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 ||DaggerDagger

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

Received for publication, October 11, 2000, and in revised form, November 8, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 beta (1'right-arrow6)-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-alpha 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

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.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- E. coli INValpha F' and DH5alpha 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.

Bacterial Culture Conditions-- Encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of NMB (PBCC7232-NMB Delta 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.

E. coli strains GM2163(dam-), DH5alpha and INValpha 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).

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 INValpha 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).

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 INValpha 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 INValpha F' and was used to generate a meningococcal acetate auxotroph.

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-NMBDelta siaA-D).

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 alpha -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.

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 -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.

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 >= 80%.

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 × 10-4 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

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 >= 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.

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).



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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.


                              
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Table I
14C-fatty acid composition of peak A and B from NMBACE 1 and NMBACE 2 
14C-fatty acids were measured as described under "Experimental Procedures." Data are expressed as percentages of total 14C-fatty acids. Values shown represent the means ± S.E. of determinations from three independent samples.

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 >= 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.

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 (Delta 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.

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.



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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.


                              
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Table II
Effect of added LBP and sCD14 on interactions of purified aggregates and complexes containing 14C-LOS with HUVEC
Incubations (0.6 ml) contained 5 ng/ml of 14C-LOS (from NMBACE 1) ± LBP (100 ng/ml) and sCD14 (250 ng/ml), as indicated. Cell activation (LOS-dependent accumulation of extracellular IL-8) and cell association of 14C-LOS were measured after 20 h of incubation as described under "Experimental Procedures." 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., where indicated, of three or more determinations. Peak C generated from aggregates of LOS from NMBACE2 has closely similar functional properties to that of peak C from NMBACE1 shown here.

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).



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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.

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.



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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

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 (Delta (aroP-aceEF)Delta (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).

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.



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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.


    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-NMBDelta siaA-D.


    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.

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.

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.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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