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Journal of Bacteriology, May 2009, p. 3311-3320, Vol. 191, No. 10
0021-9193/09/$08.00+0     doi:10.1128/JB.01433-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Structural Characterization of an Oligosaccharide Made by Neisseria sicca

Ellen T. O'Connor,¹ Hui Zhou,² Kevin Bullock,² Karen V. Swanson,^1,3 J. McLeod Griffiss,³ Vernon N. Reinhold,² Clinton J. Miller,¹ and Daniel C. Stein^1*

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742,¹ Department of Chemistry, University of New Hampshire, Durham, New Hampshire 03824,² Department of Laboratory Medicine and Veterans Affairs Medical Center, University of California San Francisco, San Francisco, California 94121³

Received 13 October 2008/ Accepted 27 February 2009

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Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization—time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-D-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its β-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) β-L-rhamnose (1-3) β-N-acetyl-D-glucosamine (1-] with an N-acetyl-D-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.

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Commensal Neisseria strains colonize the human respiratory tract. Frequent interspecific genetic exchange between commensal Neisseria strains and the pathogens Neisseria meningitidis and Neisseria gonorrhoeae occurs (13). It is thought the commensal organisms serve as reservoirs for antibiotic resistance genes (20). The similarity between the gene complements of the commensals and pathogens suggests that the virulence of the pathogenic Neisseria spp. may not result from the genes that they possess but rather from a "genetic personality" which is a result of combinations of these genes, sequence variations that alter the function of gene products, the presence of genes for which a virulence phenotype has not yet been identified, and/or differences in the regulation of genes (25).

Lipooligosaccharide (LOS) is an important neisserial virulence determinant consisting of an oligosaccharide (OS) component attached to lipid A via 3-deoxy-2-keto-D-manno-octulosonic acid (Kdo). The structures of a sufficient number of neisserial LOS molecules have been determined to form a coherent yet incomplete picture of the structural diversity of their LOS (Fig. 1). The different LOS structures have a conserved core with two Kdo molecules, two heptose (Hep) molecules, and one N-acetylhexosamine (HexNAc) molecule and vary in the composition and size of the OS attached to one Hep (HepI; -chain variation) and in the attachment of an OS or phosphoethanolamine to the other Hep (HepII; β-chain variation) or by addition of a galactose to the N-acetylglucosamine (GlcNAc) found on HepII (-chain extension) (3, 6, 7, 9-11). This structural motif is different from that of lipopolysaccharide (LPS) of other types of bacteria, which contains an O antigen composed of a repeating sugar polymer, typically consisting of four to seven sugars (26). No one has reported the presence of an O antigen in pathogenic strains of Neisseria.

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FIG. 1. Structrual diversity of the sugar backbone of LOS isolated from pathogenic Neisseria spp. The various LOS structures that have been identified in N. gonorrhoeae or N. meningitidis are shown.

A few studies have analyzed the structure of LOS produced by commensal Neisseria strains, and the data indicate that the LOS heterogeneity is greater than the heterogeneity in the gonococcus and meningococcus (21). Commensal Neisseria strains are capable of producing LOS molecules that are structurally different from the molecules in the known Neisseria repertoire in that they fail to bind monoclonal antibodies specific for LOS epitopes characteristic of the gonococcus and meningococcus (1). They also can lack some of the LOS biosynthesis genes found in N. meningitidis and N. gonorrhoeae (1, 33). These findings suggest that alternative LOS structures are present in commensal Neisseria strains. Sandlin and Stein (21) identified a strain of Neisseria sicca that expressed an unusual glycolipid that appeared based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to be analogous to LPS made by enteric bacteria. A poly-N-acetyllactosamine repeat was seen in some strains of the gonococcus (10), and we postulated that the repeating carbohydrate found in N. sicca could be a variant of this structure.

A few studies have shown that both Neisseria and Haemophilus strains have the ability to extend their LOS by adding lactosamine repeats to form polylactosamine; these strains seem to possess increased virulence (4, 10, 22). When N. gonorrhoeae MS11mkC was used to inoculate healthy male volunteers, 100% of the volunteers developed urethritis, compared to an infectivity rate of 40% for strains expressing a truncated LOS (23). Fresh isolates from the volunteers who had contracted urethritis produced LOS molecules with N-acetyllactosamine repeats, whereas the isolates in the original inoculum expressed paraglobsyl and gangliosyl LOS (23, 24). However, the number of disaccharide repeats was limited to two or three (10). Analogous results were generated when the infectivity of Haemophilus ducreyi, a causative agent of genital ulcers, was studied; infectious strains made LOS with a few lactosamine repeats (4, 22).

N. sicca is normally not pathogenic in healthy adults. A previous study indicated that N. sicca 4320 expressed a molecule with a repeating carbohydrate structure that appeared to be similar to the O-antigen structure (21). Because such molecules have not been found in pathogenic Neisseria strains, we analyzed the structure of the repeating carbohydrate unit and showed that it is a repeating disaccharide that is novel to the genus Neisseria.

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Bacterial strains and culture conditions. N. sicca 4320 was obtained from the culture collection of the late Herman Schneider, Walter Reed Army Institute of Research, and was reported to have caused a fatal case of bacterial endocarditis. It was grown in phosphate-buffered gonococcal medium (Difco) supplemented with 20 mM D-glucose and growth supplements (30) either in broth with addition of 0.042% NaHCO₃ or on agar in a CO₂ incubator at 37°C.

Chemicals, reagents, and enzymes. All chemicals used in this study were reagent grade or better and were purchased from Sigma Chemical Co. (St. Louis, MO), unless otherwise specified. Tris-Tricine gels (16.5%) and running buffer were obtained from Bio-Rad Laboratories (Richmond, CA). β-N-Acetylhexosaminidase was purchased from New England Biolabs (Beverly, MA). The lectin GS-II was purchased from EY Labs (San Mateo, CA).

LPS-LOS purification and SDS-PAGE. LPS-LOS was purified from broth-grown cells by the hot phenol-water method, followed by lyophilization (29), or from agar plate cultures as described by Hitchcock and Brown (8). LPS-LOS was diluted in lysing buffer, and the suspension was boiled for 10 min immediately before SDS-PAGE gels were loaded. Approximately 0.1 µg of LOS or 1 µg of LPS was subjected to SDS-PAGE on a 16.5% Tris-Tricine gel in Tris-Tricine running buffer at 30 mA for 2 h. The gel was fixed for 18 h in 40% ethanol-5% acetic acid, and glycolipids were visualized by silver staining (28). LOS made by N. gonorrhoeae strain F62 and its LgtAlpt3::Tn5 mutant were used as LOS size markers for SDS-PAGE comparisons. These two strains and their LOS have been described previously (17).

Lectin and Western blotting. LPS-LOS was transferred onto Immobilon-P polyvinylidene difluoride membranes (0.45 µm; Millipore) using a Criterion blotter at a constant 100 V for 20 min. For detection of lectin binding, membranes were dried for 1 h at 37°C, blocked in 1% casein (hydrolyzed with 1 N NaOH and neutralized with HCl to pH 7.5) for 1 h, and incubated for 18 h at 4°C with lectin GS-II at a concentration of 10 µg/ml. Membranes were washed three times with horseradish peroxidase-conjugated GS-II buffer (0.01 M phosphate, 0.15 M NaCl, 0.5 mM CaCl₂; pH 7.4) and incubated with developer (4.48 mM 4-chloro-1-naphthol, 0.006% H₂O₂, 50 mM Tris). For binding to a polyclonal N. sicca antibody, membranes were blocked for 18 h in phosphate-buffered saline (PBS) containing 1% gelatin (Sigma) and 0.1% Tween 20 (Fisher Scientific). The membranes were incubated with N. sicca antibody for 90 min, washed three times with PBS containing 0.1% Tween 20, and incubated with horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (Upstate) for 1 h. Membranes were then washed three times with PBS containing 0.1% Tween 20 and incubated with developer.

Production of N. sicca antibody. For generation of polyclonal serum specific for N. sicca 4320 LPS-LOS, New Zealand White rabbits (2 to 2.5 kg) were immunized intraperitoneally with 50 µg of LOS-LPS purified from N. sicca 4320 three times at 2-week intervals. Serum was collected 14 days after the last immunization and stored at –20°C until it was used.

MALDI MS of O-deacylated LOS and LPS. LPS-LOS mixtures were O deacylated prior to matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) (10). Anhydrous hydrazine (200 µl) was added to 0.5 mg of LPS-LOS and incubated at 37°C with periodic vortexing for 20 min. O-deacylated glycolipids were precipitated with –20°C acetone and centrifuged at 12,000 x g for 20 min. The pellet was washed with cold acetone and resuspended in H₂O to a final concentration of 2 µg/µl. Samples were desalted with cation-exchange beads (Dowex 50X) and then combined with 100 mM 2,5-dihydrobenzoic acid in methanol (MeOH). Negative-ion MALDI MS was performed in linear mode with delayed extraction using a Voyager Elite time of flight (TOF) instrument equipped with a 337-nm nitrogen laser (PerSeptive Biosystems, Framingham, MA). Analyses were performed with a 150-ns time delay and a grid voltage that was 92 to 94% of the full acceleration voltage (20 kV) and with external calibration.

MALDI-TOF and ESI MS of permethylated LOS-LPS. Samples (500 µg) of LPS-LOS were combined with 1 µg of β-cyclodextrin standard; 200 µl of 1% acetic acid in water was added to the samples, and the mixtures were heated to 100°C for 1 h to separate glycans from lipids. After centrifugation, the liquid layers were saved and dried by vacuum centrifugation. Samples were derivatized with a pyrazole reducing-end protecting tag by adding 20 µl of anhydrous hydrazine to each dried sample. After vacuum centrifugation, 50 µl of 10% 2,4-pentanedione in water was added, and a cyclic pyrazole protecting group was formed during vacuum centrifugation. Both samples were methylated, dried, and reconstituted in MeOH for MALDI-TOF- and electrospray ionization (ESI)-MS analyses (16).

Glycosidase digestion of repeating carbohydrate unit. LPS-LOS (600 ng) was digested for 18 h with β-N-acetylhexosaminidase at 37°C (10, 15, 27, 32). Dilutions of the digested glycoses, along with identical amounts of undigested N. sicca 4320 LPS-LOS, were subjected to SDS-PAGE on a 16.5% Tris-Tricine gel, as described above.

Analysis of the composition by GC-MS. N. sicca 4320 LOS and LPS were hydrolyzed to monosaccharide components. HCl (1 N) in anhydrous MeOH was added to dried samples of N. sicca 4320 LOS-LPS and the L-rhamnose, L-fucose, and D-GlcNAc standards. These samples were flushed with N₂ prior to incubation at 80°C for 16 to 24 h. After evaporation to dryness under N₂ at 35 to 40°C, MeOH was added to eliminate HCl. MeOH (200 µl), pyridine (20 µl), and acetic anhydride (20 µl) were added, and the tubes were vortexed and held at room temperature for 20 min. Samples were evaporated to dryness under N₂ at 35 to 40°C before addition of toluene and acetic acid in excess acetic anhydride. To the methylated glycosides Tri-Sil (200 µl) was added, and the tubes were flushed with N₂ and placed at 80°C for 20 to 30 min. After rapid cooling to 20 to 22°C, the tubes were again evaporated to dryness under N₂ at 35 to 40°C. The remaining white residue was washed twice with 100 µl of n-hexane. The combined washes were used for gas chromatography (GC)-MS analysis.

MSⁿ analysis. Two 500-µg samples of purified LPS-LOS were combined with 1 µg of a β-cyclodextrin standard. Acetic acid in water (200 µl of a 1% solution) was added to the samples, and the preparations were heated at 100°C for 1 h to separate the glycan from the lipid. After centrifugation, the liquid layers were saved and dried by vacuum centrifugation. Samples were reconstituted in 100 µl MeOH and analyzed by ESI by nanospray ionization MS/MSⁿ with a Finnigan LTQ Classic equipped with a nanospray ionization source at a flow rate of 0.5 µl/min and a spray voltage of 1.3 kV.

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SDS-PAGE analysis of N. sicca 4320 extracts. A proteinase K-treated whole-cell lysate of N. sicca was electrophoresed through an SDS-PAGE gel and stained with silver (Fig. 2). Figure 2A shows a pattern of repeating bands that is characteristic of LPS but not of neisserial LOS. The SDS-PAGE gel in Fig. 2B shows that the predominant low-molecular-mass LOS molecule from N. sicca 4320 has a mobility comparable to that of the m/z 2426 N. gonorrhoeae F62LgtAlpt3::Tn5 LOS (17). The repeating OS bands in the top portion of the gel were consistent with those seen when LPS is analyzed, suggesting that N. sicca produces a glycolipid similar to LPS.

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FIG. 2. Analysis of N. sicca 4320 LPS-LOS. (A) LPS-LOS was electrophoresed through a 16.5% SDS-PAGE gel and silver stained. The prominent high-mobility band is the LOS molecule, whereas the low-mobility bands represent successive additions of LPS O antigen. (B) Resolution of the low-molecular-mass LOS region. The lanes contained LOS-LPS purified from N. sicca 4320 (lane 1), N. gonorrhoeae F62 (lane 2), and N. gonorrhoeae F62LgtAlpt3::Tn5 (20) (lane 3).

MALDI-TOF analysis of N. sicca 4320 glycolipids. To determine the structure of the carbohydrate-containing component, it was first purified by the hot phenol-water method (29). Negative-ion MALDI-TOF MS was performed with O-deacylated purified glycolipids. The spectrum acquired (Fig. 3) has three prominent peaks. The peak at m/z 951.50 is consistent with an O-deacylated, N-diacylated, diphosphoryl neisserial LOS lipoidal moiety (10). Based on the composition of other neisserial LOS, the peak at m/z 2414.8 is likely to correspond to (Hex)₂(Hep)₃(phosphoethanolamine)₁(Kdo)₂ (lipoidal moiety), and the ions at m/z 2222.65 could differ by a single Hep (192 Da). The masses of the larger molecules are consistent with electrophoretic retention of the less abundant, slower-migrating band on the SDS-PAGE gel (Fig. 2B), whereas the m/z 2222.65 ions are consistent with the faster-migrating band. These data suggest that Hep is the terminal sugar in the LOS of N. sicca 4320. The data demonstrate that while N. sicca 4320 expresses a low-molecular-mass glycolipid whose SDS-PAGE mobility is similar to that of known neisserial LOS, its structure is more typical of the three-Hep LOS expressed by Haemophilus species (4).

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FIG. 3. MALDI-TOF analysis showing that N. sicca 4320 produces LOS and LPS. The spectrum is the spectrum for purified O-deacylated LOS and LPS of N. sicca 4320. The masses of the abundant fragments are indicated above the corresponding peaks. Asterisks indicate a series of molecular ions separated by a constant m/z 349 mass difference. The arrow indicates a component with a mass lower than that of the neisserial lipid.

Examination of the strain 4320 spectrum also shows the presence of a series of peaks differing by an apparent mass of 349 Da that could correspond to a disaccharide composed of HexNAc (203 Da) and deoxyhexose (dHex) (146 Da). Each of the series of peaks differing by m/z 349 in the MALDI spectrum could correspond to one band in the ladder pattern on the SDS-PAGE gel (Fig. 2A). The spectrum in Fig. 3 contains a peak at m/z 851, at a mass less than and apparently unrelated to that of the peak at m/z 951.50 for the diphosphoryl deacylated lipid A, and this suggested that the repeating units could have been cleaved from the lipid during hydrazinolysis. Accordingly, the presence of the series of peaks representing molecules with masses lower than those of the molecules postulated to produce the LOS peaks at m/z 2222.65 and m/z 2414 also suggests that these repeating carbohydrate units are anchored to a novel lipid or by a novel chemical linkage that is more susceptible to hydrazinolysis.

Determination of composition of the disaccharide repeat. To determine the components of the repeating unit, MALDI-TOF and ESI MS were performed with the glycoses released by acid hydrolysis after permethylation. The MALDI-TOF MS spectrum is shown in Fig. 4. In the spectrum between m/z 2400 and m/z 4800, a region expected to be free of peaks for core glycoses, there is a series of peaks at m/z 2651.1, 3069.9, 3490.1, 3909.2, 4328.6, and 4747.9 that differ by m/z 419. A difference of 419 Da is consistent with the mass of a HexNAc-dHex disaccharide with addition of five methyl groups.

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FIG. 4. MALDI-TOF analysis showing that N. sicca 4320 LPS contains a dissacharide repeat. The spectrum shows the profile of N. sicca 4320 LPS produced by MALDI MS. The position of the β-cyclodextrin standard is indicated, and the masses of the abundant fragments are indicated above the corresponding peaks. The region from m/z 2400 to m/z 4800 is enlarged to provide a clear view of the peaks representing the repeating carbohydrate.

To establish further the components of the m/z 419 repeating unit, ESI MS was performed after acid hydolysis, permethylation, and pyrazole derivatization (Fig. 5). The spectrum confirmed the presence of the m/z 419 repeating unit, along with fragments with mass differences that were consistent with the loss of a dHex or a HexNAc. For example, the ions at m/z 1809.8 correspond to four repeating units linked to pyrazole; the mass of the ions at m/z 1635.8 is 174 Da less (corresponding to loss of a methylated dHex), and it is 245 Da (the mass of methylated HexNAc) greater than the mass of the ions at m/z 1390.8. This is consistent with three repeating units. Similarly, the mass of the ions at m/z 1216.7 is consistent with loss of methylated dHex from the ions at m/z 1390.8 and addition of a methylated HexNAc to the ions corresponding to a two-disaccharide repeating unit ion at m/z 971.8.

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FIG. 5. ESI-MS analysis showing a dHex-HexNAc repeat. The spectrum shows the results of ESI MS of the pyrazole-derivatized methylated polysaccharide. The masses of the abundant fragments are indicated above the corresponding peaks. Structures representing the m/z 971.8 and 1809.8 ions also are shown, and the dHex and HexNAc components are identified as rhamnose and GlcNAc, respectively.

Since the carbohydrate repeat was apparently released from its membrane anchor by both hydrazine and acid hydrolysis, we could not definitively establish whether it is linked to lipid A or some other molecule. All attempts to purify the high-molecular-mass subunits by removing the low-molecular-mass units were unsuccessful. The presence of this relatively high-molecular-mass repeating unit component and the carbohydrate composition of the disaccharide unit are novel and have not been reported previously for Neisseria.

Identification of the terminal sugar of the disaccharide repeat. To determine if HexNAc was at the nonreducing terminus of the disaccharide, glycosidase digestion was performed (Fig. 6) with the enzyme β-N-acetylhexosaminidase, which specifically cleaves nonreducing terminal β-D-N-acetyl-D-galactosamine and -glucosamine residues from OSs. After incubation of N. sicca 4320 OSs with this enzyme, the digested products, alongside untreated molecules, were electrophoresed through an SDS-PAGE gel and silver stained. Based on the MS data, each band of the ladder is apparently larger than the one below it by one disaccharide repeating unit. When lanes 1 (undigested LPS-LOS) and 2 (digested LPS-LOS) are compared, a shift in the mobility of the molecules after digestion is evident, to a position midway between the positions of the bands for the undigested molecules. These data suggest that the OS was digested by the enzyme and hence that the nonreducing terminus of each repeat is a HexNAc linked in the β-configuration. While commercially available glycosidases can contain other contaminating glycosidases, the digestion specificity of the glycosidase employed suggests that the nonreducing terminus is either D-N-acetylgalactosamine or D-GlcNAc.

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FIG. 6. Exoglycosidase digestion of N. sicca 4320 LPS with N-acetylhexosaminidase. (A) Lane 1 shows the SDS-PAGE profile of N. sicca 4320 LOS and LPS after digestion with β-N-acetylhexosaminidase. Lane 2 contained the same preparation, but it was not digested. Preparations were run on a 16.5% Tris-Tricine polyacrylamide gel and then silver stained. The arrows indicate bands demonstrating the shift in mobility that occurred upon addition of the glycosidase. (B) Western analysis of N. sicca 4320 LPS-LOS. Blot 1 is a Western blot of LPS-LOS isolated from N. sicca 4320 separated on a 16.5% Tris-Tricine SDS-PAGE gel using lectin GS-II, which specifically recognizes terminal N-acetyl-D-glucosamine. Blot 2 is a Western blot of N. sicca 4320 LOS and LPS after digestion with β-N-acetylhexosaminidase. (C) Western blot with anti-N. sicca antibody of LPS-LOS isolated from N. sicca 4320 after separation on a 16.5% Tris-Tricine SDS-PAGE gel.

The lectin GS-II is specific for nonreducing terminal - or β-GlcNAc residues. Western blot analysis (Fig. 6B, blot 1) shows that the lectin bound to the N. sicca OS but not to OS that had been digested with enzyme (Fig. 2B, lane 2), supporting the conclusion that the nonreducing terminus of the O repeat is GlcNAc. Hexosaminidase digestion did not affect the electrophoretic mobility of N. sicca 4320 core LOS (Fig. 6A), nor did the GS-II lectin bind to the core LOS (Fig. 6B, blot 1). These data support the conclusion that there is no terminal β-GlcNAc in the core LOS component of N. sicca.

Polyclonal antibody raised against N. sicca glycolipids bound to both the disaccharide repeat component and the LOS (Fig. 6B, blot 2). Since the immunoblotting with the polyclonal antibody was performed on the same membrane as the immunoblotting with the lectin, this result shows that the failure of the lectin to bind was not due to the failure of the LOS-LPS to bind to the membrane.

To determine the identity of the other monosaccharide in the N. sicca 4320 disaccharide, GC-MS was performed after acid hydrolysis, reduction, and O trimethylsilylation. GC-MS spectra for potential monosaccharide components were generated as standards. As shown in Fig. 7, derivatized L-rhamnose had a retention time of 11.10 min, whereas the D-GlcNAc derivative had a retention time of 24.51 min and generated a different mass spectral fragmentation pattern. N. sicca 4320 LPS-LOS were hydrolyzed to their monosaccharide components and derivatized. The corresponding GC-MS spectra are shown in Fig. 7C. The major components of the glycose mixture had retention times of 11.09 and 24.51 min, and MS fragmentation of these major components created patterns that matched those of the L-Rha and D-GlcNAc standards. The MALDI-TOF, ESI, and GC-MS data and the data for SDS-PAGE with lectin binding and glycosidase digestion are all consistent with the presence of N. sicca 4320 disaccharide repeating units that are composed of L-Rha and with the presence of nonreducing terminal β-D-GlcNAc.

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FIG. 7. GC-MS analysis of N. sicca 4320 LPS. (A and B) Profiles of the L-rhamnose and D-GlcNAc monosaccharide controls, respectively. The retention times of these sugars along with the fragmentation patterns of the ions represented by the peaks are shown. (C) The profile in panel a shows the retention times of the monosaccharide sugars in strain 4320 LOS and LPS. The retention times of the major peaks are indicated. Panels b and c show the fragmentation patterns of the sugars represented by the peaks at 11.09 min (panel b) and 24.51 min (panel c).

The gonococcus is known to contain a cryptic rhamnose biosynthetic cluster (19). While no one has identified rhamnose in any of the LOS structures of pathogenic Neisseria, Wiseman and Caird (31) were able to detect small amounts of this sugar in glycolipid preparations from a variety of gonococcal isolates. Therefore, it is not entirely surprising that N. sicca 4320 produces rhamnose and that it has a fully functional rhamnose biosynthetic cluster.

Linkage analysis of the disaccharide repeat. To analyze the linkage between Rha and GlcNAc, the glycoses were permethylated and reduced prior to extensive fragmentation by ESI MSⁿ analysis. The ions at m/z 923 containing two disaccharide units were selected for successive fragmentation by MSⁿ analysis into smaller structures. Cleavage of these fragments produced patterns that have been shown to be characteristic of specific glycosidic bond orientations. The MS³ spectrum with fragment ions of the m/z 701 ions is shown in Fig. 8A. As shown in Fig. 8B, these molecular ions are composed of two GlcNAc molecules and one Rha. For example, fragment ion peaks at m/z 474, m/z 456, and m/z 442 are each characteristic of loss of a methylated GlcNAc from the ions at m/z 701. Corresponding peaks at m/z 268 and m/z 282 are present in the spectrum due to loss of the disaccharide unit from the ions at m/z 701. In addition, daughter ion peaks at m/z 595 and m/z 627 are present in the MS³ spectrum, and as shown in Fig. 8B, these ions are characteristic for a β(1-3) linkage to GlcNAc. Together, the data provide evidence that the disaccharide unit has an L-Rha residue that is connected to carbon 3 of GlcNAc through a β bond.

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FIG. 8. MS3 spectrum from MSn analysis of N. sicca 4320 LPS. (A) MS3 spectrum obtained after fragmentation of the m/z 701 ion. The masses of the abundant fragments are indicated above the corresponding peaks. (B) Expected structures characteristic of specific linkages generated during MS3 analysis. Me, methyl.

The orientation of the bond linking GlcNAc to rhamnose was also examined by use of fragmentation with MSⁿ analysis. The MS⁴ spectrum generated by further fragmentation of the m/z 456 ion is shown in Fig. 9A. Structures corresponding to the generated daughter ions are consistent with the expected fragmentation of this ion. For example, several cleavage products of the m/z 456 ion, including the m/z 282, m/z 300, and m/z 268 ions shown in Fig. 9B, are evident in the spectrum. These ions represent loss of Rha compared with the m/z 456 ion. Additionally, an m/z 340 ion is observed, whose structure is consistent with β(1-4) linkage of GlcNAc to Rha.

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FIG. 9. MS4 spectrum from MSn analysis of N. sicca 4320 LPS. (A) MS4 spectrum generated by fragmentation of the m/z 456 ion. The masses of the abundant fragments are indicated above the corresponding peaks. (B) Expected structures characteristic of specific linkages generated during MS4 analysis. Me, methyl.

It is interesting that N. sicca 4320 was isolated from a fatal case of endocarditis. Because this species is not normally pathogenic in humans, the production of an OS repeating unit conceivably could have contributed to the increased pathogenicity of this isolate. Further experiments are needed to determine if the gonococcus and meningococcus are also capable of synthesizing this type of novel LPS structure. It is possible that the pathogenic Neisseria strains are capable of expressing the higher-molecular-mass structure in vivo, since in vivo extension of the gonococcal MS11mkC LOS with a small number of poly-N-acetyllactosamine repeats is associated with increased pathogenicity (10). This is a possibility as this work provides documentation that Neisseria strains have the necessary genetic machinery to synthesize LPS-like structures.

 
The work described in this paper was supported in part by grants AI 24452 (D.C.S.) and AI 21620 and AI 065605 (J.M.G.) from the National Institutes of Health and by the Research Service of the Department of Veterans Affairs (J.M.G.).

We thank Connie John for her critical reading of the manuscript.

 
* Corresponding author. Mailing address: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742. Phone: (301) 405-5448. Fax: (301) 314-9489. E-mail: dcstein{at}umd.edu

Published ahead of print on 6 March 2009.

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1
1. Arking, D., Y. Tong, and D. C. Stein. 2001. Analysis of lipooligosaccharide biosynthesis in the Neisseriaceae. J. Bacteriol. 183:934-941.[Abstract/Free Full Text]
2
2. Reference deleted.
3
3. Banerjee, A., R. Wang, S. Uljohn, P. A. Rice, E. C. Gotschlich, and D. C. Stein. 1998. Identification of the gene (lgtG) encoding the lipooligosaccharide β chain synthesizing glucosyl transferase from Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. USA 95:10872-10877.[Abstract/Free Full Text]
4
4. Filiatrault, M. J., B. W. Gibson, B. Schilling, S. Sun, R. S. Munson, Jr., and A. A. Campagnari. 2000. Construction and characterization of Haemophilus ducreyi lipooligosaccharide (LOS) mutants defective in expression of heptosyltransferase III and β1,4-glucosyltransferase: identification of LOS glycoforms containing lactosamine repeats. Infect. Immun. 68:3352-3361.[Abstract/Free Full Text]
5
5. Reference deleted.
6
6. Gibson, B. W., J. W. Webb, R. Yamasaki, S. J. Fisher, A. L. Burlingame, R. E. Mandrell, H. Schneider, and J. M. Griffiss. 1989. Structure and heterogeneity of the oligosaccharides from the lipopolysaccharides of a pyocin-resistant Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. USA 86:17-21.[Abstract/Free Full Text]
7
7. Gotschlich, E. C. 1994. Genetic locus for the biosynthesis of the variable portion of Neisseria gonorrhoeae lipooligosaccharide. J. Exp. Med. 180:2181-2190.[Abstract/Free Full Text]
8
8. Hitchcock, P. J., and T. M. Brown. 1983. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J. Bacteriol. 154:269-277.[Abstract/Free Full Text]
9
9. John, C. M., J. M. Griffiss, M. A. Apicella, R. E. Mandrell, and B. W. Gibson. 1991. The structural basis for pyocin resistance in Neisseria gonorrhoeae lipooligosaccharides. J. Biol. Chem. 266:19303-19311.[Abstract/Free Full Text]
10
10. John, C. M., H. Schneider, and J. M. Griffiss. 1999. Neisseria gonorrhoeae that infect men have lipooligosaccharides with terminal N-acetyl-lactosamine repeats. J. Biol. Chem. 274:1017-1025.[Abstract/Free Full Text]
11
11. Kahler, C. M., R. W. Carlson, M. M. Rahman, L. E. Martin, and D. S. Stephens. 1996. Inner core biosynthesis of lipooligosaccharide (LOS) in Neisseria meningitidis serogroup B: identification and role in LOS assembly of the alpha1,2 N-acetylglucosamine transferase (RfaK). J. Bacteriol. 178:1265-1273.[Abstract/Free Full Text]
12
12. Reference deleted.
13
13. Linz, B., M. Schenker, P. Zhu, and M. Achtman. 2000. Frequent interspecific genetic exchange between commensal neisseriae and Neisseria meningitidis. Mol. Microbiol. 36:1049-1058.[CrossRef][Medline]
14
14. Reference deleted.
15
15. McLeod Griffiss, J., B. L. Brandt, N. B. Saunders, and W. Zollinger. 2000. Structural relationships and sialylation among meningococcal L1, L8 and L3,7 lipooligosaccharide serotypes. J. Biol. Chem. 275:9716-9724.[Abstract/Free Full Text]
16
16. Mühlecker, W., S. Gulati, D. P. McQuillen, S. Ram, P. A. Rice, and V. N. Reinhold. 1999. An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn. Glycobiology 9:157-171.[Abstract/Free Full Text]
17
17. O'Connor, E. T., A. Piekarowicz, K. V. Swanson, J. M. Griffiss, and D. C. Stein. 2006. Biochemical analysis of Lpt3, a protein responsible for phosphoethanolamine addition to lipooligosaccharide of pathogenic Neisseria. J. Bacteriol. 188:1039-1048.[Abstract/Free Full Text]
18
18. Reference deleted.
19
19. Robertson, B. D., M. Frosch, and J. P. van Putten. 1994. The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis. J. Bacteriol. 176:6915-6920.[Abstract/Free Full Text]
20
20. Saez-Nieto, J. A., R. Lujan, J. V. Martinez-Suarez, S. Berron, J. A. Vazquez, M. Vinas, and J. Campos. 1990. Neisseria lactamica and Neisseria polysaccharea as possible sources of meningococcal beta-lactam resistance by genetic transformation. Antimicrob. Agents Chemother. 34:2269-2272.[Abstract/Free Full Text]
21
21. Sandlin, R., and D. C. Stein. 1991. Structural heterogeneity of lipopolysaccharides the Neisseriaceae. FEMS Microbiol. Let. 90:69-72.[CrossRef]
22
22. Schilling, B., B. W. Gibson, M. J. Filiatrault, and A. A. Campagnari. 2002. Characterization of lipooligosaccharides from Haemophilus ducreyi containing polylactosamine repeats. J. Am. Soc. Mass Spectrom. 13:724-734.[CrossRef][Medline]
23
23. Schneider, H., A. S. Cross, R. A. Kuschner, D. N. Taylor, J. C. Sadoff, J. W. Boslego, and C. D. Deal. 1995. Experimental human gonococcal urethritis: 250 Neisseria gonorrhoeae MS11mkC are infective. J. Infect. Dis. 172:180-185.[Medline]
24
24. Schneider, H., K. A. Schmidt, D. R. Skillman, L. Van De Verg, R. L. Warren, H. J. Wylie, J. C. Sadoff, C. D. Deal, and A. S. Cross. 1996. Sialylation lessens the infectivity of Neisseria gonorrhoeae MS11mkC. J. Infect. Dis. 173:1422-1427.[Medline]
25
25. Snyder, L. A. S., and N. J. Saunders. 2006. The majority of genes in the pathogenic Neisseria species are present in non-pathogenic Neisseria lactamica, including those designated as ‘virulence genes’. BMC Genomics 7:128.[CrossRef][Medline]
26
26. Stenutz, R., A. Weintraub, and G. Widmalm. 2006. The structures of Escherichia coli O-polysaccharide antigens. FEMS Microbiol. Rev. 30:382-403.[CrossRef][Medline]
27
27. Tong, Y., D. Arking, S. Ye, B. Reinhold, V. Reinhold, and D. C. Stein. 2002. Neisseria gonorrhoeae strain PID2 simultaneously expresses six chemically related lipooligosaccharide structures. Glycobiology 12:523-533.[Abstract/Free Full Text]
28
28. Tsai, C. M., and C. E. Frasch. 1982. A sensitive silver stain for detecting lipooligosaccharide in polyacrylamide gels. Anal. Biochem. 119:115-119.[CrossRef][Medline]
29
29. Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharides: extraction with phenol-water and further applications of the procedure. Methods Carbohydr. Chem. 5:83-91.
30
30. White, L. A., and D. S. Kellogg, Jr. 1965. Neisseria gonorrhoeae identification in direct smears by a fluorescent antibody counterstain method. Appl. Microbiol. 13:171-174.[Medline]
31
31. Wiseman, G. M., and J. D. Caird. 1977. Composition of the lipopolysaccharide of Neisseria gonorrhoeae. Infect. Immun. 16:550-556.[Abstract/Free Full Text]
32
32. Yamasaki, R., H. Schneider, J. M. Griffiss, and R. Mandrell. 1988. Epitope expression of gonococcal lipooligosaccharide (LOS): importance of the lipoidal moiety for expression of an epitope that exists in the oligosaccharide moiety of LOS. Mol. Immunol. 25:799-809.[CrossRef][Medline]
33
33. Zhu, P., M. J. Klutch, M. C. Bash, R. S. Tsang, L. K. Ng, and C. M. Tsai. 2002. Genetic diversity of three lgt loci for biosynthesis of lipooligosaccharide (LOS) in Neisseria species. Microbiology 148:1833-1844.[Abstract/Free Full Text]

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Journal of Bacteriology, May 2009, p. 3311-3320, Vol. 191, No. 10
0021-9193/09/$08.00+0     doi:10.1128/JB.01433-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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