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J. Biol. Chem., Vol. 280, Issue 46, 38383-38394, November 18, 2005

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Biochemical and Functional Characterization of Membrane Blebs Purified from Neisseria meningitidis Serogroup B^*

Deborah M. B. Post, DeSheng Zhang, Joshua S. Eastvold, Athmane Teghanemt, Bradford W. Gibson¶, and Jerrold P. Weiss||**1

From the Inflammation Program, Department of Internal Medicine and the ^||Department of Microbiology, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, ^**Veterans Affairs Medical Center, Iowa City, Iowa 52246, the ^¶Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143, and The Buck Institute for Age Research, Novato, California 94945

Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBP-dependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin·sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.

Received for publication, July 22, 2005

^* This work was supported by Grants P0144642 (to J. P. W. and B. W. G.) and AI18571 and AI59372 (to J. P. W.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: University of Iowa, The Inflammation Program, 2501 Crosspark Rd., D158 MTF, Coralville, IA 52241. Tel.: 319-335-4268; Fax: 319-335-4194; E-mail: jerrold-weiss{at}uiowa.edu.

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