Composition and Antigenic Activity of the Oligosaccharide Moiety of Haemophilus influenzae Type b Lipooligosaccharide. THOMAS J. INZANA,lt* WILLIAM E.
INFECTION AND IMMUNITY, May 1985, p. 324-330 0019-9567/85/050324-07$02.00/0 Copyright C 1985, American Society for Microbiology
Vol. 48, No. 2
Composition and Antigenic Activity of the Oligosaccharide Moiety of Haemophilus influenzae Type b Lipooligosaccharide Department
THOMAS J. INZANA,lt* WILLIAM E. SEIFERT, JR.,2 AND ROBERT P. WILLIAMS1 and Immunology, Baylor College of Medicine,' and Department of Analytical Chemistry,
of Microbiology
University of Texas Medical Center,2 Houston, Texas 77030 Received 29 October 1984/Accepted 15 January 1985
The oligosaccharide moiety of the lipooligosaccharide of Haemophilus influenzae type b strain Eag was isolated from the lipid component by mild acid hydrolysis and purified by gel filtration. Fast atom bombardment-mass spectrometry indicated that the lipid-free oligosaccharide had a basic molecular weight of 1,768; polysaccharides comparable to high-molecular-weight 0 side chains were not found. Glucose, galactose, galactosamine, heptose, 3-deoxy-D-manno-2-octulosonic acid (KDO), ethanolamine, and phosphate were identified in the lipid-free oligosaccharide by colorimetric assays, gas chromatography-mass spectrometry, or an amino acid analyzer. The presence of KDO was not clearly established by a thiobarbituric acid assay or by growth inhibition by a diazaborine derivative thought to block KDO synthesis. However, the semicarbizide assay and gas chromatography-mass spectrometry confirmed the presence of KDO. Lectin precipitation by Eag lipooligosaccharide in gels indicated that P-D-galactose was present and that some of this monosaccharide was a terminal, nonreducing residue linked to N-acetyl-D-galactosamine. The lipid-free oligosaccharide was antigenic and completely inhibited lipooligosaccharide antibody (predominantly immunoglobulin G [IgG] and IgM) in an enzyme-linked immunosorbent assay, whereas the solubilized lipid A moiety did not. H. influenzae type b lipid-free oligosaccharide differed from core oligosaccharide of Salmonella lipooligosaccharide by the presence of galactosamine and a smaller percentage of heptose and KDO.
less than 1% of a 3-deoxy-D-manno-2-octulosonic acid (KDO)-like molecule (13). Neither dideoxyhexose nor 6-deoxyhexose, common to enteric 0 chain polysaccharides (27), were found. Additional evidence that H. influenzae LOS does not contain polymerized 0 side chains and that it is similar in molecular weight to rough Salmonella LOS was indicated by gel electrophoresis (19, 47). However, H. influenzae type b LOS cross-reacts serologically with, and contains fatty acids common to, lipid A of Salmonella (13, 21, 26). Thus, the lipid components of H. influenzae LOS and enteric LPS appear to be similar, whereas the carbohydrate moieties may be substantially different. Before the role of H. influenzae type b LOS in virulence or in pathogenesis can be evaluated, a more detailed analysis of the oligosaccharide moiety is needed. The aim of this investigation was to determine the molecular weight, composition, and antigenic activity of the oligosaccharide moiety from the LOS of a commonly studied strain of H. influenzae type b.
Capsular polysaccharide, outer membrane proteins, and lipooligosaccharide (LOS) comprise the major surface antigens of Haemophilus influenzae type b. Each of these components is capable of inducing bactericidal antibodies (2, 5, 24). However, only antibodies to the capsule and to some outer membrane proteins protect against bacteremia and meningitis (2, 34, 39). The role of LOS in H. influenzae type b disease has not been established. However, H. influenzae LOS is a biologically active endotoxin (13), and antibodies to LOS may prevent mortality (29, 45) but not disease (5, 39, 40). Thorough investigation of the chemistry and immunological properties of the polysaccharide moiety of lipopolysaccharide (LPS) of members of the family Enterobacteriaceae, such as Salmonella species, has aided understanding the serological identification, virulence, and host parasite interactions of these bacteria (38). The 0 side chain repeating units of enteric LPS account for antigenic specificity (27), activation of the alternative complement pathway (9, 32), and protection of the cell against host defense systems and adverse environmental conditions (8, 10, 38); and it acts as a barrier to antibiotics and other antibacterial agents (10, 43). Although biological activity resides in the lipid moiety (14), mutants of enteric pathogens that lack 0 side chains are relatively avirulent and have not been isolated in nature (38). A detailed analysis of the oligosaccharide moiety of H. influenzae LOS has not been completed. Flesher and Insel (13) have found intact H. influenzae LOS to be composed of fatty acids (29%), carbohydrate (30%), and phosphate (4.7%). The sugars identified in the LOS of one H. influenzae type b strain were glucose, galactose, glucosamine, heptose, and
MATERIALS AND METHODS
Bacteria, culture conditions, and isolation of LOS. H. influenzae type b strain Eag was isolated from a patient with meningitis (6) and grown with shaking at 37°C in brain heart infusion broth supplemented with factors X and V (6) to late-logarithmic phase. The cells from 10 liters of culture were harvested by centrifugation (10,000 x g for 15 min), washed once in phosphate-buffered saline (PBS; pH 7.4), and lyophilized. The cells were suspended in 170 ml of 50 mM sodium phosphate buffer (pH 7.0) containing 5 mM EDTA and stirred in a Waring blender at top speed for 2 min. Enzyme digestion, followed by hot phenol-water extraction, was performed as described by Johnson and Perry (22). Residual phenol was removed from the pooled aqueous
* Corresponding author. t Present address: Department of Veterinary Microbiology-Pathology, Washington State University, Pullman, WA 99164-7040.
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phases by dialysis against distilled water, and the crude LOS was lyophilized. The white, fluffy material was suspended in distilled water to 8% and heated to 65°C to disperse aggregates. Centrifugation at 12,000 x g for 10 min, followed by a second centrifugation at 40,000 x g for 15 min, was used to remove particulate matter. Repeated ultracentrifugation of the LOS in distilled water (105,000 x g for 3 h) was done until the content of capsular polysaccharide, protein, and nucleic acid in the LOS was less than 1%, as determined by a radioantigen inhibition assay (35), an assay by the method of Lowry et al. (25), and spectrophotometric absorbance of the supernatants at 280 and 260 nm, respectively. Supernatants containing appreciable quantities of heptose, determined by colorimetric assay (44), were recentrifuged to recover any residual LOS. Smooth LOS from Salmonella typhimurium was obtained from Sigma Chemical Co., St. Louis, Mo., and rough LOS from Salmonella minnesota R345 was obtained from List Biological Laboratories, Inc., Campbell, Calif. Isolation of lipid-free oligosaccharide. Portions of H. influenzae LOS and S. minnesota R345 LOS were each made 1% (wt/vol) in 1% (vol/vol) acetic acid and heated to 100°C for 3 h. The resulting white precipitate was separated by centrifugation at 2,000 x g for 10 min. A small amount of chloroform was added to the supernatant to extract residual lipid. After vigorous mixing, the two phases were separated by centrifugation at 8,000 x g for 15 min, and the clear aqueous phase was lyophilized. The lipid was washed three times with distilled water, dried, and stored at -70°C. The lyophilized, lipid-free oligosaccharide (LFO) was taken up in 2 ml of distilled water and chromatographed on a column of Sephadex G-50 (3 by 90 cm) equilibrated with 0.1% acetic acid. The carbohydrate-positive fractions (12) were pooled, lyophilized, taken up in a small amount of distilled water, and desalted on a column of Sephadex G-10 (3.5 by 10 cm) equilibrated with double-distilled water. Gel diffusion of LOS and lectins. Lectins derived from Ricinus communis A, peanut, soybean, concanavalin A, Dolichos biflorus, and wheat germ were kindly provided by Peter Z. Allen, Department of Microbiology, University of Rochester Medical Center, Rochester, N.Y. The origin of these lectins has been described previously (3). The reported specificity (16) of the lectins used in this study was confirmed previously by inhibition of bacterial agglutination with increasing amounts of various sugars (3). Gel diffusion was performed in 1% agar in PBS containing 0.025% sodium azide, as described by Allen et al. (3). LOS solutions (1 mg/ml) were made up in 0.25% desoxycholate (Sigma) in saline. Lectin solutions contained 1 mg of lectin per ml in saline. Hog gastric mucin (provided by P. Z. Allen) was used as a positive control. Antiserum. Antiserum to Eag LOS was produced by an adult New Zealand White rabbit that was inoculated subcutaneously with 25 p.g of LOS mixed at a ratio of 1:1 with Freund complete adjuvant. Intramuscular booster immunizations were performed at weekly intervals with increasing doses of LOS (twofold increments up to 200 j,g) for 6 weeks. Blood was obtained by cardiac puncture 1 week after the final immunization. Serum was assayed for LOS antibody by enzyme-linked immunosorbent assay (ELISA), as described by Insel et al. (18). Reagents, microtiter plates, and other materials used in the ELISA have been previously described (18). Analytical methods. The LFO from Eag LOS or S. minnesota R345 LOS was dissolved in 2 M HCI (1 mg/ml) and incubated in a Teflon-capped Pyrex tube at 100°C for 4 h.
OLIGOSACCHARIDE OF H. INFLUENZAE b LOS
325
The samples were dried under reduced pressure over NaOH and P205. Neutral sugars were silylated before analysis by gas chromatography-mass spectrometry (GC-MS): the samples were dissolved in a small amount of water-methanol (1:1) and transferred to a 1-ml Teflon-capped, conical-bottom reaction vial and dried; the residue was then derivatized with 20 ,u of bis(trimethylsilyl)trifluoroacetamide (Pierce Chemical Co.) and 10 RI of pyridine at 60°C for 30 min. The derivatization mixture was then analyzed by electron-impact GC-MS with a Finnigan 3200 GC-MS interfaced to an INCOS data system (San Jose, Calif.). Chromatographic separation was achieved with a glass column (1.8 m by 2 mm [inner diameter]) packed with either 3% OV-1 or 3% OV-101 on 100/120 mesh Supelcoport (Supelco, Inc., Bellefonte, Pa.). The injector temperature was maintained at 230°C. After an initial 1 min, the column oven was programmed linearly from 100 to 290°C at 12°C/min. At least two preparations of each LFO were separately hydrolyzed, silylated, and analyzed. Heptose was assayed by the procedure described by Wright and Rebers (44). The thiobarbituric acid (TBA) assay by Karkhanis et al. (23) and the semicarbizide assay (28) were used for KDO determination. Uronic acids were assayed by the procedure reported by Dische (11). Phosphate was measured by the method of Ames (4). Pure standards for each assay were obtained from Sigma. For analysis of aminosugars and ethanolamine, Eag LFO was hydrolyzed in 4 M HCI for 10 h at 100°C. The samples were neutralized, dried (as described above), taken up in 1 ml of lithium citrate (pH 2.2), and analyzed on an amino acid analyzer (model D-400; Dionex, Sunnyvale, Calif.), which was set up for analyzing physiological fluids with the ninhydrin detection system. Data were recorded on a reporting integrator (model 3390; Hewlett-Packard Co., Palo Alto, Calif.). Total aminohexose was determined from hydrolyzed samples by the method of Morgan and Elson (31). Fast atom bombardment-mass spectrometry was performed on Eag LFO as described by Martin et al. (30). Briefly, the LFO was dissolved in water-glycerol (1:1), and 0.5 pul was applied to the probe tip of a double-focusing mass spectrometer (MAT 731; Varian, Los Altos, Calif.), which was modified for fast atom bombardment by mounting a neutral atom gun (1311N; Ion Tech LTD., Teddington, England) on the electron impact/field ionization/field desorption ion source. The neutral gas used was xenon. Spectra were recorded oscillographically in both the positive and negative ionization mode during magnetic scans at a resolution of 1:2,000. RESULTS Physical properties of Eag LOS and molecular weight of the LFO. The solubility of lyophilized Eag LOS decreased with increasing ionic strength of the solvent; at 1 mg/ml, Eag LOS was soluble in distilled water after warming to 45°C, was soluble in PBS after heating to 100°C or after sonication, but was not soluble in Veronal-buffered saline under any of the conditions described above. S. typhimurium smooth LPS was readily soluble in all of the solvents described above, whereas the solubility of S. minnesota R345 LOS was similar to that of Eag LOS. A total of 95 mg of Eag LFO was obtained from 225 mg of LOS. Thus, the lipid-to-oligosaccharide ratio was about 1.36:1, explaining its relatively poor solubility. Gel filtration of the LFO on Sephadex G-50 resulted in one carbohydrate-positive peak (Ka,, 0.35), confirming results of earlier analyses (19, 47) that H. influenzae
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type b LOS lacks heterologous 0 side chain repeating units (33). Fast atom bombardment-mass spectrometry demonstrated quasimolecular ions for H. influenzae type b LFO with mass-to-charge ratios (mlz) of 1,769, 1,791, 1,811, and 1,833, each of which represent different molecular weight species of the LFO plus a proton (H+). These m/z peaks may correspond to a basic compound of molecular weight 1,768 and an acetylated compound of molecular weight 1,810, both of which may have also contained a sodium ion (molecular weights of 1,790 and 1,832, respectively). Thus, the molecular weight of Eag LFO was slightly less than that of the core oligosaccharide from Salmonella R345 LOS (27). Composition of Eag LFO. The major components identified in Eag LFO and their molar ratios are shown in Table 1. Glucose (28%), galactose (18%), galactosamine (20%), heptose (13%), KDO (13%), ethanolamine (3%), and phosphate (4%) accounted for 99% of the LFO by weight. Nine sugars, one phosphate molecule, and one ethanolamine molecule were identified in Eag LFO. Uronic acids were not found. GC-MS was used for the analysis of neutral sugars from Eag LFO and, for comparative purposes, from R345 LFO. The gas chromatographic spectrum of each LFO is shown in Fig. 1. The heptose peaks from Eag LFO and R345 LFO were indistinguishable, but peaks of the L-glycerol-D-glucoheptose standard were substantially different (data not shown). The TBA assay for KDO was weakly positive with Eag LFO (0.3% [wt/wt]). Furthermore, the D-glucoheptose standard (100 ,ug), but not hexoses or hexosamines, was positive by the TBA assay, forming a chromophore with an identical absorbance maximum as KDO. Therefore, it was not clear whether the weak reaction of Eag LFO in the TBA assay was due to cross-reaction by heptose or from degradation of KDO (standard KDO was unstable when stored longer than 24 h in water, losing up to 90% of its colorimetric reactivity in the TBA assay). A diazaborine derivative, 1,2-dihydro-1hydroxy-6-methyl-2 - (propanesufonyl) - thieno (3 ,2-D) (1,2,3)diazaborine (no. 84.474), kindly donated by Sandoz Pharmaceuticals, Inc., Vienna, Austria, thought to block KDO synthesis and inhibit the growth of gram-negative organisms that contain KDO (17), did not inhibit the growth of H. influenzae type b strain Eag at concentrations inhibitory to Escherichia coli (5 ,ug/ml) (Fig. 2). However, higher concentrations of the diazaborine compound (at least 50 jig/ml) were inhibitory to strain Eag (data not shown), and therefore diazaborine growth inhibition could not confirm the presence or absence of KDO in this strain. The semicarbizide assay, however, indicated that Eag LFO contained 13% KDO and that R345 LFO contained 34% KDO (R345 LFO contains nine monosaccharides, three of which are KDO) (27). FurTABLE 1. Composition of the LFO derived from the LOS of H. infliuenzae type b strain Eag Component
Glucose Galactose
Galactosamine Heptose 3-Deoxy-D-ttianno-2-octulosonic acid Ethanolamine Phosphate
Percent
(by weight)' 28 18 20 13
Molar ratio
13 3 4
1.1 1.2 0.9
3.1 2.0 1.9 1.2
" Percentages were determined by colorimetric assay. GC-MS. or an amino acid analyzer, as described in the text.
A.S. minnesota R345
B.
H.influenzae type b
gic
hep 210
KDO'
154
FIG. 1. Reconstructed ion chromatogram from the GC-MS analysis of trimethylsilated hydrolysates of the LFO from S. minnesota R345 (A) or the LFO from H. influenzae type b strain Eag (B). Chromatographic separation was achieved with a glass column (1.8 m by 2 mm [inner diameter]) packed with 3% OV-101 on 100/120 mesh Supelcoport. Identification of components was made by comparing the mass spectra and peak retention times with those of monosaccharide standards prepared as for each LFO. Peak values represent the spectrum number. Abbreviations: glc, glucose; gal, galactose; hep, heptose.
thermore, 100 jig of heptose, hexose, or hexosamine did not cause substantial absorbance in the semicarbizide assay. When standard KDO was analyzed directly by GC-MS, one major peak was found that eluted after the heptose standard. However, when KDO was treated similarly as LFO, the mass spectrum showed fragmentation patterns with several faster eluting peaks, most of which overlapped in the glucose-galactose-heptose region. One small, early peak unique to KDO, however, was seen in R345 LFO, Eag LFO (Fig. 1) and hydrolyzed KDO (data not shown). An amino acid analyzer was used to determine that about 20% of the LFO contained galactosamine, with trace amounts of glucosamine and cysteine. In addition, a substantial amount of ethanolamine (3%) was clearly identified. The composition of Eag LFO and R345 LFO were similar except for the qualitative difference in galactosamine. Furthermore, the heptose-KDO region of R345 LFO accounts for more than 50% of this oligosaccharide (27), whereas it only accounted for about 28% in Eag LFO. Interaction of Eag LOS with lectins by gel diffusion. Limited structural information on the sugars in Eag LOS was obtained by lectin precipitation of LOS in gels (Fig. 3; see legend for lectin specificity). Lectins derived from R. communis A and peanut precipitated Eag LOS, indicating the presence of 3-D-galactose, and that at least some of this sugar was linked to N-acetyl-D-galactosamine as a terminal, nonreducing residue. Interaction of the LOS with lectin from soybean, but not from D. biflorus, suggested that N-acetylD-galactosamine was accessible for lectin binding in the ,B configuration only. The galactosamine identified in the LFO with an amino acid analyzer, therefore, was likely to be N-acetylated. Although glucose was identified in the LFO, lectin from concanavalin A failed to precipitate Eag LOS in
OLIGOSACCHARIDE OF H. INFLUENZAE b LOS
VOL. 48, 1985
30.0
10.0
c E
5.0
0 0)
3.0
%-
~._
0
0
a
1.0
0.
0
0.5
0.3 0.1
0 1
2 3 4 5 Time (hrs.)
6
7
FIG. 2. Effect of diazaborine derivative no. 84.474 on growth of E. coli or H. influenzae type b strain Eag. Cells were grown in brain heart infusion broth supplemented with factors X and V alone (0) or in the same broth containing 5 ,ug of diazaborine derivative no. 84.474 per ml (0) to late-logarithmic phase. Symbols: ----, E. coli;
327
lower than the molecular weight of S. minnesota R345 LFO (27). Gel electrophoresis of LOSs from a variety of strains of typable and untypable H. influenzae demonstrate compounds that are microheterogeneous and of low molecular weight (19). Thus, a lipid-oligosaccharide molecule appears to be standard for H. influenzae, rather than the lipid-oligosaccharide-O side-chain polymer of members of the family Enterobacteriaceae (27). In our laboratory, the isolation of H. influenzae type b strain Eag LOS was more effective with hot phenol-water than with phenol-chloroform-petroleum ether (unpublished data), indicating that Eag LOS was relatively hydrophilic when extracted from whole cells. However, after purification, lyophilized Eag LOS was only slightly more soluble in aqueous solutions than S. minnesota R345 LOS. It is possible that interaction of H. influenzae type b LOS with other surface components (e.g., capsular polysaccharide or outer membrane proteins) enhances its solubility after crude extraction. After purification, however, the decreased solubility of Eag LOS was more characteristic of Salmonella LOS without 0 antigen (27). The composition of a purified oligosaccharide moiety from H. influenzae LOS has not been previously analyzed. Glucose, galactose, heptose, and phosphate were previously identified in Eag LOS (13); we also identified these components in Eag LFO. In addition, our GC-MS profile for the heptose in Eag LFO was indistinguishable from the heptose in R345 LFO, suggesting that L-glycerol-D-mannoheptose is the heptose isomer present in Eag LOS (27). Our results and those of others show very small quantities of KDO (13, 46) or none at all (37) to be present in H. influenzae LOS by the TBA assay after hydrolysis with 0.2 N H2SO4. Recently, Parr and Bryan have reported that
-, H. influenzae type b Eag.
gels. Likewise, lectin from wheat germ failed to demonstrate the presence or availability of N-acetyl-D-glucosamine in Eag LOS. Antibody response to Eag LOS and antigenic activity of the LFO. Immunization of a rabbit with Eag LOS stimulated ELISA antibody titers of 1:8192 for immunoglobulin G (IgG) and IgM and 1:128 for IgA, which were determined with monospecific, alkaline phosphatase-conjugated goat antibody to rabbit IgG, IgM, or IgA. In this assay, a 1:250 dilution of rabbit anti-Eag LOS serum, followed by a 1:50 dilution of polyspecific, conjugated goat immunoglobulin to rabbit IgG, IgM, and IgA, resulted in an absorbance of 1.83 at 405 nm. The anti-LOS activity of the serum was completely inhibited with LFO or LOS, but not with solubilized Eag lipid A (Table 2). Although the LOS was two to four times more inhibitory than LFO, the completeness of inhibition by LFO and the lack of substantial inhibition by the lipid moiety suggested that most, if not all, of the antibody to LOS was directed to the oligosaccharide moiety. DISCUSSION We studied the molecular weight, composition, and antigenic activity of the oligosaccharide moiety of H. influenzae type b Eag LOS. The molecular weight of Eag LFO was found to be relatively low (1,768 to 1,810); this is slightly
FIG. 3. Precipitation of lectins by purified LOS from H. influenzae type b strain Eag in gel diffusion. Lectins were made up at 1 mg/ml in saline, and LOS (center well) was made up at 1 mg/ml in saline containing 0.25% sodium desoxycholate. Sources and specificity of lectins used were as follows: concanavalin A (CON A), a-D-mannose > a-D-glucose; D. biflorus (DB), N-acetyl-a-Dglucosamine > N-acetyl-p-D-glucosamine; R. communis A (RC), P-D-galactose > a-D-galactose; soybean (S), N-acetyl-a-Dgalactosamine 2 N-acetyl-p-D-galactosamine; peanut (P), P-Dgalactose (1-*3)-N-acetyl-D-galactosamine; wheat germ (WG), (Nacetyl-ct-D-glucosamine)3 (16).
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hydrolysis of H. influenzae LOS in 4 N HCl results in an increase in detection of KDO by the TBA assay (36). However, the TBA assay has been found to be inadequate for quantitating KDO (41). Furthermore, we found that heptose can cross-react in the TBA assay and may therefore result in misinterpretation of the amount of KDO present. Growth inhibition by diazaborine derivative no. 84.474 also gave equivocal results. GC-MS has been used successfully to identify KDO in Neisseria LOS after methanolysis and trimethylsilylation (20) and in some Vibrionaceae LPS after methanolysis and acetylation (7). Because KDO is acid-labile and degrades during hydrolysis with a strong acid (e.g., 2 M HCl, 100°C), fragmentation patterns may result that overlap with other glycosides (7, 20). Nonetheless, KDO could be identified in Eag LFO by comparing GC-MS profiles from pure KDO, R345 LFO, and Eag LFO after hydrolysis and silylation; one small, distinct peak for KDO (probably a decomposition product) was present in all samples. In addition, the semicarbizide assay has been used to identify a substantial amount of KDO in Rhodopseudomonas sphaeroides LOS under conditions in which the TBA assay identified only a minor quantity (42). Our results indicated that the semicarbizide assay was sensitive and specific for KDO; one molecule of KDO per Eag LFO was estimated based on this assay. Galactosamine and ethanolamine were identified in Eag LOS with an amino acid analyzer. We found trace amounts of glucosamine in the LFO, whereas it has been identified as the major aminosugar in Eag LOS by thin-layer chromatography (13). Because the previous work was concerned with the composition of the entire LOS, it is likely that glucosamine is a component of the lipid A moiety, as it is in Salmonella species (27), and was present in Eag LFO owing to incomplete hydrolysis. Galactosamine was probably not previously identified because the Rf value of the ninhydrinpositive spot for hydrolyzed Eag LOS overlapped with the Rf values for the glucosamine and galactosamine standards, which were relatively close together (13). Lectin precipitation by LOS has been useful in providing limited information on oligosaccharide composition and structure (1, 3). Although glucose was present in greatest quantity in Eag LFO, lectin from concanavalin A did not
Absorbance'
precipitate the LOS by gel diffusion. Therefore, glucose was either in the P configuration or was unavailable for lectin binding owing to steric hindrance. Interaction of Eag LOS with lectin from R. communis A indicated that P-D-galactose was present and accessible for lectin binding. Selective interaction of Eag LOS with soybean lectin, but not with lectin from D. biflorus, suggested that N-acetyl-D-galactosamine was also present in the D configuration. Furthermore, precipitation of peanut lectin by Eag LOS suggested that some of the galactose residues were terminal, nonreducing sugars linked to N-acetyl-D-galactosamine. The absence of 0 side chains in Salmonella species is associated with a lack of virulence (38). However, virulent strains of H. influenzae do not have 0 chain polysaccharide, and in Eag the oligosaccharide core appeared to consist of nine monosaccharides, phosphate, and ethanolamine. Results of recent studies indicate that alteration in the structure or composition of H. influenzae LOS (as determined by polyacrylamide gel electrophoresis) by transformation with cloned DNA may be associated with attenuation of virulence and with increased sensitivity to bactericidal serum (A. Zwahlen, L. G. Rubin, C. J. Connelly, T. J. Inzana, and E. R. Moxon, in press). Further investigation is required to determine if strain-to-strain variation in LOS structure may influence variation in virulence. The polysaccharide moiety is known to account for the antigenic specificity of LPS of members of the family Enterobacteriaceae (27). In deep rough mutants (containing only KDO and lipid A), however, antibodies can be made to lipid A (15). Therefore, it was not surprising that IgG and IgM antibodies made to H. influenzae type b LOS could be completely inhibited by homologous LOS or LFO, but not by homologous solubilized lipid A. Of interest was that more LFO than LOS was required to completely inhibit anti-LOS activity. It is possible that acetyl groups or other acid-labile groups may have been removed during the hydrolysis and that these groups influenced the antigenic behavior of the oligosaccharide. Alternatively, owing to its nature to aggregate (27), the LOS may be more active antigenically than a simple oligosaccharide. Antibody to rough E. coli LOS has been reported to be capable of preventing mortality in mice owing to H. influenzae type b (28) and endotoxic shock due to H. influenzae bacteremia in humans (45). Additional investigation of the biological activity and antigenic nature of H. influenzae LOS may provide better care for patients suffering from its endotoxic properties (13, 14).
0.28 ± 0.04
ACKNOWLEDGMENTS
TABLE 2. Inhibition of an ELISA" for Eag LOS antibody by homologous LOS, LFO, or solubilized lipid A" Serum and inhibitor
Normal rabbit serum None Anti-LOS rabbit serum None Lipid A LOS LOS LOS LOS LFO LFO LFO LFO
Concn (p.g/ml)
1.83 ± 0.03 1.66 ± 0.10 400 0.42 ± 0.04 12.5 0.36 ± 0.02 25 0.30 ± 0.05 50 0.27 ± 0.04 100 0.80 + 0.08 12.5 0.50 ± 0.06 50 0.32 ± 0.03 200 0.24 ± 0.03 400 " Checkerboard titration was performed to determine the optimal concentration of LOS (1 ,tg), anti-LOS serum (1:250), and goat-anti-rabbit conjugate
(1:50). LOS, LFO, or lipid A was incubated with the anti-LOS serum for 1 h at
0'C. ELISA was performed as described by Insel et al. (18). " Lipid A (1 mg/ml in water) was solubilized with 0.5 ,ul of triethylamine (Sigma) per ml. ' Mean + standard error of the mean of triplicate samples.
We thank Porter Anderson for helpful discussion during the course of this work, Peter Z. Allen and Richard A. Insel for materials and suggestions, and Catherine Costello for performing the fast atom bombardment-mass spectrometry. Fast atom bombardment-mass spectrometry was done at the Massachusetts Institute of Technology and was supported by Public Health Service grant no. RR00317 from the National Institutes of Health to K. Biemann. Part of this work was carried out at the Departments of Microbiology and Pediatrics, University of Rochester Medical Center, Rochester, N.Y. This work was supported, in part, by U.S. Public Health Service training grant A107145 from the National Institute of Allergy and Infectious Diseases. LITERATURE CITED 1. Ahamed, N. M., J. Radziejewska-Lebrect, C. Widemann, and H. Mayer. 1980. Reactivity of isolated lipopolysaccharides of en-
OLIGOSACCHARIDE OF H. INFLUENZAE b LOS
VOL. 48, 1985 terobacterial R mutants with complete or incomplete core structures with lectins. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. Reihe A 247:468-482. 2. Alexander, H. F. 1965. The Hemophilus group, p. 724-741. In R. J. Dubois and J. G. Hirsh (ed.), Bacterial and mycotic infections in man. J. B. Lippincott Co., Philadelphia. 3. Allen, P. Z., M. C. Connelly, and M. A. Apicella. 1980. Interaction of lectins with Neisseria gonorrhoeae. Can. J. Microbiol. 26:468-474. 4. Ames, B. N. 1966. Assay of inorganic phosphate, total phosphate, and phosphatases. Methods Enzymol. 8:115-118. 5. Anderson, P., A. Flesher, S. Shaw, A. L. Harding, and D. H. Smith. 1980. Phenotypic and genotypic variation in the susceptibility of Haemophilus influenzae type b to antibodies to somatic antigens. J. Clin. Invest. 65:885-891. 6. Anderson, P., R. B. Johnston, Jr., and D. H. Smith. 1972. Human serum activities against Haemophilus influenzae, type b. J. Clin. Invest. 51:31-38. 7. Banoub, J. H., D. H. Shaw, and F. Michon. 1983. Hydrolytic release and identification by G.L.C.-M.S., of 3-deoxy-D-manno2-octulosonic acid in the lipopolysaccharides isolated from bacteria of the Vibrionaceae. Carbohydr. Res. 123:117-122. 8. Bennett, G. M., A. Seaver, and P. H. Calcott. 1981. Effect of defined lipopolysaccharide core defects on resistance of Salmonella typhimurium to freezing and thawing and other stresses. Appl. Environ. Microbiol. 42:843-849. 9. Bjornson, A. B., and H. S. Bjornson. 1977. Activation of complement by opportunistic pathogens and chemotypes of Salmonella minnesota. Infect. Immun. 16:748-753. 10. Costerton, J. W., J. M. Ingram, and K.-J. Cheng. 1974. Structure and function of the cell envelope of gram-negative bacteria. Bacteriol. Rev. 38:87-110. 11. Dische, Z. 1947. A new specific color reaction of hexuronic acids. J. Biol. Chem. 167:189-198. 12. Dubois, M., K. A. Hamilton, P. A. Rebers, and F. Smith. 1956. Colorimetric method for determination of sugars and related substances. Anal. Chem. 28:350-356. 13. Flesher, A. R., and R. A. Insel. 1978. Characterization of lipopolysaccharide of Haemophilus influenzae. J. Infect. Dis. 138:719-730. 14. Galanos, C., 0. Luderitz, E. T. Rietschel, and 0. Westphal. 1977. Newer aspects of the chemistry and biology of bacterial lipopolysaccharides, with special reference to their lipid A component, p. 239-335. In T. W. Goodwin (ed.), International review of biochemistry. Biochemistry of lipids II, vol. 14. University Park Press, Baltimore. 15. Galanos, C., 0. Luderitz, and 0. Westphal. 1971. Preparation and properties of antisera against the lipid-A component of bacterial lipopolysaccharides. Eur. J. Biochem. 24:116-122. 16. Goldstein, I. J., and C. E. Hayes. 1978. The lectin: carbohydrate-binding proteins of plants and animals. Adv. Carbohydr. Chem. Biochem. 35:127-340. 17. Hogenauer, G., and M. Woisetschlfiger. 1981. A diazaborine derivative inhibits lipopolysaccharide biosynthesis. Nature (London) 293:662-664. 18. Insel, R. A., R. Anderson, M. R. Loeb, and D. H. Smith. 1981. A polysaccharide-protein complex from Haemophilus influenzae type b. II. Human antibodies to its somatic components. J. Infect. Dis. 144:521-529. 19. Inzana, T. J. 1983. Electrophoretic heterogeneity and interstrain variation of the lipopolysaccharide of Haemophilus influenzae. J. Infect. Dis. 148:492-499. 20. Jantzen, E., K. Bryan, and K. Bovre. 1976. Cellular monosaccharide patterns of Neisseriaceae. Acta Pathol. Microbiol. Scand. Sect. B 84:177-188. 21. Johns, M. A., S. C. Bruins, and W. R. McCabe. 1977. Immunization with R mutants of Salmonella minnesota 1I. Serological response to lipid A and the lipopolysaccharide of Re mutants. Infect. Immun. 17:9-15. 22. Johnson, K. G., and M. B. Perry. 1976. Improved techniques for the preparation of bacterial lipopolysaccharides. Can. J. Microbiol. 22:29-34. 23. Karkhanis, Y. D., J. A. Zeltner, J. J. Jackson, and D. J. Carlo.
24.
25. 26.
27.
28. 29.
30. 31. 32. 33.
34.
35.
36.
37. 38.
39.
40.
41.
42. 43.
329
1978. A new and improved microassay to determine 2-keto-3deoxyoctonate in lipopolysaccharide of gram-negative bacteria. Anal. Biochem. 85:595-601. Lam, J. S., D. M. Granoff, J. R. Gilsdorf, and J. W. Costerton. 1980. Immunogenicity of outer membrane derivatives of Haemophilus influenzae type b. Curr. Microbiol. 3:359-364. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Luderitz, O., C. Galanos, V. Lehmann, M. Nurminen, E. T. Reitschel, G. Rosenfelder, M. Simon, and 0. Westphal. 1973. Lipid A: chemical structure and biological activity. J. Infect. Dis. 128(Suppl.):S17-S29. Luderitz, O., 0. Westphal, A. M. Staub, and H. Nikaido. 1971. Isolation and chemical and immunological characterization of bacterial lipopolysaccharides, p. 145-234. In G. Weinbaum, S. Kadis, and S. J. Ajl (ed.), Microbial toxins IV. Academic Press, Inc., New York. MacGee, J., and M. Doudoroff. 1954. A new phosphorylated intermediate in glucose oxidation. J. Biol. Chem. 210:617-626. Marks, M. I., E. J. Ziegler, H. Douglas, L. B. Corbeil, and A. I. Braude. 1982. Induction of immunity against lethal Haemophilus influenzae type b infection by Escherichia coli core lipopolysaccharide. J. Clin. Invest. 69:742-749. Martin, S. A., C. E. Costello, and K. Biemann. 1982. Optimization of experimental procedures for fast atom bombardment mass spectrometry. Anal. Chem. 54:2362-2368. Morgan, W. R. J., and L. A. Elson. 1934. A colorimetric method for the determination of N-acetyl-glucosamine and N-acetylchondrosamine. Biochem. J. 28:988-995. Morrison, D. C., and L. F. Kline. 1977. Activation of the classical and properdin pathways of complement by bacterial lipopolysaccharides (LPS). J. Immunol. 118:362-368. Muller-Seitz, E., B. Jann, and K. Jann. 1968. Degradation studies on the lipopolysaccharide from E. coli 071: K?:H12. Separation and investigation of 0-specific and core polysaccharides. FEBS Lett. 1:311-314. Munson, R. S., Jr., J. L. Shenep, S. J. Barenkamp, and D. M. Granoff. 1983. Purification and comparison of outer membrane protein P2 from Haemophilus influenzae type b isolates. J. Clin. Invest. 72:677-685. O'Reilley, R. J., P. Anderson, D. L. Ingram, G. Peter, and D. H. Smith. 1975. Circulating polyribophosphate in Haemophilus influenzae type b meningitis. Correlation with clinical course and antibody response. J. Clin. Invest. 56:1012-1022. Parr, T. R., and L. E. Bryan. 1984. Lipopolysaccharide composition of three strains of Haemophilus influenzae. Can. J. Microbiol. 30:1184-1187. Raichvarg, D., C. Brossard, and J. Agneray. 1979. Chemical composition and biological activities of a phenol-water extract from Haemophilus influenzae type a. Infect. Immun. 26:415-421. Roantree, R. J. 1971. The relationship of lipopolysaccharide structure to bacterial virulence, p. 1-37. In S. Kadis, G. Weinbaum, and S. J. Ajl (ed.), Microbial toxins V. Academic Press, Inc., New York. Shenep, J. L., R. S. Munson, Jr., S. J. Barenkamp, and D. M. Granoff. 1983. Further studies of the role of noncapsular antibody in protection against experimental Haemophilus influenzae type b bacteremia. Infect. Immun. 42:257-263. Shenep, J. L., R. S. Munson, Jr., and D. M. Granoff. 1982. Human antibody responses to lipopolysaccharide after meningitis due to Haemophilus influenzae type b. J. Infect. Dis. 145:181-190. Strain, S. M., S. W. Fesik, and I. M. Armitage. 1983. Characterization of lipopolysaccharide from a heptoseless mutant of Escherichia coli by carbon 13 nuclear magnetic resonance. J. Biol. Chem. 258:2906-2910. Strittmatter, W., J. Weckesser, P. V. Salimath, and C. Galanos. 1983. Nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides ATCC 17023. J. Bacteriol. 155:153-158. Tamaki, S., T. Sato, and M. Matsuhashi. 1971. Role of lipopolysaccharides in antibiotic resistance and bacteriophage adsorption of Escherichia coli K-12. J. Bacteriol. 105:968-975.
330
INZANA, SEIFERT, AND WILLIAMS
44. Wright, B., and P. A. Rebers. 1972. Procedure for determining heptose and hexose in lipopolysaccharides. Anal. Biochem. 49:307-319. 45. Ziegler, E. J., J. A. McCutchan, J. Fierer, M. P. Glauser, J. C. Sudoff, H. Douglas, and A. I. Braude. 1982. Treatment of gram-negative bacteremia and shock with human antiserum to a mutant Escherichia coli. N. Engl. J. Med. 307:1225-1230.
INFECT. IMMUN. 46. Zoon, K. C., and J. Scocca. 1975. Constitution of the cell envelope of Haemophilus influenzae in relation to competence for genetic transformation. J. Bacteriol. 123:666-677. 47. Zwahlen, A., J. A. Winkelstein, and E. R. Moxon. 1983. Surface determinants of Haemophilus influenzae pathogenicity: comparative virulence of capsular transformants in normal and complement-depleted rats. J. Infect. Dis. 148:385-394.
