JOURNAL OF BACTERIOLOGY, May 2009, p. 3311–3320 Vol. 191, No. 100021-9193/09/$08.00�0 doi:10.1128/JB.01433-08Copyright © 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,3Vernon N. Reinhold,2 Clinton J. Miller,1 and Daniel C. Stein1*

Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 207421; Department ofChemistry, University of New Hampshire, Durham, New Hampshire 038242; and 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

Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysac-charide. Using matrix-assisted laser desorption ionization—time of flight and electrospray ionizationmass 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 andestablished its �-anomeric configuration. Mass spectrometry analysis and lectin binding were used toverify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4)�-L-rhamnose (1-3) �-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 genusNeisseria.

Commensal Neisseria strains colonize the human respiratorytract. Frequent interspecific genetic exchange between com-mensal Neisseria strains and the pathogens Neisseria meningi-tidis and Neisseria gonorrhoeae occurs (13). It is thought thecommensal organisms serve as reservoirs for antibiotic resis-tance genes (20). The similarity between the gene comple-ments of the commensals and pathogens suggests that thevirulence of the pathogenic Neisseria spp. may not result fromthe genes that they possess but rather from a “genetic person-ality” which is a result of combinations of these genes, se-quence variations that alter the function of gene products, thepresence of genes for which a virulence phenotype has not yetbeen identified, and/or differences in the regulation of genes(25).

Lipooligosaccharide (LOS) is an important neisserial viru-lence determinant consisting of an oligosaccharide (OS) com-ponent attached to lipid A via 3-deoxy-2-keto-D-manno-octu-losonic acid (Kdo). The structures of a sufficient number ofneisserial LOS molecules have been determined to form acoherent yet incomplete picture of the structural diversity oftheir LOS (Fig. 1). The different LOS structures have a con-served core with two Kdo molecules, two heptose (Hep) mol-ecules, and one N-acetylhexosamine (HexNAc) molecule andvary in the composition and size of the OS attached to oneHep (HepI; �-chain variation) and in the attachment of anOS or phosphoethanolamine to the other Hep (HepII;�-chain variation) or by addition of a galactose to the N-acetylglucosamine (GlcNAc) found on HepII (�-chain ex-tension) (3, 6, 7, 9–11). This structural motif is different

from that of lipopolysaccharide (LPS) of other types ofbacteria, which contains an O antigen composed of a re-peating sugar polymer, typically consisting of four to sevensugars (26). No one has reported the presence of an Oantigen in pathogenic strains of Neisseria.

A few studies have analyzed the structure of LOS pro-duced by commensal Neisseria strains, and the data indicatethat the LOS heterogeneity is greater than the heterogeneityin the gonococcus and meningococcus (21). CommensalNeisseria strains are capable of producing LOS moleculesthat are structurally different from the molecules in theknown Neisseria repertoire in that they fail to bind mono-clonal antibodies specific for LOS epitopes characteristic ofthe gonococcus and meningococcus (1). They also can lacksome of the LOS biosynthesis genes found in N. meningitidisand N. gonorrhoeae (1, 33). These findings suggest that al-ternative LOS structures are present in commensal Neisseriastrains. Sandlin and Stein (21) identified a strain of Neisseriasicca that expressed an unusual glycolipid that appearedbased on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) to be analogous to LPS made byenteric bacteria. A poly-N-acetyllactosamine repeat wasseen in some strains of the gonococcus (10), and we postu-lated that the repeating carbohydrate found in N. siccacould be a variant of this structure.

A few studies have shown that both Neisseria and Haemophi-lus strains have the ability to extend their LOS by addinglactosamine repeats to form polylactosamine; these strainsseem to possess increased virulence (4, 10, 22). When N. gonor-rhoeae MS11mkC was used to inoculate healthy male volun-teers, 100% of the volunteers developed urethritis, comparedto an infectivity rate of 40% for strains expressing a truncatedLOS (23). Fresh isolates from the volunteers who had con-tracted urethritis produced LOS molecules with N-acetyllac-tosamine repeats, whereas the isolates in the original inoculum

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

� Published ahead of print on 6 March 2009.

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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 ofHaemophilus ducreyi, a causative agent of genital ulcers, wasstudied; infectious strains made LOS with a few lactosaminerepeats (4, 22).

N. sicca is normally not pathogenic in healthy adults. Aprevious study indicated that N. sicca 4320 expressed a mole-cule with a repeating carbohydrate structure that appeared tobe similar to the O-antigen structure (21). Because such mol-ecules have not been found in pathogenic Neisseria strains, weanalyzed the structure of the repeating carbohydrate unit andshowed that it is a repeating disaccharide that is novel to thegenus Neisseria.

MATERIALS AND METHODS

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

Chemicals, reagents, and enzymes. All chemicals used in this study werereagent grade or better and were purchased from Sigma Chemical Co. (St. Louis,MO), unless otherwise specified. Tris-Tricine gels (16.5%) and running bufferwere obtained from Bio-Rad Laboratories (Richmond, CA). �-N-Acetylhex-osaminidase was purchased from New England Biolabs (Beverly, MA). Thelectin 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), orfrom agar plate cultures as described by Hitchcock and Brown (8). LPS-LOS wasdiluted in lysing buffer, and the suspension was boiled for 10 min immediatelybefore SDS-PAGE gels were loaded. Approximately 0.1 �g of LOS or 1 �g ofLPS was subjected to SDS-PAGE on a 16.5% Tris-Tricine gel in Tris-Tricinerunning 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 byN. gonorrhoeae strain F62 and its �LgtA�lpt3::Tn5 mutant were used as LOSsize markers for SDS-PAGE comparisons. These two strains and their LOS havebeen described previously (17).

Lectin and Western blotting. LPS-LOS was transferred onto Immobilon-Ppolyvinylidene difluoride membranes (0.45 �m; Millipore) using a Criterionblotter 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 with1 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 �g/ml. Membraneswere washed three times with horseradish peroxidase-conjugated GS-IIbuffer (0.01 M phosphate, 0.15 M NaCl, 0.5 mM CaCl2; pH 7.4) and incu-bated with developer (4.48 mM 4-chloro-1-naphthol, 0.006% H2O2, 50 mMTris). For binding to a polyclonal N. sicca antibody, membranes were blockedfor �18 h in phosphate-buffered saline (PBS) containing 1% gelatin (Sigma)and 0.1% Tween 20 (Fisher Scientific). The membranes were incubated withN. 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 washedthree times with PBS containing 0.1% Tween 20 and incubated with devel-oper.

Production of N. sicca antibody. For generation of polyclonal serum specificfor N. sicca 4320 LPS-LOS, New Zealand White rabbits (2 to 2.5 kg) wereimmunized intraperitoneally with 50 �g of LOS-LPS purified from N. sicca 4320three times at 2-week intervals. Serum was collected 14 days after the lastimmunization and stored at �20°C until it was used.

MALDI MS of O-deacylated LOS and LPS. LPS-LOS mixtures were O deac-ylated prior to matrix-assisted laser desorption ionization (MALDI) mass spec-trometry (MS) (10). Anhydrous hydrazine (200 �l) was added to 0.5 mg ofLPS-LOS and incubated at 37°C with periodic vortexing for 20 min. O-deacyl-ated glycolipids were precipitated with �20°C acetone and centrifuged at12,000 g for 20 min. The pellet was washed with cold acetone and resuspendedin H2O to a final concentration of 2 �g/�l. Samples were desalted with cation-exchange beads (Dowex 50X) and then combined with 100 mM 2,5-dihydroben-zoic acid in methanol (MeOH). Negative-ion MALDI MS was performed inlinear 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 gridvoltage that was 92 to 94% of the full acceleration voltage (20 kV) and withexternal calibration.

MALDI-TOF and ESI MS of permethylated LOS-LPS. Samples (500 �g) ofLPS-LOS were combined with 1 �g of �-cyclodextrin standard; 200 �l of 1%acetic acid in water was added to the samples, and the mixtures were heated to100°C for 1 h to separate glycans from lipids. After centrifugation, the liquidlayers were saved and dried by vacuum centrifugation. Samples were derivatizedwith a pyrazole reducing-end protecting tag by adding 20 �l of anhydrous hy-drazine to each dried sample. After vacuum centrifugation, 50 �l of 10% 2,4-pentanedione in water was added, and a cyclic pyrazole protecting group wasformed during vacuum centrifugation. Both samples were methylated, dried, andreconstituted in MeOH for MALDI-TOF- and electrospray ionization (ESI)-MSanalyses (16).

Glycosidase digestion of repeating carbohydrate unit. LPS-LOS (600 ng) wasdigested for �18 h with �-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.

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

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Analysis of the composition by GC-MS. N. sicca 4320 LOS and LPS werehydrolyzed to monosaccharide components. HCl (1 N) in anhydrous MeOH wasadded 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 incuba-tion 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 �l), pyridine (20 �l), and aceticanhydride (20 �l) were added, and the tubes were vortexed and held at roomtemperature for 20 min. Samples were evaporated to dryness under N2 at 35 to40°C before addition of toluene and acetic acid in excess acetic anhydride. To themethylated glycosides Tri-Sil (200 �l) was added, and the tubes were flushed withN2 and placed at 80°C for 20 to 30 min. After rapid cooling to 20 to 22°C, thetubes were again evaporated to dryness under N2 at 35 to 40°C. The remainingwhite residue was washed twice with 100 �l of n-hexane. The combined washeswere used for gas chromatography (GC)-MS analysis.

MSn analysis. Two 500-�g samples of purified LPS-LOS were combined with1 �g of a �-cyclodextrin standard. Acetic acid in water (200 �l of a 1% solution)was added to the samples, and the preparations were heated at 100°C for 1 h toseparate the glycan from the lipid. After centrifugation, the liquid layers weresaved and dried by vacuum centrifugation. Samples were reconstituted in 100 �lMeOH and analyzed by ESI by nanospray ionization MS/MSn with a FinniganLTQ Classic equipped with a nanospray ionization source at a flow rate of 0.5�l/min and a spray voltage of 1.3 kV.

RESULTS AND DISCUSSION

SDS-PAGE analysis of N. sicca 4320 extracts. A protein-ase K-treated whole-cell lysate of N. sicca was electropho-resed through an SDS-PAGE gel and stained with silver(Fig. 2). Figure 2A shows a pattern of repeating bands thatis characteristic of LPS but not of neisserial LOS. The SDS-PAGE gel in Fig. 2B shows that the predominant low-mo-lecular-mass LOS molecule from N. sicca 4320 has a mobil-ity comparable to that of the m/z 2426 N. gonorrhoeaeF62�LgtAlpt3::Tn5 LOS (17). The repeating OS bands inthe top portion of the gel were consistent with those seenwhen LPS is analyzed, suggesting that N. sicca produces aglycolipid similar to LPS.

MALDI-TOF analysis of N. sicca 4320 glycolipids. To deter-mine the structure of the carbohydrate-containing component,

FIG. 2. Analysis of N. sicca 4320 LPS-LOS. (A) LPS-LOS waselectrophoresed through a 16.5% SDS-PAGE gel and silver stained.The prominent high-mobility band is the LOS molecule, whereas thelow-mobility bands represent successive additions of LPS O antigen.(B) Resolution of the low-molecular-mass LOS region. The lanescontained LOS-LPS purified from N. sicca 4320 (lane 1), N. gonor-rhoeae F62 (lane 2), and N. gonorrhoeae F62�LgtAlpt3::Tn5 (20)(lane 3).

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

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it was first purified by the hot phenol-water method (29). Neg-ative-ion MALDI-TOF MS was performed with O-deacylatedpurified glycolipids. The spectrum acquired (Fig. 3) has threeprominent peaks. The peak at m/z 951.50 is consistent with anO-deacylated, N-diacylated, diphosphoryl neisserial LOS lipoi-dal moiety (10). Based on the composition of other neisserialLOS, the peak at m/z 2414.8 is likely to correspond to(Hex)2(Hep)3(phosphoethanolamine)1(Kdo)2 (lipoidal moi-ety), and the ions at m/z 2222.65 could differ by a single Hep(192 Da). The masses of the larger molecules are consistentwith electrophoretic retention of the less abundant, slower-migrating band on the SDS-PAGE gel (Fig. 2B), whereas them/z 2222.65 ions are consistent with the faster-migrating band.These data suggest that Hep is the terminal sugar in the LOSof N. sicca 4320. The data demonstrate that while N. sicca 4320expresses a low-molecular-mass glycolipid whose SDS-PAGEmobility is similar to that of known neisserial LOS, its structureis more typical of the three-Hep LOS expressed by Haemophi-lus species (4).

Examination of the strain 4320 spectrum also shows thepresence of a series of peaks differing by an apparent massof 349 Da that could correspond to a disaccharide composedof HexNAc (203 Da) and deoxyhexose (dHex) (146 Da).

Each of the series of peaks differing by m/z 349 in theMALDI spectrum could correspond to one band in theladder pattern on the SDS-PAGE gel (Fig. 2A). The spec-trum in Fig. 3 contains a peak at m/z 851, at a mass less thanand apparently unrelated to that of the peak at m/z 951.50for the diphosphoryl deacylated lipid A, and this suggestedthat the repeating units could have been cleaved from thelipid during hydrazinolysis. Accordingly, the presence of theseries of peaks representing molecules with masses lowerthan those of the molecules postulated to produce the LOSpeaks at m/z 2222.65 and m/z 2414 also suggests that theserepeating carbohydrate units are anchored to a novel lipidor by a novel chemical linkage that is more susceptible tohydrazinolysis.

Determination of composition of the disaccharide repeat.To determine the components of the repeating unit,MALDI-TOF and ESI MS were performed with the glycosesreleased by acid hydrolysis after permethylation. TheMALDI-TOF MS spectrum is shown in Fig. 4. In the spec-trum between m/z 2400 and m/z 4800, a region expected tobe free of peaks for core glycoses, there is a series of peaksat m/z 2651.1, 3069.9, 3490.1, 3909.2, 4328.6, and 4747.9 thatdiffer by m/z 419. A difference of 419 Da is consistent with

FIG. 4. MALDI-TOF analysis showing that N. sicca 4320 LPS contains a dissacharide repeat. The spectrum shows the profile of N. sicca 4320LPS produced by MALDI MS. The position of the �-cyclodextrin standard is indicated, and the masses of the abundant fragments are indicatedabove 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 repeatingcarbohydrate.

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the mass of a HexNAc-dHex disaccharide with addition offive methyl groups.

To establish further the components of the m/z 419 re-peating unit, ESI MS was performed after acid hydolysis,permethylation, and pyrazole derivatization (Fig. 5). Thespectrum confirmed the presence of the m/z 419 repeatingunit, along with fragments with mass differences that wereconsistent with the loss of a dHex or a HexNAc. For exam-ple, the ions at m/z 1809.8 correspond to four repeatingunits linked to pyrazole; the mass of the ions at m/z 1635.8is 174 Da less (corresponding to loss of a methylated dHex),and it is 245 Da (the mass of methylated HexNAc) greaterthan the mass of the ions at m/z 1390.8. This is consistentwith three repeating units. Similarly, the mass of the ions atm/z 1216.7 is consistent with loss of methylated dHex fromthe ions at m/z 1390.8 and addition of a methylated HexNActo the ions corresponding to a two-disaccharide repeatingunit ion at m/z 971.8.

Since the carbohydrate repeat was apparently released fromits membrane anchor by both hydrazine and acid hydrolysis, wecould not definitively establish whether it is linked to lipid A orsome other molecule. All attempts to purify the high-molecu-lar-mass subunits by removing the low-molecular-mass unitswere unsuccessful. The presence of this relatively high-molec-ular-mass repeating unit component and the carbohydratecomposition of the disaccharide unit are novel and have notbeen reported previously for Neisseria.

Identification of the terminal sugar of the disaccharide re-peat. To determine if HexNAc was at the nonreducing termi-nus of the disaccharide, glycosidase digestion was performed(Fig. 6) with the enzyme �-N-acetylhexosaminidase, which spe-cifically cleaves nonreducing terminal �-D-N-acetyl-D-galac-

FIG. 5. ESI-MS analysis showing a dHex-HexNAc repeat. The spectrum shows the results of ESI MS of the pyrazole-derivatized methylatedpolysaccharide. The masses of the abundant fragments are indicated above the corresponding peaks. Structures representing the m/z 971.8 and1809.8 ions also are shown, and the dHex and HexNAc components are identified as rhamnose and GlcNAc, respectively.

FIG. 6. Exoglycosidase digestion of N. sicca 4320 LPS withN-acetylhexosaminidase. (A) Lane 1 shows the SDS-PAGE profile ofN. sicca 4320 LOS and LPS after digestion with �-N-acetylhexosamini-dase. Lane 2 contained the same preparation, but it was not digested.Preparations were run on a 16.5% Tris-Tricine polyacrylamide gel andthen silver stained. The arrows indicate bands demonstrating the shiftin mobility that occurred upon addition of the glycosidase. (B) Westernanalysis of N. sicca 4320 LPS-LOS. Blot 1 is a Western blot of LPS-LOSisolated from N. sicca 4320 separated on a 16.5% Tris-Tricine SDS-PAGEgel 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 afterdigestion with �-N-acetylhexosaminidase. (C) Western blot with anti-N.sicca antibody of LPS-LOS isolated from N. sicca 4320 after separation ona 16.5% Tris-Tricine SDS-PAGE gel.

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tosamine and -glucosamine residues from OSs. After incuba-tion of N. sicca 4320 OSs with this enzyme, the digestedproducts, alongside untreated molecules, were electropho-resed through an SDS-PAGE gel and silver stained. Basedon the MS data, each band of the ladder is apparently largerthan 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 moleculesafter digestion is evident, to a position midway between thepositions of the bands for the undigested molecules. Thesedata suggest that the OS was digested by the enzyme andhence that the nonreducing terminus of each repeat is aHexNAc linked in the �-configuration. While commerciallyavailable glycosidases can contain other contaminating gly-cosidases, the digestion specificity of the glycosidase em-ployed suggests that the nonreducing terminus is either D-N-acetylgalactosamine or D-GlcNAc.

The lectin GS-II is specific for nonreducing terminal �- or�-GlcNAc residues. Western blot analysis (Fig. 6B, blot 1)shows that the lectin bound to the N. sicca OS but not to OSthat had been digested with enzyme (Fig. 2B, lane 2), support-ing the conclusion that the nonreducing terminus of the Orepeat is GlcNAc. Hexosaminidase digestion did not affect theelectrophoretic 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�-GlcNAc in the core LOS component of N. sicca.

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

To determine the identity of the other monosaccharide inthe N. sicca 4320 disaccharide, GC-MS was performed afteracid hydrolysis, reduction, and O trimethylsilylation. GC-MS spectra for potential monosaccharide components weregenerated as standards. As shown in Fig. 7, derivatizedL-rhamnose had a retention time of 11.10 min, whereas theD-GlcNAc derivative had a retention time of 24.51 min andgenerated a different mass spectral fragmentation pattern.N. sicca 4320 LPS-LOS were hydrolyzed to their monosac-charide components and derivatized. The correspondingGC-MS spectra are shown in Fig. 7C. The major compo-nents of the glycose mixture had retention times of 11.09and 24.51 min, and MS fragmentation of these major com-ponents created patterns that matched those of the L-Rhaand D-GlcNAc standards. The MALDI-TOF, ESI, andGC-MS data and the data for SDS-PAGE with lectin bind-ing and glycosidase digestion are all consistent with thepresence of N. sicca 4320 disaccharide repeating units that

are composed of L-Rha and with the presence of nonreduc-ing terminal �-D-GlcNAc.

The gonococcus is known to contain a cryptic rhamnosebiosynthetic cluster (19). While no one has identified rham-nose in any of the LOS structures of pathogenic Neisseria,Wiseman and Caird (31) were able to detect small amounts ofthis sugar in glycolipid preparations from a variety of gonococ-cal isolates. Therefore, it is not entirely surprising that N. sicca4320 produces rhamnose and that it has a fully functionalrhamnose biosynthetic cluster.

Linkage analysis of the disaccharide repeat. To analyze thelinkage between Rha and GlcNAc, the glycoses were per-methylated and reduced prior to extensive fragmentation byESI MSn analysis. The ions at m/z 923 containing two disac-charide units were selected for successive fragmentation byMSn analysis into smaller structures. Cleavage of these frag-ments produced patterns that have been shown to be charac-teristic of specific glycosidic bond orientations. The MS3 spec-trum with fragment ions of the m/z 701 ions is shown in Fig.8A. As shown in Fig. 8B, these molecular ions are composed oftwo GlcNAc molecules and one Rha. For example, fragmention peaks at m/z 474, m/z 456, and m/z 442 are each charac-teristic of loss of a methylated GlcNAc from the ions at m/z701. Corresponding peaks at m/z 268 and m/z 282 are presentin the spectrum due to loss of the disaccharide unit from theions at m/z 701. In addition, daughter ion peaks at m/z 595 andm/z 627 are present in the MS3 spectrum, and as shown in Fig.8B, these ions are characteristic for a �(1-3) linkage toGlcNAc. Together, the data provide evidence that the disac-charide unit has an L-Rha residue that is connected to carbon3 of GlcNAc through a � bond.

The orientation of the bond linking GlcNAc to rhamnosewas also examined by use of fragmentation with MSn anal-ysis. The MS4 spectrum generated by further fragmentationof the m/z 456 ion is shown in Fig. 9A. Structures corre-sponding to the generated daughter ions are consistent withthe expected fragmentation of this ion. For example, severalcleavage 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 inthe spectrum. These ions represent loss of Rha comparedwith the m/z 456 ion. Additionally, an m/z 340 ion is ob-served, whose structure is consistent with �(1-4) linkage ofGlcNAc to Rha.

It is interesting that N. sicca 4320 was isolated from a fatalcase of endocarditis. Because this species is not normallypathogenic in humans, the production of an OS repeating unitconceivably could have contributed to the increased pathoge-nicity of this isolate. Further experiments are needed to deter-mine if the gonococcus and meningococcus are also capable ofsynthesizing this type of novel LPS structure. It is possible thatthe pathogenic Neisseria strains are capable of expressing thehigher-molecular-mass structure in vivo, since in vivo extension

FIG. 7. GC-MS analysis of N. sicca 4320 LPS. (A and B) Profiles of the L-rhamnose and D-GlcNAc monosaccharide controls, respectively. Theretention times of these sugars along with the fragmentation patterns of the ions represented by the peaks are shown. (C) The profile in panel ashows the retention times of the monosaccharide sugars in strain 4320 LOS and LPS. The retention times of the major peaks are indicated. Panelsb 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).

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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 massesof the abundant fragments are indicated above the corresponding peaks. (B) Expected structures characteristic of specific linkages generatedduring MS3 analysis. Me, methyl.

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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 massesof the abundant fragments are indicated above the corresponding peaks. (B) Expected structures characteristic of specific linkages generatedduring MS4 analysis. Me, methyl.

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of the gonococcal MS11mkC LOS with a small number ofpoly-N-acetyllactosamine repeats is associated with increasedpathogenicity (10). This is a possibility as this work providesdocumentation that Neisseria strains have the necessary geneticmachinery to synthesize LPS-like structures.

ACKNOWLEDGMENTS

The work described in this paper was supported in part by grants AI24452 (D.C.S.) and AI 21620 and AI 065605 (J.M.G.) from the Na-tional Institutes of Health and by the Research Service of the Depart-ment of Veterans Affairs (J.M.G.).

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

REFERENCES

1. Arking, D., Y. Tong, and D. C. Stein. 2001. Analysis of lipooligosaccharidebiosynthesis in the Neisseriaceae. J. Bacteriol. 183:934–941.

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