From the Departments of Microbiology,
¶ Biochemistry, and
Medicine, Division of Infectious
Diseases, The Inflammation Program, University of Iowa and
Veterans' Administration Medical Center, Iowa City, Iowa 52242
Received for publication, October 11, 2000, and in revised form, November 8, 2000
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ABSTRACT |
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To facilitate studies of the molecular
determinants of host-meningococcal lipooligosaccharide (endotoxin)
interactions at patho-physiologically relevant endotoxin concentrations
(i.e. Lipooligosaccharide (LOS;
endotoxin)1 is a specialized
bacterial glycolipid that is structurally related to lipopolysaccharide (LPS) and is expressed on the outer leaflet of the outer membrane of
certain Gram-negative bacteria (1, 2). LPS and LOS activation of
eukaryotic cells is facilitated by their interaction with specific host
proteins that bind the conserved endotoxin lipid A moiety (3, 4).
Meningococcal lipid A consists of an
O-phosphorylethanolamine-substituted The most sensitive responses of mammals to LOS involve the serum
protein, LPS-binding protein (LBP), and either membrane-bound or
extracellular soluble forms of CD14 (mCD14 and sCD14, respectively) (3,
14-18). LBP is a 60-kDa glycoprotein that binds to lipid A and
catalyzes the interaction of LOS and LPS with CD14 (14, 19-21). The
mCD14 is a glycosylphophatidylinositol-linked glycoprotein expressed on
the surface of myeloid-derived cells (22-24) and certain other cell
types (25-27). sCD14 is a circulating glycoprotein involved in
activation of endothelial cells and other cell types by LOS and LPS
(28, 29). Subsequent activation of host cells involves other host cell
proteins including Toll-like receptors (e.g. TLR-4; Refs. 30
and 31) and other membrane-associated proteins (3, 32). In addition,
interaction of endotoxin with other host proteins (e.g.
lipoproteins (33, 34), bactericidal/permeability-increasing protein
(35), and scavenger receptors (36)) may be critical for the clearance
and detoxification and, hence, regulation of the host response to endotoxin.
Better understanding of the factors regulating meningococcal
mobilization of LOS and the interactions of LOS with defined host
proteins and cells would be greatly facilitated by an efficient method
of labeling LOS. In this report we describe a method for high specific
radiolabeling of the FA moieties of meningococcal lipid A, similar to a
previously published method for radiolabeling Escherichia
coli LPS to high specific activity (37). This method involves the
generation of an acetate auxotroph by mutagenesis of the pyruvate
dehydrogenase (PDH) coding region. The E1 component of the PDH complex
is encoded by the aceE gene in E. coli
(pdhA in Neisseria spp.; Ref. 38) and takes part
in the overall conversion of pyruvate to acetyl-CoA and
CO2. The chief obstacle to efficient radiolabeling of LOS
lipid moieties is the production of nonradiolabeled acetyl-CoA from
pyruvate and the incorporation of this pyruvate-derived acetate into
nascent lipid chains. Here we describe the generation of meningococcal
acetate auxotrophs. These mutant strains have been used to produce
radiolabeled LOS of high specific radioactivity for the purpose of
studying the physical characteristics of purified LOS and the
relationship of the physical properties of LOS to its biological
activity toward mammalian cells.
Materials--
E. coli INV Bacterial Culture Conditions--
Encapsulated Neisseria
meningitidis serogroup B strain NMB and an acapsular mutant of NMB
(PBCC7232-NMB
E. coli strains GM2163(dam-), DH5 Cloning the Meningococcal pdhA Gene--
The University of
Oklahoma gonococcal FA1090 genome data base was searched using
the BLASTp algorithm (42). The putative gonococcal gene encoding the
PDH E1 component (pdhA) was identified by sequence homology
with E. coli AceE (GenBankTM accession 2506964)
and Hemeophilus influenzae Rd putative AceE (GenBankTM accession 1574164). Oligonucleotide primers
(primer 1: 5'-GAA GGA CGG GCA AGA CCA GAT; primer 2: 5'-GCA TTC CGG GCG
ACC AAA ACA) were used to generate a 2,080-base pair polymerase chain reaction product from gonococcal FA1090 genomic DNA, which contained the putative pdhA open reading frame. The amplification
product was ligated to the cloning vector, pCR2.1, and the resulting
product (pACE1) was used to transform E. coli strain
INV
The insert within pACE1 was isolated by EcoRI digestion
followed by agarose gel filtration and affinity spin column
purification and ligated to EcoRI-digested pBlueScript SK
cloning vector. Transformants of E. coli INV Generation of Meningococcal Acetate
Auxotrophs--
Transformation of the naturally competent meningococci
has been described previously (44, 45). In brief, meningococcal strain,
NMB, was incubated with pACE4 (~1 × 107
colony-forming units/50 ng DNA) on GC agar for 4 h at
37 °C and 5% CO2/95% atmosphere. The resulting
transformants were incubated on supplemented brain-heart infusion
medium agar (see "Bacterial Culture Conditions"). Genomic
DNA preparations from selected colony-purified transformants were
analyzed by polymerase chain reaction using primers 1 and 2 (above) to
verify the presence of the Km cassette within the putative
pdhA open reading frame (data not shown). The Km-resistant,
Ap-sensitive NMB transformant NMBACE1 was chosen for further study.
This same method was used to generate an acetate auxotroph
(NMBACE2) of the acapsular NMB mutant (PBCC-
7232-NMB Assay of Acetate Requirements for Bacterial Growth--
Acetate
auxotrophy of NMBACE1 and NMBACE2 was established by measuring
bacterial growth in MDM supplemented with 0-5 mM sodium acetate. Bacterial growth was measured as increasing turbidity using a
Klett-Summerson Photoelectric Colorimeter (green filter) (Manostat, New
York, NY).
Assay of Bacterial PDH Activity--
Lysates of bacteria were
analyzed for PDH activity by measuring the conversion of NAD to NADH
(A340 nm) in the presence of pyruvate and
cocarboxylase. Bacteria were grown to mid-log phase
(A660 nm = 0.4) in supplemented MDM with 3.5 mM acetate. The bacteria were washed and resuspended in 50 mM MOPS, pH 7.0, containing 1 mM
MgCl2 and lysed in a French pressure cell at 10,000 p.s.i.
(SLM Aminco). Lysates were cleared by centrifugation, and the resulting
supernatants were assayed for PDH activity as described (46).
Metabolic Labeling of LOS during Growth of Meningococci
with [1,2-14C]Acetate: Quantitation of
[14C]3-OH-FA--
Meningococci were harvested from
supplemented GC agar, resuspended in MDM containing 1.5 mM
sodium acetate to a density of A660 nm = 0.05, and incubated to late log phase. Incubations contained 2 µCi/ml of
[1,2-14C]sodium acetate to measure synthesis of
14C-LOS during bacterial growth or 160 µCi/ml of
[1,2-14C]sodium acetate to prepare 14C-LOS of
high specific radioactivity. Synthesis of 14C-LOS was
monitored by measuring incorporation of [14C]acetate into
3-OH-fatty acids that are unique to LOS (5, 47, 48). Whole cells were
treated sequentially with 4 N HCl and 4 N NaOH
at 90 °C to release ester- and amide-linked FA and then extracted
with chloroform/methanol to recover labeled 3-OH- and nonhydroxylated
FA in the chloroform phase (39). Nonhydroxylated- and 3-OH-FA were
separated by TLC (0.25-mm silica gel G HPTLC; Analtech, Newark, DE)
using petroleum ether/diethyl ether/glacial acetic acid (70:30:1 v/v/v)
as the solvent system. Individual FA were resolved by reverse phase TLC
(0.2 mm HPTLC, RP-18; Merck) using acetonitrile/acetic acid (1:1 v/v)
as the solvent system (39) and identified by comigration with purified
14C-FA. The relative amounts of labeled 3-OH-FA and
nonhydroxylated FA were determined by image analysis (PhosphorImager;
Molecular Dynamics, Sunnyvale, CA) using a tritium screen that
permitted quantitation of as little as 200 cpm after 24 h of
exposure. From these data, the absolute amount of radiolabeled 3-OH-FA
could be calculated ((14C cpm in chloroform phase after
acid/base treatment) × (fraction of 14C-labeled
species in chloroform phase migrating as 3-OH-FA during TLC)).
14C-Labeled species were measured in a Beckman LS 5000TD
liquid scintillation counter (Beckman Instruments, Inc., Fullerton,
CA). No free [14C]3-OH-FA was detected without acid/base
treatment, indicating that these FA were covalently linked to LOS.
Chemical Analysis of 3-OH-FA by GC-MS--
The amount of 3-OH-FA
in various purified 14C-LOS preparations was measured by
GC-MS using a gas chromatograph with a DBXLB column (30 m × 0.25 mm × 0.25 µm; J & W Scientific) connected to a Hewlett
Packard Mass-ENGINE. Samples were prepared from bacteria metabolically
labeled with 1 µCi/ml [14C]acetate in 2 mM
sodium acetate to ensure that recovered FA contained mainly nonisotopic
carbon. Samples were chemically hydrolyzed and extracted as described
above. Recovered free FA in the chloroform phase were spiked with 15:0
to provide an internal standard. Perfluorobenzyl derivatives were
prepared by reaction of FA with Purification of 14C-LOS--
14C-LOS was
purified from radiolabeled bacteria by a modification of the hot
phenol-water method. In brief, bacteria were harvested after growth to
late log phase in MDM containing [14C]acetate,
resuspended in 0.1-0.4 vol of lysozyme (2 mg/ml), and incubated
overnight with gentle shaking at room temperature. Nuclease (2.5 units/ml) was added, and the sample was incubated with gentle shaking
(200 rpm) for 3 h at 37 °C. After addition of an equal volume
of hot phenol (65 °C), the sample was incubated at 65 °C for 20 min with vigorous shaking and then placed on ice for 15 min. The
aqueous and phenol phases were separated by centrifugation at 4000 rpm
for 5 min. After removal of the aqueous phase, the remaining phenol
phase was re-extracted twice with an equal volume of water. The
recovered aqueous phases were combined, and LOS was precipitated by the
addition of 0.1 vol of 3 M sodium acetate and 2 vol of 95%
ethanol. After overnight incubation at Sephacryl S500 Chromatography--
Aliquots of resuspended
and sonicated 14C-LOS were diluted in HEPES-buffered (10 mM, pH 7.4) Hanks' balanced salts solution (HBSS with
divalent cations) supplemented with 0.1-1% human serum albumin and
incubated at 37 °C for 15 min before gel filtration chromatography.
Samples containing from 4 to 8 µg of LOS were applied in 0.2-0.5-ml
samples on a 1.5 cm × 17.5 cm column of Sephacryl S500 HR
equilibrated in the same buffered solution. Fractions (1 ml) were
collected at a flow rate of 0.5 ml/min at room temperature. Aliquots of
the collected 14C-LOS fractions were analyzed by liquid
scintillation counting. Recoveries of the radiolabeled LOS ranged from
70 to 90%. To preclude contamination of purified LOS preparations, all
solutions were pyrogen-free and sterile. The glass columns and
connecting tubing were either autoclaved or washed extensively with
70% ethanol. After chromatography, selected fractions were pooled and
passed through syringe sterile filters (pore size, 0.22 µm) with
greater than 90% recovery of labeled material in the sterile filtrate. Fractions were stored under sterile conditions at 4 °C for up to 3 months with no detectable changes in chromatographic or functional properties.
The column was calibrated using 14C-oleate-labeled E. coli PL-2 and [14C]acetate as markers of the void
(10-11 ml) and inclusion volumes (25-26 ml), respectively, in column
buffer with or without 0.1% albumin. Additional standards included
blue dextran (estimated molecular mass, 2000 kDa), thyroglobulin (650 and 1300 kDa, monomers and dimers, respectively), ferritin (440 kDa),
catalase (232 kDa), and aldolase (158 kDa). Elution profiles of
bacteria, blue dextran, and acetate were unaffected by the presence of
albumin in the column buffer.
Compositional Analysis of Purified
14C-LOS--
14C-FA composition of purified
14C-LOS was determined as described above. Quantitation of
14C-fatty acids, resolved by reverse phase TLC, was
achieved by image analysis. Dilute samples were lyophilized before
chemical hydrolysis. SDS-PAGE of 14C-LOS was carried out in
16% acrylamide containing glycerol. 14C-LOS was visualized
by autoradiography. To permit analysis of samples containing as little
as 50 ng LOS/ml, selected fractions from Sephacryl S500 chromatography
were concentrated by ultrafiltration using a Centricon-100 and
rechromatographed on Sephacryl S200 in HEPES-buffered HBSS to deplete
fractions containing LOS of albumin. Recovered LOS-containing fractions
were desalted using Centricon-100. Recoveries of 14C-LOS at
each step was Human Cells--
HUVEC were routinely cultured on
collagen-coated plasticware (Costar, Cambridge, MA) at 37 °C, 5%
CO2, and 95% relative humidity in endothelial cell basal
medium supplemented with 5% fetal bovine serum, 12 µg/ml bovine
brain extract, 10 ng/ml human endothelial growth factor, 1 µg/ml
hydrocortisone, and 50 µg/ml gentamicin. Cells were subcultured and
grown to confluence (~4-5 days). Cell monolayers were then washed
twice with warm HBSS to remove traces of serum before adding
experimental media. Experiments were done with cells between passages 2 and 6.
Human polymorphonuclear leukocytes (PMN) and peripheral blood
mononuclear cells (PBMC) were obtained from heparinized venous blood
from healthy volunteers as described (49) and stored on ice in sterile
pyrogen-free HBSS supplemented with 10 mM
D-glucose until use (<60 min). Purified PMN contained
<1% monocytes and PBMC contained <2% PMN.
Assay of HUVEC Activation--
Cells in 48-well tissue culture
plates were incubated for 20 h at 37 °C, 5% CO2,
and 95% humidity in Dulbecco's minimal essential medium and 0.1%
albumin with various concentrations of 14C-LOS ± LBP
(0.2 µg/ml) and sCD14 (0.5 µg/ml). Activation of HUVEC was
monitored by measuring accumulation of extracellular IL-8 by
enzyme-linked immunosorbent assay as described (50). Equivalent responses were observed with 0.1-0.5 µg/ml of LBP and 0.25-1.0 µg/ml sCD14.
Assay of Association of [14C-LOS with
HUVEC--
Cells were grown to confluence in 6- or 12-well tissue
culture dishes, washed to remove serum, and incubated in Dulbecco's minimal essential medium and 0.1% albumin with
14C-LOS-containing aggregates (3-5 ng/ml; ~2000-3000
cpm) ± LBP (0.1 µg/ml) and sCD14 (0.25 µg/ml) for 20 h
at 37 °C in 5% CO2. After the incubation, the
extracellular medium was removed, and the cells were washed three times
with 10 mM HEPES buffer-HBSS (pH 7.4) containing 0.1%
albumin. The adherent cells were dislodged by scraping with a rubber
policeman and transferred to a fresh tube. After sedimentation, the
cells were lysed in 200 µl of 2% SDS for 10 min at 37 °C.
Aliquots of the recovered supernatants, washes, and cell lysates were
analyzed by scintillation counting. The percent cell association of
14C-LOS was calculated as (cpm recovered in cell
lysates)/(total cpm recovered) × 100. Total cpm recovery was
routinely >90%. Adsorption of added 14C-LOS to tissue
culture wells not containing cells was <3%, and no
14C-LOS was recovered after scraping of plates in the
absence of cells.
Assays of Human Leukocyte Activation--
Purified human PMN
(760,000 PMN/well) were incubated for 60 min at 37 °C in 96-well
Optiplates with or without 14C-LOS and/or LBP (100 ng/ml)
in HEPES-buffered HBSS (pH 7.4) containing 0.1% albumin and 1 × 10 Generation of Acetate Auxotroph of NMB (NMBACE1)--
To
facilitate metabolic labeling of meningococcal LOS to high specific
radioactivity, we sought to generate an acetate auxotroph of NMB. PDH
activity is essential for conversion of pyruvate to acetyl-CoA and
CO2 and thought to be encoded in meningococci within a
single open reading frame (pdhA) (38). We generated a mutant of NMB, NMBACE1, by allelic exchange of the wild-type pdhA
gene of NMB with a copy of pdhA disrupted by insertion of a
Kmr cassette.
Growth of the parent strain, NMB, in MDM is unaffected by addition of
up to 10 mM sodium acetate (data not shown). In contrast, growth of NMBACE1 in MDM depended on the addition of acetate (Fig. 1A), demonstrating that
NMBACE1 is an acetate auxotroph. Maximal growth of NMBACE1 in this
medium required Enhanced Metabolic Labeling of LOS in NMBACE1--
To determine
whether acetate auxotrophy permitted greater metabolic radiolabeling of
LOS, NMB and NMBACE1 were grown to maximal density in medium
supplemented with 1.5 mM sodium acetate including 2 µCi/ml [1,2-14C]sodium acetate. Metabolic labeling of
LOS was measured as formation of [14C]3-OH-FA that are
unique to lipid A of LOS (5). All material containing
[14C]3-OH-FA was recovered at the interphase along with
intact LOS after Bligh/Dyer extraction of radiolabeled bacterial
suspensions (41). Free [14C]3-OH-FA was detected only
after chemical hydrolysis (i.e. acid/base treatment) of
bacteria to release the fatty acids from ester and amide linkages in
lipid A (data not shown and Ref. 41). Metabolic radiolabeling of
3-OH-FA (i.e. LOS) was nearly 10 times greater (8.8 ± 1.8; n = 3) in NMBACE1 than in NMB.
Isolation of [14C-LOS-containing Aggregates of
Different Size and Composition--
To generate
14C-labeled LOS of high specific radioactivity, NMBACE1 was
biosynthetically labeled in continuous culture using [1,2-14C]acetate as the only exogenous source of acetate.
After harvesting the bacteria in late log phase, 14C-LOS
was purified by a modification of the hot phenol-water method (65). Gel sieving chromatography utilizing Sephacryl S500
resolved two major peaks of radiolabeled material (Fig.
2A). Analysis of the eluted
radiolabeled material by chemical hydrolysis and extraction suggested
that the major fractions within each individual peak (i.e.
fractions 11-13 within peak A and fractions 19-23 within peak B) were
of similar composition. However, the chemical content of peaks A and B
differed (Fig. 2B). After acid/base treatment, nearly all
radiolabeled material from peak A was recovered in the chloroform phase
and migrated, during reverse phase TLC, as mainly 3-OH-12:0, 3-OH-14:0,
and 12:0 in proportions (Fig.
3A and Table
I) that matched the previously determined
FA composition of NMB LOS (5, 47).
In contrast, after acid/base treatment, nearly equal amounts of
the radiolabeled species within peak B partitioned into the chloroform
and water-methanol phases (Fig. 2B). Labeled species in the
chloroform phase included mainly those FA present uniquely in LOS (Fig.
3A). In addition, a small amount of labeled material comigrating with palmitic acid was observed in peak B but not peak A
(Fig. 3A and Table I). The polysialic acid capsule of NMB is
a polymer of N-acetylneuraminic acid linked to dipalmitin (52). Both N-acetylneuraminic acid and palmitate would be
labeled during growth with [14C]acetate and, after
chemical hydrolysis and extraction, would partition into the
water-methanol (]14C[(poly)N-acetylneuraminic
acid) and chloroform ([14C]palmitate) phases,
respectively. Thus, the composition of peak B suggests the presence of
14C-LOS and 14C-capsule that coelute during gel
filtration chromatography through Sephacryl S500. Rechromatography of
the peak fractions comprising peak A and peak B (Fig. 2A)
each produced single, symmetric labeled peaks representing either peak
A (Fig. 2C) or peak B (Fig. 2D), respectively.
The chromatographic as well as functional properties (see below) of
peaks A and B do not change during storage at 4 °C for at least 3 months.
By gel sieving and FA analysis, peak A is essentially equivalent to NMB
LOS extracted and purified by the hot phenol-water method and
sedimented by ultracentrifugation. Peak A corresponds to aggregates of
essentially pure NMB LOS with an apparent molecular mass of Recovery of 14C-LOS-containing Aggregates of
Different Size Does Not Depend on the Presence of Sialylated LOS or
Polysialic Acid Capsule--
The probable presence of capsular
polysaccharide in peak B from NMBACE1 raised the possibility that the
capsule influenced the physical properties of aggregates containing
LOS. To address this possibility, we constructed an acetate auxotroph
(NMBACE2) from PBCC7232-NMB ( Interactions of 14C-LOS with Cultured Endothelial Cells
Are Inhibited by the Presence of Capsule but Unaffected by the Size of
the Aggregates--
The purification of physically distinct aggregates
of LOS in sterile medium compatible with sensitive bioassays permitted comparison of the functional properties of LOS presented in different aggregate forms. All LOS-containing aggregates triggered
dose-dependent activation of the synthesis and secretion of
IL-8 by HUVECs (Fig. 5) that was much
greater in the presence of added LBP and sCD14 (Table
II and data not shown). However, when
equivalent amounts of LOS were added, peak A from NMBACE1 was nearly
five times more potent than peak B from NMBACE1 in eliciting IL-8
release (Fig. 5, left panel). In contrast, LOS from
peak A and B derived from NMBACE2 were nearly equipotent toward HUVEC
and closely similar in activity to that of LOS in peak A from NMBACE1
(Fig. 5, left panel). Differences in activity paralleled
differences in association of 14C-LOS from these aggregates
with HUVECs (Fig. 5B). These findings indicate that the
ability of purified meningococcal LOS to interact with and activate
endothelial cells is inhibited by the presence of capsular
polysaccharide but unaffected by differences in the size of aggregates
containing LOS.
Differences in Activation of HUVECs by LOS Aggregates Correlates
with LBP/sCD14-dependent Disaggregation of LOS--
LBP-
and sCD14-dependent disaggregation of endotoxin may be
important in endotoxin-induced activation of cells such as endothelial cells that lack the glycosylphosphatidylinisitol-linked membrane-bound form of CD14 (mCD14) (3, 18, 53). Therefore, differences in the ability
of LOS-containing aggregates to activate HUVECs could be explained by
differences in the susceptibility of LOS within different aggregates to
be extracted by LBP and sCD14. To test this possibility, we compared
LBP and sCD14-dependent disaggregation of LOS in peak A and
B from NMBACE1 and 2 by utilizing gel sieving to monitor LOS
disaggregation. Incubations at 37 °C of LOS aggregates ± LBP
and/or sCD14 and subsequent column chromatography were carried out in
cell culture medium to mimic as closely as possible the conditions of
the bioassays. After incubation for 15 min with both LBP (500 ng/ml)
and sCD14 (5 µg/ml), nearly all LOS within peak A of NMBACE1 (Fig.
6A) and within peaks A and B
from NMBACE2 (Fig. 6, C and D) was transformed to
a form of lower apparent molecular size (i.e. peak C) as
shown by its slower elution through Sephacryl S500. Incubation with
either protein alone produced no apparent disaggregation of LOS (Fig.
6A and data not shown). Similar effects of LBP and sCD14
were seen over a broad range of LOS concentrations (10-400 ng/ml) and
incubation times (15 min to 24 h) (data not shown), suggesting
that peak C represents the final product of the concerted action of LBP and sCD14 on aggregates of LOS. Peak C derived from peak A of NMBACE1
and peak C from peaks A and B of NMBACE2 were chromatographically indistinguishable and coeluted with proteins with molecular masses of
~1.5-2.5 × 105 Da (Fig. 6). In contrast to LOS
within peak A and B, disaggregated LOS within peak C bound to and
activated HUVECs in the absence of freshly added LBP and sCD14 (Table
II).
LOS within peak B from NMBACE1 was relatively resistant to
disaggregation by LBP and sCD14 (Fig. 6B).
14C-FA analysis of collected fractions confirmed the
limited disaggregation of LOS within peak B by LBP and sCD14 (data not
shown). These findings suggest that the ability of LBP and sCD14 to
disaggregate and deliver LOS to HUVECs leading to cell activation is
not dependent on the size of aggregates containing LOS but is inhibited
by the presence of capsular polysaccharide.
Activation of Leukocytes by LOS-containing Aggregates Is Affected
by the Size of the Aggregates but Not by the Presence of
Capsule--
Potent activation by endotoxin of cells containing mCD14
(e.g. monocytes, PMN) normally requires only LBP and, hence,
not disaggregation (Fig. 6A) of endotoxin before delivery of
endotoxin to these host cells. We therefore compared
LBP-dependent activation of leukocytes by the different
LOS-containing aggregates from NMBACE1 and 2. At comparable doses of
LOS, peak B from both NMBACE1 and NMBACE2 activated PMN and PBMC
(monocytes) more strongly than peak A (Fig.
7). The greater potency of peak B was
manifest both in the induction of synthesis and secretion of IL-8 from
monocytes (Fig. 7, A and B) and in the rapid
activation of the respiratory burst oxidase of PMN (measured as
lucigenin-enhanced chemiluminescence; Fig. 7C). In all
cases, activation was LBP-dependent and inhibited by BPI
and by a neutralizing mAb (MY-4) to CD14 (data not shown), indicating
that cell activation was LOS-dependent and mediated through
mCD14. The relatively low activity of LOS in peak A toward these cells
could be greatly amplified by pretreatment with LBP and sCD14 to form
peak C (Fig. 7A). These findings indicate that, in contrast
to activation of endothelial cells, activation of leukocytes by
purified LOS is affected by the size of the aggregates in which LOS is
presented but not by the presence of capsule.
We have demonstrated that by mutating the pdhA gene of
N. meningitidis strain NMB, LOS can be metabolically
radiolabeled to high specific radioactivity with
[14C]acetate during continuous culture. This method was
employed previously for high specific radiolabeling of LPS (37) in
E. coli strain LCD25
( By gel filtration chromatography of metabolically labeled LOS, we have
identified and separated two major subpopulations of LOS existing in
different aggregation forms. This has permitted assessment of the
effect of aggregation state on the biochemical reactivity and
biological activity of LOS. The broad resolving range of Sephacryl S500
was particularly important as aggregates exist that vary in apparent
molecular mass from ~1-20 × 106 Da and bioactive
complexes containing LOS (peak C; Fig. 5) are ~105 Da.
Chromatography was performed in buffered salts solution containing physiological concentrations of divalent cations and albumin to approximate the host extracellular environment and permit the examination of the interaction of fractionated LOS with isolated host
proteins and cells under similar conditions. Recoveries of LOS from
this resin were high (>80%). Recovery was similar with or without
albumin for LOS from NMBACE1, but 1% albumin was needed for high
recovery of LOS from NMBACE2, possibly reflecting the greater
hydrophobicity of nonsialylated LOS.
Previous studies have revealed that the responsiveness to
endotoxin of cells lacking mCD14 (e.g. endothelial cells) is
sCD14-dependent (18, 28, 29). Soluble CD14 alone or much
more efficiently in concert with LBP can disaggregate LPS (18, 19, 54),
and the small complexes of sCD14 and LPS that are formed can directly activate cells (19, 55). From these observations, it has been inferred
that these complexes represent the bioactive form of LPS and the means
by which sCD14 promotes endotoxin action. Our findings support and
extend this view. Disaggregation of LOS by LBP and sCD14 correlated
closely with the ability of an aggregate containing LOS to activate
HUVEC. In contrast to these earlier studies, we assayed disaggregation
and cell activation under similar experimental conditions, increasing
the likelihood that the disaggregation of LOS we detected was
functionally important. In contrast to the aggregates containing LOS
purified by Sephacryl S500 chromatography (i.e. peak A and
B), disaggregated LOS within peak C did not need freshly added LBP and
sCD14 to activate cells (Table II). These properties are consistent
with the interpretaion that peak C is the bioactive form of LOS toward
HUVECs and, hence, that differences in its formation account for
differences in delivery and bioactivity toward endothelial cells of LOS
from different aggregates (Fig. 8). LOS
within peak C is associated with sCD14 but not
LBP3; studies are in progress
to fully define the composition and stoichiometry of these
complexes.
10 ng/ml), we have generated acetate auxotrophs
NMBACE1 from encapsulated Neisseria meningitidis (serogroup
B, strain NMB) and NMBACE2 from an isogenic bacterial mutant lacking
the polysialic acid capsule. Growth of the auxotrophs in medium
containing [14C]acetate yielded
14C-lipooligosaccharides containing ~600 cpm/ng. Gel
sieving resolved 14C-lipooligosaccharide-containing
aggregates with an estimated molecular mass of
20 × 106 Da (peak A) and ~1 × 106 Da (peak
B) from both strains. Lipooligosaccharides in peaks A and B had the
same fatty acid composition and SDS-polyacrylamide gel
electrophoresis profile. 14C-Labeled capsule
copurified with 14C-lipooligosaccharides in peak B from
NMBACE1, whereas the other aggregates contained only
14C-lipooligosaccharide. For all aggregates,
lipopolysaccharide-binding protein and soluble CD14-induced delivery of
lipooligosaccharides to endothelial cells and cell activation
correlated with disaggregation of lipooligosaccharides. These processes
were inhibited by the presence of capsule but unaffected by the size of
the aggregates. In contrast, endotoxin activation of cells containing
membrane CD14 was unaffected by capsule but diminished when endotoxin
was presented in larger aggregates. These findings demonstrate that the
physical presentation of lipooligosaccharide, including possible interactions with capsule, affect the ability of meningococcal endotoxin to interact with and activate specific host targets.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(1'
6)-linked
D-glucosamine backbone containing symmetrically arranged
ester- and amide-linked 3-hydroxy fatty acids (3-OH FA) (5). Among its
reported effects, LOS promotes the release of cytokines such as IL-1,
IL-6, IL-8, and tumor necrosis factor-
from mammalian cells and
induces the activation of host cells such as macrophages and
neutrophils and endothelial cells (6-9). These responses are important
for mobilization of host defenses against invading Gram-negative
bacteria but can also lead to many of the most severe pathologic
consequences of uncontrolled invasive Gram-negative bacterial
infections. Thus, the severity of outcome in meningococcemia appears
closely correlated to plasma levels of LOS (10-13). The unusually
acute progression of invasive meningococcal disease may be linked to
both the high levels of LOS in host tissues and the intrinsically
potent effects of LOS toward host leukocytes and
endothelium.2
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
F' and DH5
were
obtained from Invitrogen (San Diego, CA) and Life Technologies,
Inc., respectively. Plasmids pCR2.1 pBlueScript cloning vector
were purchased from Invitrogen and Stratagene (La Jolla, CA),
respectively. Bacteriological media were obtained from Difco
Laboratories, Inc. (Detroit, MI). Recombinant bactericidal/permeability-increasing protein (rBPI21), LBP,
and sCD14 were obtained from XOMA Corp. (Berkeley, CA).
14C-Labeled fatty acids were either purchased from Amersham
Pharmacia Biotech (myristic, palmitic, and oleic acids, 14:0, 16:0,
and 18:1) or purified from 14C-labeled E. coli
LPS as previously described (39). [1,2-14C]Acetic acid
sodium salt (110 mCi/mmol) was purchased from Moravek Biochemicals,
Inc. (Brea, CA). Unlabeled FA used as standards for gas
chromatography-mass spectrometry (GC-MS) (12:0, 14:0, 15:0, 16:0,
3-OH-12:0, and 3-OH-14:0) were obtained from Matreya, Inc. (Pleasant
Gap, PA). Sephacryl S-500 HR and a protein molecular weight standards
kit were purchased from Amersham Pharmacia Biotech. Centricon-100
filters were from Amicon (Beverley, MA). Neutralizing anti-CD14 mAb
(MY-4) and an isotype matched irrelevant mAb were from Coulter Corp.
(Miami, FL). Human umbilical vein endothelial cells (HUVEC),
endothelial cell basal medium, fetal bovine serum, bovine brain
extract, human endothelial growth factor, hydrocortisone, and
gentamicin were from Clonetics (San Francisco, CA). Bovine type 1 collagen was obtained from Collaborative Research Products. 96-well
Optiplates were from Anthos Labtec Instruments (New Castle, DE), and
lucigenin was from Sigma.
siaA-D) were routinely cultured at 37 °C
in 5% CO2/95% atmosphere on GC agar supplemented with 1×
isovitalex. The latter strain was a gift from Wyeth-Lederle Vaccines
and Pediatrics (West Henrietta, NY). Meningococcal transformants were
selected and maintained on brain-heart infusion medium agar supplemented with 2.5% fetal calf serum, 1× isovitalex, 10 mM sodium acetate, and 45 µg/ml kanamycin (Km).
Radiolabeling and acetate titration experiments were carried out in
Morse's defined broth medium (MDM) (40) supplemented with 1- isovitalex, 10 mM sodium bicarbonate, and, as indicated,
various concentrations of sodium acetate.
and INV
F'
were routinely cultured on LB agar at 37 °C or in LB broth with 200 rpm agitation at 37 °C. E. coli transformants were
selected and maintained with 100 µg/ml ampicillin (Ap) and/or 40 µg/ml Km where indicated. E. coli PL-2 was grown in
nutrient broth supplemented with [14C]oleic acid (1 µCi/ml) to label bacterial phospholipids as previously described
(41).
F' by the manufacturer's protocol. Transformants were selected
on LB agar with Ap, and pACE1 was isolated from a colony-purified
transformant by alkaline lysis followed by purification using an
affinity spin column (Qiagen Inc., Valencia, CA).
F' were
selected on Ap-containing LB agar, and plasmid pACE2 was isolated as
described above. This plasmid was used to transform the Dam
methylase-deficient E. coli strain, GM2163. The plasmid was
purified from a resulting transformant and digested with
NruI that cleaved the insert DNA once within the putative
pdhA open reading frame. A Km resistance cassette within
pBSL86 (43) was isolated by HincII digestion and agarose gel
filtration, as described above, and ligated to the unique NruI restriction site in the pACE2 insert. The resulting
plasmid, pACE4, was isolated as described above from a Km- and
Ap-resistant bacterial colony following transformation of E. coli INV
F' and was used to generate a meningococcal acetate auxotroph.
siaA-D).
-bromo-2,3,4,5,6-pentafluoro-toluene and N,N-diisopropylethylamine in acetonitrile at
40 °C for 1 h. Derivatized FA were separated from excess
reagents by TLC using a silica gel G plate developed in ethyl
acetate/methanol (98:2 v/v). Eluted derivatized FA were dried under
nitrogen and dissolved in a 1:1 mixture of
N,O-bis(trimethylsilyl)trifluoroacetamide and
acetonitrile and incubated at 40 °C for 1 h to produce
trimethyl silane derivatives. After evaporation of solvent, the sample
was resuspended in 10-25 µl of isooctane and injected (1-4 µl)
for GC-MS analysis. FA were eluted using a temperature gradient from 70 to 250 °C developed at a rate of ~10 °C/min. Derivatized FA species were ionized by negative ion chemical ionization using methane
gas. Species from experimental samples were identified and quantified
by comparison with elution of known amounts (20-500 pg) of appropriate
FA standards. Data were acquired and processed using HP Chemstation.
Recoveries of derivatized 14C-FA in isooctane (before
application to GC) and of the internal standard (15:0) (after GC) were
monitored to correct for losses of material during sample preparation
(recovery was >70%). The amount of LOS in samples from which 3-OH-FA
were derived was estimated on the basis of the known mass and
composition of NMB LOS and of the 3-OH-FA present within LOS (5, 47).
By SDS-PAGE and silver staining or autoradiography, nearly all LOS
contained the full-length oligosaccharide chain. The specific
radioactivity of 14C-LOS was calculated from image and
GC-MS analyses and corrected for dilution in the growth medium of
[14C]acetate with unlabeled acetate to determine the
specific radioactivity of 14C-LOS derived from acetate
auxotrophs metabolically labeled with [14C]acetate alone.
20 °C, the sample was spun
at 10,000 × g to sediment 14C-LOS. After
removal of the supernatant, the pellet was washed three times with
ice-cold 95% ethanol. The remaining pellet was dried at 37 °C,
resuspended in cold distilled water to an estimated concentration of
100 µg LOS/ml, and sonicated at room temperature for 15 min in a
water bath sonicator.
80%.
4 M lucigenin. An oxidative response of PMN
was measured indirectly as lucigenin-enhanced chemiluminescence using a
LUCY1 version VI.5 luminometer (Anthos Labtec Instruments) as
previously described (51). The role of mCD14 in LOS-induced PMN
chemiluminescence was tested by preincubation of PMN for 15 min at
4 °C with neutralizing anti-CD14 mAb (My-4; 10 µg/ml) or an
irrelevant isotype-matched control mAb. Induction of synthesis and
secretion of IL-8 by PBMC was measured as described above (see
" Assay of HUVEC Activation") except that incubations were
for only 2 h and did not contain sCD14.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
3.5 mM acetate. As judged by bacterial
colony-forming ability, NMBACE1 remained viable through stationary
phase at 3.5 and 5.0 mM acetate. High concentrations of
sodium acetate (
40 mM) inhibited bacterial growth. PDH
activity was detected in lysates of the parent strain (NMB) but not of NMBACE1 (Fig. 1B). These data show that acetate auxotrophy
was successfully achieved by disrupting the meningococcal
pdhA gene.
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Fig. 1.
NMBACE1 is an acetate auxotroph.
A shows growth of bacteria in MDM supplemented with
increasing concentrations of sodium acetate. Bacterial growth is
expressed as increasing culture turbidity
(A550 nm) as a function of time. B
shows PDHase activity of bacterial lysates measured as described under
"Experimental Procedures." Data shown correspond to results of one
experiment that was representative of three or more separate
experiments.
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Fig. 2.
Purification of
14C-LOS-containing aggregates from NMBACE1 by Sephacryl
S500 chromatography. A, fractionation of
[14C]acetate species recovered from NMBACE1 after hot
phenol-water extraction and ethanol precipitation. B,
recovery of 14C-labeled species in chloroform and
water-methanol phases after chemical hydrolysis and extraction of the
indicated fractions. C and D, Rechromatography of
an aliquot (0.2 ml) of pooled fractions 11-13 (peak A) and 19-23
(peak B), respectively. All data shown represent the mean of results of
three separate experiments. Data are expressed as percentages of cpm
recovered; total recovery was >70%. Similar results were obtained by
chromatography of from 50 ng to 25 µg of LOS/ml.
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Fig. 3.
Autoradiograms of reverse phase TLC
(A) and SDS-PAGE analyses (B) of
14C-labeled material in peak A and B from NMBACE 1 and
NMBACE2. Note that neither glycoform of LOS from NMBACE2 contained
sialic acid. Pk, peak.
14C-fatty acid composition of peak A and B from NMBACE 1 and
NMBACE 2
20 × 106 Da. In contrast, Peak B represents smaller
aggregates of estimated Mr of ~1 × 106 Da containing 14C-LOS and apparently
14C-capsule. Essentially all 14C-labeled
material in peak A was LOS, whereas 14C-LOS represented
about 40% of the labeled material within peak B. 14C-LOS
in peaks A and B are indistinguishable by SDS-PAGE/autoradiography (Fig. 3B); each contain mainly full-length, sialylated
species. Estimation of the mass and radiolabeling of LOS within peaks A and B by assay of 3-OH-FA indicated that the specific radioactivity of
14C-LOS in both peaks was ~600 cpm/ng.
siaA-D), an isogenic mutant of NMB that
lacks the ability to produce sialylated LOS and capsule. NMBACE2
resembled NMBACE1 in its dependence on acetate for growth and the
specific radioactivity of metabolically labeled 14C-LOS
but, as expected, produced only nonsialylated glycoforms of LOS (Fig.
3B). Sephacryl S500 chromatography of purified
14C-LOS from NMBACE2 yielded two major populations of
14C-labeled aggregates with elution profiles similar to
peaks A and B from NMBACE1 (Fig. 4,
A, C, and D, compared with Fig. 2, A, C, and D). However, in marked
contrast to peak A and B from NMBACE1, the composition of
14C-labeled material within peak A and B from NMBACE2 was
essentially the same. After chemical hydrolysis and extraction, there
were almost no 14C-labeled species in the recovered
water/methanol phase from either peak A or peak B (Fig. 4B)
and no [14C]palmitate in the chloroform phase (Fig.
3A and Table I). These findings confirm that the
radiolabeled material within peak B from NMBACE1, in addition to LOS,
is sialylated material (i.e. capsule). However, the
existence of physically diverse aggregates of LOS does not depend on
the presence of either sialylated forms of LOS or of capsular
polysaccharide. As in NMBACE1, 14C-LOS in peak A and B from
NMBACE2 are indistinguishable by SDS-PAGE (Fig. 3B).
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Fig. 4.
Purification of
14C-LOS-containing aggregates from NMBACE2 by Sephacryl
S-500 chromatography. See legend to Fig. 2. Column buffer
contained 1% human serum albumin.
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Fig. 5.
Comparison of interactions with endothelial
cells of purified aggregates containing 14C-LOS from
NMBACE1 and NMBACE2. Cell activation and association of
14C-LOS with cells was measured as described under
"Experimental Procedures." All samples shown contained LBP (100 ng/ml) and sCD14 (250 ng/ml). The amount of LOS added was calculated
from the experimentally determined specific activity of LOS (see
"Experimental Procedures") and the percentages of radiolabeled
material in each aggregate population that corresponded to
14C-LOS. Incubations to assay cell association of
14C-LOS contained 5 ng of 14C-LOS/ml in 0.6 ml.
Levels of extracellular IL-8 in the absence of added LOS were <0.1
ng/ml and were subtracted so that only LOS-dependent
accumulation of IL-8 is shown. Data shown represent the means ± S.E. of the data from three or more experiments, each in
duplicate.
Effect of added LBP and sCD14 on interactions of purified aggregates
and complexes containing 14C-LOS with HUVEC
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Fig. 6.
Sephacryl S-500 chromatography of purified
aggregates containing 14C-LOS: effect of LBP and/or
sCD14. Samples (14C-LOS (100 ng/ml) ± LBP (300 ng/ml) and/or sCD14 (5 µg/ml)) were incubated for 15 min at 37 °C
in HEPES (10 mM, pH 7.4)-buffered HBSS containing divalent
cations and supplemented with 1% human serum albumin before being
applied to the column as described under "Experimental Procedures."
Recovery of 14C-labeled material in collected fractions is
expressed as a percentage of total cpm recovered (total recovery,
>70%). Results shown are representative of three or more experiments.
A, peak A, NMBACE1. B, peak B, NMBACE1.
C, peak A, NMBACE2. D, peak B, NMBACE2. Note that
the elution of peak C derived from either peak A of NMBACE1 or peak A
and B from NMBACE2 was nearly the same as that of aldolase, mouse
IgG1 and catalase.
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Fig. 7.
Dose-dependent activation of PBMC
(A and B) and PMN
(C) by purified aggregates and complexes containing
14C-LOS. Cell activation was measured as described
under "Experimental Procedures." Accumulation of secreted IL-8 by
activated PBMC (A and B) is expressed as ng/ml of
IL-8. Lucigenin-enhanced chemiluminescence by activated PMN
(C) is expressed as percentage of the response induced by 1 ng/ml of LOS in peak (Pk) B from NMBACE 1. LOS used in
experiments shown in A and C are from NMBACE 1 and from NMBACE2 for experiments shown in B. Responses of
cells without added LOS were <2% of maximum LOS-induced responses and
have been subtracted. Results shown represent the means ± S.E. of
more than three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(aroP-aceEF)
(gltA::kan)) derived
from strain K12. The purpose of mutagenesis of gltA in
E. coli LCD25 was to prevent acetate metabolism via the
trichloroacetic acid cycle. Although we were unable to generate
a viable meningococcal pdhA/gltA double mutant, the
tricarboxylic acid cycle is repressed when Neisseriae are
grown in the presence of glucose (e.g. MDM) and, thus, is
less important in their overall acetate metabolism. The single mutation
in pdhA permitted generation of radiolabeled LOS in NMBACE1
and NMBACE2 with up to 10-fold greater specific radioactivity than that
achieved in wild-type NMB. In all other respects (e.g. electrophoretic migration in SDS-PAGE, FA composition, bioactivity) LOS
produced by NMBACE1 and NMB were indistinguishable. NMBACE2 LOS was
distinguishable only by the lack of sialic acid-containing LOS species
(Fig. 3B). By use of [14C]acetate as the sole
source of acetate, we generated radiolabeled LOS of sufficiently high
specific radioactivity to permit study of the interactions of
meningococcal LOS with defined host targets at LOS concentrations
(~1-10 ng/ml) present in patients with severe invasive meningococcal
disease (10).
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Fig. 8.
Model of LBP and CD14-dependent
interactions of purified meningococcal endotoxin with host cells.
Note that the precise composition of disaggregated endotoxin after
interaction with LBP and CD14 (membrane or soluble) remains to be
determined. E, a single endotoxin molecule;
Eagg, aggregated endotoxin; AOAH,
acyloxyacylhydrolase.
Potent activation by endotoxin of cells containing mCD14 (e.g. PMN, monocytes) generally requires LBP but not sCD14. Our gel sieving studies show that LBP alone, under conditions that closely simulate the extracellular environment present in bioassays, does not induce disaggregation of LOS present in either peak A or B (Fig. 6A and data not shown). Thus, cell activation under these conditions is apparently mediated by delivery of LOS to these cells in aggregated, not disaggregated, form (Fig. 8). However, once bound to mCD14, transfer of a single molecule of endotoxin from CD14 to downstream acceptors (e.g. TLR4 and/or MD-2) may still be needed for cell activation (Fig. 8). The concentration of aggregates of endotoxin at the cell surface may also facilitate delivery of endotoxin to host cell acceptors linked to internalization and detoxification rather than cell activation (Refs. 18 and 56 and Fig. 8). The potency of endotoxin bound to mCD14 may thus depend on the relative rates of endotoxin disaggregation by CD14 versus delivery of aggregated endotoxin to targets that are not coupled to cell activation (Fig. 8). Although binding of aggregates of LBP-treated LOS to (m)CD144 and their disaggregation by (s)CD14 (Fig. 6, C and D) seem unaffected by aggregate size, bulk endotoxin internalization is faster with larger aggregates (57). This difference could explain the lower activity we observed for LOS in peak A toward monocytes and PMN and the much higher activity of the same LOS when presented to these cells in already disaggregated form (i.e. peak C) (Fig. 7). The use of cell lines with abnormally high levels of mCD14 expression may explain why effects of aggregation size on cell activation were not previously observed (57). The absence of an effect of aggregation size on LOS activity toward endothelial cells (Fig. 5C) may mean that competing internalization pathways do not exist in these cells or, more likely (56), that binding and disaggregation by sCD14 occur much more readily than delivery of extracellular LBP-treated LOS to these alternative cellular targets.
We do not know the precise structural and/or compositional bases of the different aggregation states of LOS manifest as peaks A and B from NMBACE1 and 2. No difference in FA composition (Fig. 3A and Table I)), oligosaccharide chain structure (as inferred by SDS-PAGE; Fig. 3B), or associated cations (data not shown) is apparent between LOS within peaks A and B. These data also strongly suggest that other acylated bacterial products (e.g. palmitoylated lipoproteins; Ref. 58) are not present. The presence of LOS-containing aggregates of similar size from NMBACE1 and 2 (Figs. 2 and 4) also indicates that sialylation of LOS does not affect the aggregation state of LOS. Sialylated LOS residing within the bacterial envelope can have important effects on interactions of meningococci with specific host targets (48, 59, 60). However, the nearly identical functional properties observed for LOS within peak A from NMBACE1 and 2 (Figs. 5-7) suggests that once LOS is extracted and purified from the bacterial envelope, its interactions with LBP and CD14 are unaffected by sialylation. The presence of polysialic acid capsule also did not affect the apparent aggregation state of LOS (compare peaks B from NMBACE1 and NMBACE2) but reduced the ability of LBP and sCD14 to disaggregate LOS and trigger LOS-dependent activation of endothelial cells (Figs. 5 and 6). Radiochemical analysis (Fig. 2 and Table I) suggested that nearly 10% of the molecules within peak B from NMBACE1 are capsular polysaccharides containing a dipalmityl anchor and, on average, ~200 residues of sialic acid. Copurification of LOS and capsular polysaccharide in peak B during chromatography in Sephacryl S500 (Fig. 2B) as well as in Sephacryl S200 and during ultracentrifugation5 suggest a physical association between LOS and capsule that could sterically impede interactions of LOS with LBP and sCD14. Such an association between LOS and capsule is likely on the surface of intact NMB. The presence of capsule may, therefore, affect not only cell-cell interactions between host and bacterium (52, 61, 62) but also extracellular interactions important in LOS-dependent host cell activation.
In summary, bacterial mutagenesis and gel sieving have been
utilized to generate and resolve physically distinct species containing LOS of high specific radioactivity. This isolation has permitted examination of physical determinants of specific LOS-host interactions and an assessment of the relation of these interactions to host cell
activation by LOS. The extension of these approaches to other defined
meningococcal genotypes, including variants in LOS and capsule
structure, should facilitate further characterization of the structural
determinants and consequences of the interactions of LOS with specific
biological targets. Our findings demonstrate the importance of the
physical context of LOS presentation and underscore the need to
progress from studies using purified LOS or LPS to an examination of
the fate and activities of membrane-associated endotoxin as it is
presented naturally to the host. This may be especially important for
meningococci in which elaboration and extrusion of LOS-containing
membranes (blebs; Refs. 63 and 64) may explain the high levels of
disseminating endotoxin that are characteristic of severe invasive
meningococcal disease (10). The methods described in this study,
including efficient metabolic labeling and detection of radiolabeled
LOS in complex environments, should now make it possible to examine
in vitro the elaboration and functional interactions of
membrane-associated LOS.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. William Nauseef for
valuable assistance in review and critique of the manuscript and Jan
Renee for assistance in chemiluminescence experiments. We also thank
Xoma Corp. (Berkeley, CA) for providing recombinant LBP, sCD14, and
rBPI21 and Wyeth-Lederle Vaccines and Pediatrics (West
Hendrietta, NY) for supplying the meningococcal capsule biosynthesis
mutant, PBCC7232-NMBsiaA-D.
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FOOTNOTES |
---|
* This work was supported by United States Public Health Service Grants DK05472 and PO14462 (to J. W.) and PO1AI44642 and R01AI145728 (to M. A. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by National Institutes of Health Postdoctoral Training Fellowship AI07343.
** Present address: Washington University, Dept. Molecular Biology, 660 South Euclid Ave., Box 8230, St. Louis, MO 63110.
To whom correspondence should be addressed: Dept. of Medicine,
University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242. Tel.: 319-384-8622; Fax: 319-356-4600; E-mail:
jerrold-weiss@uiowa.edu.
Published, JBC Papers in Press, November 17, 2000, DOI 10.1074/jbc.M009273200
2 L. Stoll, unpublished observations.
3 A. Teghanemt, unpublished observations.
4 J. Hume, unpublished observations.
5 D.-S. Zhang, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: LOS, lipooligosaccharide; 3-OH-FA, 3-hydroxy-fatty acid; FA, fatty acid(s); HBSS, Hanks' balanced salts solution; HUVEC, human umbilical vein endothelial cells; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; mCD 14, membrane-bound CD14; MDM, Morse's defined medium; PDH, pyruvate dehydrogenase; PBMC, peripheral blood mononuclear cells; PMN, polymorphonuclear leukocytes; sCD14, soluble CD14; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; GC, gas chromatography; MS, mass spectrometry; mAb, monoclonal antibody; Ap, ampicillin; Km, kanamycin; MOPS, 4-morpholinepropanesulfonic acid.
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REFERENCES |
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