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Originally published In Press as doi:10.1074/jbc.M009273200 on November 17, 2000

J. Biol. Chem., Vol. 276, Issue 8, 5883-5891, February 23, 2001
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Construction of Acetate Auxotrophs of Neisseria meningitidis to Study Host-Meningococcal Endotoxin Interactions*

Peter C. GiardinaDagger §, Theresa Gioannini||, Benjamin A. BuscherDagger **, Anthony ZaleskiDagger , De-Shang Zheng||, Lynn Stoll||, Athmane Teghanemt||, Michael A. ApicellaDagger , and Jerrold WeissDagger ||Dagger Dagger

From the Departments of Dagger  Microbiology,  Biochemistry, and || Medicine, Division of Infectious Diseases, The Inflammation Program, University of Iowa and Veterans' Administration Medical Center, Iowa City, Iowa 52242

To facilitate studies of the molecular determinants of host-meningococcal lipooligosaccharide (endotoxin) interactions at patho-physiologically relevant endotoxin concentrations (i.e. <= 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.

§ 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.

Dagger Dagger 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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