higher molecular weight (Fig. 2B).
All strains showed levels of capsule expression that were not
significantly different (P ? 0.05) from that of the wild-type
strain, NMB (Fig. 2C), as determined by a whole-cell ELISA
(34). Electron microscopy analysis showed that all strains were
piliated (Fig. 2D).
Mutations in LOS change the capacity of meningococci to
adhere to and invade epithelial cells. An assay to assess adhe-
sion of the wild-type strain and LOS mutants to the epithelial
cell line Detroit 562 revealed that mutations resulting in a
truncated structure of LOS modulated the capacity of the
meningococci to associate with these cells (Fig. 3A). The wild-
type strain NMB showed a level of adherence to Detroit 562
cells significantly lower than those of the mutants (P ? 0.05),
with approximately five associated bacteria per cell at 2 h
postinfection. Although the NMB strain appeared to express
less PilC (Fig. 2B), it is unlikely that this difference alone
mediated the decrease in adhesion, since the mutant strains
expressed similar levels of PilC but also had different capacities
to adhere. The pgm [Hep2(GlcNAc) 3-deoxy-D-manno-2-octu-
losonic acid (KDO2)-lipid A] and gmhB (KDO2-lipid A) mu-
tants demonstrated that between 50 and 80 bacteria adhered to
each cell, which was a level significantly enhanced (P ? 0.05)
compared to that seen for the rfaK mutant (Hep2-KDO2-lipid
A), which adhered at levels of approximately 20 bacteria per
cell. The percentage of associated meningococci that entered
and survived within cells was also examined using gentamicin
to kill extracellular meningococci. The wild-type strain showed
a steady increase in bacterial counts with time, with signifi-
cantly greater numbers of bacteria within cells at 3, 4, and 5 h
(P ? 0.05). The pgm and rfaK mutants showed decreased
FIG. 2. Phenotypic analysis of the wild-type meningococcal strain NMB and the pgm, rfaK, and gmhB LOS mutants. (A) Bacterial strains
showed similar growth rates, measured as optical density at 600 nm, in cell medium. (B) Lipooligosaccharide structure was assessed by Tricine
SDS-PAGE, and PilC and opacity proteins were examined by Western blotting using PilC-specific and Opa-specific antisera, respectively.
(C) Capsule expression was measured by ELISA, and the unencapsulated strain M7 was used as a negative control for capsule expression.
(D) Transmission electron microscopy of whole bacterial cells showed expression of pili.
1362PLANT ET AL.INFECT. IMMUN.

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bacterial counts compared to the wild-type strain at the early
time points (Fig. 3B), but at 6 h postinfection, the numbers of
these mutants in epithelial cells were not different from that of
the parent. The gmhB mutant was unable to enter and survive
within epithelial cells. The differences in intracellular numbers
of bacteria were not related to differences in sensitivity to
gentamicin (data not shown).
LOS structure affects the pathogenicity of meningococci and
mortality in a mouse model of infection. The pathogenicities of
the wild-type strain and LOS mutants were further examined
in a mouse model of meningococcal infection (9). Adult CD46
transgenic mice, which have increased susceptibility to menin-
gococci in the absence of additional supplements, such as iron,
were infected intraperitoneally with 3 ? 108meningococci/
mouse. The wild-type strain and the rfaK mutant showed sim-
ilar mortalities, with significant fatality noted for the wild-type
strain at 4 days postinfection and for the rfaK mutant at 3 days
postinfection (Fig. 4). The pgm mutant showed a mortality
significantly lower than the that of the wild-type strain and that
of the rfaK mutant. The gmhB mutant was avirulent in the
mouse model.
LOS structure affects the levels of meningococci in the blood-
stream. At 1 and 4 h postinfection, mice infected with the wild-
type strain NMB and the rfaK mutant had bacterial blood counts
(P ? 0.05) higher than those for to the pgm and gmhB mutant-
infected mice (Fig. 5). At 24 h postinfection, no significant differ-
ence in bacteremia levels in the pgm mutant- and rfaK mutant-
infected mice was observed, but the mice infected with the wild-
type strain still showed significantly enhanced bacteremia (P ?
0.05). Hence, it appears that a correlation between the level of
bacteremia at the early time points and mouse mortality exists.
Meningococci were detected in the CSF of all mice infected with
the wild-type strain NMB and in 12.5% of the mice infected with
the pgm mutant and 25% of mice infected with the rfaK mutant
(data not shown). No bacteria were detected in the CSF of mice
infected with the gmhB mutant. These data suggest that LOS
structure, including a requirement for inner core heptose, affects
the level of bacteremia seen following infection and the ability of
meningococci to enter into the CSF. It also suggests that the
mortality associated with the rfaK mutant is caused by sepsis
rather than meningitis, whereas the mortality associated with the
wild-type strain is probably due to both sepsis and meningitis.
In vitro survival in whole mouse blood. In order to evaluate
if the wild-type strain and the LOS mutants differed in their
abilities to survive in blood, bacteria were incubated with hep-
arin-treated mouse blood in vitro. As shown in Fig. 6, each of
the mutations resulting in truncation of meningococcal LOS
structure was associated with a decrease in the capacity of
meningococci to survive in whole blood taken from CD46
mice. These data suggest that the wild-type LOS structure best
promoted the survival of meningococci in blood and correlated
with the persistent bacteremia of the wild-type strain in the
mouse model. However, pathogenesis in the mouse model was
not solely mediated by the capacity to survive in blood, since no
significant differences were observed in the survival rates of the
LOS mutants in the mouse blood in vitro (P ? 0.05), in con-
trast to the differences observed in mouse mortality rates.
Mouse mortality is associated with high serum cytokine levels.
Meningococcal sepsis-associated death is often due to hypoten-
sion and organ failure characteristic of septic shock, which occurs
as a result of uncontrolled release of proinflammatory cytokines,
such as IL-6 and TNF. Host cytokine responses were analyzed by
measuring cytokine levels in mouse serum 24 h postinfection by
ELISA.AsshowninFig.7A,IL-6increasedsignificantlyinserum
after i.p. challenge with the wild-type strain NMB and the rfaK
mutant. IL-6 was not elevated in sera of mice at 24 h after
FIG. 3. Adhesion to (A) and invasion into (B) Detroit 562 epithe-
lial cells by the wild-type strain NMB and the meningococcal LOS
mutants. Viable counts of adherent meningococci were determined 2 h
after infection, and the percentages of adhered bacteria that invaded
were determined after 3, 4, 5, and 6 h. Significant increases in adhesion
over that of the wild-type strain are indicated with asterisks (P ? 0.05).
FIG. 4. Survival of mice following i.p. infection of CD46 transgenic
mice (n ? 17) with the wild-type NMB strain and the LOS mutants. The
infection dose was 3 ? 108CFU/mouse. Survival of mice was monitored
over a 6-day period. Significant mortality was determined using Fisher’s
exact test and is indicated with an asterisk (P ? 0.05).
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infection with the pgm mutant or the gmhB mutant. The wild-type
strain, the rfaK mutant, and the gmhB mutant induced serum
TNF (Fig. 7B). TNF was not induced in serum following i.p.
infection of the pgm mutant. Cytokine levels did not correlate
with a shift of neutrophil levels in blood samples following chal-
lenge (Fig. 7C), since all strains induced comparable influxes of
neutrophils into the blood at 8 h postinfection. Interestingly, the
gmhB mutant, which was avirulent in the mouse model, did in-
duce TNF but did not induce IL-6 and also caused increased
neutrophil infiltration after infection.
DISCUSSION
LOS, or endotoxin, including the ability to express a full
repertoire of LOS structures, is an important virulence factor
involved in multiple steps in the pathogenesis of N. meningitidis
and is a significant contributor to the pathologies of meningo-
coccal disease (2–4, 15, 23, 36). In this study, wild-type menin-
gococci of serogroup B strain NMB (LOS immunotype L2/4)
and mutants of this strain with defined mutations in enzymes
required for LOS assembly (12, 15, 25, 29, 34, 41) were used to
further determine the role of the LOS structure in meningo-
coccal virulence. Variations of meningococcal LOS ? chain
and inner core structures are known to occur at high frequen-
cies (15) and to influence pathogenesis. Immunotypes with
full-length, sialylated ? chain L3/7/9 and L2/4 immunotypes are
those most frequently associated with invasive disease, while
LOS with a truncated ? chain {L8 immunotype [Gal?-Glc?-
Hep2(GlcNAc, Glc?) KDO2-lipid A]} are more often found
on carrier isolates (11). However, recent evidence indicates the
L2/4 immunotype (strain NMB) has the genetic capability to
express all other immunotypes (13).
Piliation is known to be critically important in the initial
steps of the adhesion cascade of pathogenic Neisseria spp. (21).
The wild-type parent and all mutants showed similar levels of
piliation; however, the wild-type strain had slightly decreased
PilC expression compared to the mutant strains. Other impor-
tant meningococcal surface components influencing adherence
and invasion are capsule, Opa, and Opc (2, 5, 38). The parent
and mutants expressed the same amounts of capsule, although
the Opa repertoire expressed by all the mutants was different
from that expressed by the parent strain. To analyze the inter-
action between bacteria and host cells, in vitro adhesion and
FIG. 5. Bacterial counts (CFU/ml) in blood of CD46 transgenic mice challenged i.p. with wild-type strain NMB and meningococcal LOS
mutants. Blood samples were collected 1, 4, 8, and 24 h following infection and spread on GC plates for a viable count. Significant decreases in
bacteremia compared to that seen with the wild-type strain have been calculated using a Mann-Whitney test and are indicated with asterisks (P
? 0.05).
FIG. 6. Survival of the wild-type strain NMB and meningococcal
LOS mutants in whole mouse blood. Bacterial survival in whole blood
from CD46 transgenic mice was determined 3 to 12 h after infection by
plating bacteria on GC agar and counting CFU. Significant decreases
in survival from the level of the wild-type strain have been calculated
using a Mann-Whitney test and are indicated with asterisks (P ? 0.05).
1364PLANT ET AL.INFECT. IMMUN.