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J. Biol. Chem., Vol. 276, Issue 8, 5883-5891, February 23, 2001
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From the Departments of To facilitate studies of the molecular
determinants of host-meningococcal lipooligosaccharide (endotoxin)
interactions at patho-physiologically relevant endotoxin concentrations
(i.e.
Construction of Acetate Auxotrophs of Neisseria
meningitidis to Study Host-Meningococcal Endotoxin
Interactions*
§,
,**,,,,,, and
Microbiology,
¶ Biochemistry, and Medicine, Division of Infectious
Diseases, The Inflammation Program, University of Iowa and
Veterans' Administration Medical Center, Iowa City, Iowa 52242
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.
*
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.
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.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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