Glycobiology, 1999, Vol. 9, No. 12 1371-1380
© 1999 Oxford University Press
The structural heterogeneityof the lipooligosaccharide (LOS) expressed by pathogenic non-typeable Haemophilus influenzae strain NTHi 9274
Complex Carbohydrate Research Center, The University ofGeorgia, 220 Riverbend Road, Athens, GA, USA, 2Laboratoryof Immunology, National Institute on Deafness and Other CommunicationDisorders, S. Research Court, Rockville, MD 20850, USA,and 3Center for Biologics Evaluationand Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda,MD 20892, USA
Received on April 20, 1999. revisedon June 10, 1999; accepted on June 10, 1999.
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Nontypeable Haemophilus influenzae (NTHi)is an important pathogen responsible for otitis media in childrenand of pneumonitis in adults with depressed resistance. NTHi is acapsularand, therefore, capsular polysaccharide-based vaccines are ineffectivefor preventing infections by this pathogen. Recently it was foundthat a detoxified lipooligosaccharide (LOS) conjugatefrom NTHi 9274 induced bactericidal antibodies effective againsta large number of NTHi isolates, and conferred protection againstNTHi otitis media in chinchillas (X.-X.Gu et al.,1996, Infect. Immun., 64, 4047–4053;X.-X.Gu et al., 1997., Infect. Immun., 65, 4488–4493). In this paper we reportthe chemical characterization of the LOS from NTHi 9274LOS. NTHi is capable of expressing a heterogenous population ofLOS exhibited by multiple oligosaccharide (OS) epitopes. OSs released fromthe LOS of NTHi 9274 by mild acid hydrolysis were purified usingBio-Gel P4 gel permeation chromatography. The OSs were characterizedby glycosyl composition analysis, glycosyl linkage analysis, nuclearmagnetic resonance spectroscopy (NMR), fast atom bombardment massspectrometry (FAB-MS), matrix-assisted laser desorptiontime of flight mass spectrometry (MALDITOF-MS), and tandem MS/MS.At least 17 different OS molecules were observed. These containedvariable glycosyl residues, phosphate (P), and phosphoethanolamine(PEA) substituents. These molecules contained either three, four,or five hexoses, and all contained four heptosyl residues. The fourheptosyl residues consisted of one D,D-Hep andthree L,D-Hep. Dephosphorylation of the OSs withaqueous 48% hydrofluoric acid (HF) reduced the number ofmolecules to about to seven; Hex1-7Hep4Kdo1.Of these seven, Hex2Hep4Kdo1, Hex3Hep4Kdo1,and Hex4Hep4Kdo1 were the majorconstituents. Thus, this NTHi LOS preparation is very heterogeneous,and contains structures different from those previously publishedfor Haemophilus influenzae. The tandem MS/MSanalysis and glycosyl linkage data suggest that the LOS oligosaccharides havethe following structures:
Hex-(1
4)-[Glc]0-4-(14)-D,D-Hep-(16)-Glc-(14)-L,D-Hep-(15)-Kdo
3
1
L,D-Hep
2
1
(Hex)0–1-(12)-L,D-Hep
where Hex is either a Glc or Gal residue.
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Introduction |
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Haemophilus influenzae (Hi) is a Gram-negativepathogen found in the human upper respiratory tract in both encapsulated (Hitype a-f) and unencapsulated (non-typeable) forms (27
Turk,1984; 22Risberg et al.,1997). In developed countries, serotype b capsular strainscause bacterial meningitis in infants and young children. Nontypeable Haemophilus influenzae (NTHi) strains are foundin the nasopharynx of healthy carriers (26Turk,1981), and have recently been recognized to be importanthuman pathogens. These bacterial strains are responsible for otitis mediaand respiratory tract infections (1Bluestoneand Klein, 1983). Since NTHi does not produce a capsularpolysaccharide, its pathogenicity is mediated by the bacterial outermembrane components, such as proteins and lipooligosaccharides (LOSs)(2Capagnari et al., 1987; 16Patrick et al., 1987).
NTHi does not produce a lipopolysaccharide (LPS) with an O-antigenpolysaccharide. Like other H.influenzae, NTHi expressesheterogenous populations of LOSs which differ by the addition ordeletion of glycoses, phosphates (P), and phosphoethanolamine (PEA)substituents (2Capagnari et al.,1987; 16Patrick et al.,1987, 1989; 18Phillips et al., 1992). Expression of these heterogeneousoligosaccharide (OS) epitopes in NTHi is important in host–pathogeninteractions and therefore is an essential target for regulatingthe virulence potential of this organism (18Phillips et al., 1992). Mouse monoclonal antibodies toNTHi 9274 LOS bound to 90 % of 100 NTHi clinical isolateswhich indicates that at least a number of the various NTHi 9274LOS structures must be relatively conserved among these isolates(8Gu et al., 1996a).Furthermore, these monoclonal antibodies were bactericidal to 30% ofthese isolates (unpublished observations). In addition, detoxifiedLOS conjugates from strain 9274 induced bactericidal antibodiesagainst both homologous and heterologous strains in rabbits (9Gu et al., 1996b), and conferredprotection against NTHi otitis media in chinchillas (10Gu et al., 1997).
The LOSs of several H.influenzae serotype band NTHi strains have been previously studied (18Phillips et al., 1992, 1993; 13Masoud et al., 1997). Glucose (Glc), galactose(Gal), L-glycero-D-manno-heptose (L,D-Hep), and 3-deoxy-D-manno-2-octulosonic acid (Kdo)are the common sugar constituents found in these LOSs. In addition,some LOSs contain N-acetylglucosamine(GlcNAc). Phosphate, PEA, and acetyl groups are also found as commonnonglycosidic constituents. The single Kdo residue present in H.influenzae LOSs has been found to be phosphorylatedin both the serotype b strain (11Helander et al., 1988; 19Phillips et al., 1993) and in an NTHi strain (18Phillips et al., 1992).Previous structural studies (18Phillips et al., 1992, 1993; 13Masoud et al., 1997) showed that 
-L,D-Hepp-(12)--L,D-Hepp-(13)--L,D-Hepp-(15)-Kdop (i.e., HepIII-HepII-HepI-Kdo) is a common LOSinner core element for H.influenzae strains. Structuralvariation of these LOSs is due to the various OSs attached to HepI,HepII, and/or HepIII (13Masoud et al., 1997), to the phosphate and phosphoethanolamine(PEA) substituents on HepII and HepIII (13Masoud et al., 1997), and, more recently, toa phosphocholine-glucosyl moiety attached to HepIII (22Risberg et al., 1997). Unlike other H.influenzae, thisreport shows that the inner core region of NTHi 9274 LOS containsfour heptosyl and one hexosyl residues. Three of the heptosyl residuesare L,D-Hep (HepI, II, and III), and the fourth isa D-glycero-D-manno-heptosyl residue, D,D-Hep(HepIV). Additional OSs of various sizes are attached to HepIIIand/or HepIV. The presence or absence of phosphate andphosphoethanolamine (PEA) groups also contributes theheterogeneity of NTHi 9274 LOS.
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Results |
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Initial analysis of NTHi 9274 LOS
Glycosyl composition analysis of NTHi 9274 LOS preparation showsthat it contains Glc, Gal, L,D-Hep, D,D-Hep,GlcNAc (derived from the lipid A), and Kdo. Mild acid hydrolysisof LOS with dilute acid released OSs and insoluble lipid A. The OSseluted from a Bio-Gel P4 column as a single peak just after thevoid volume. The glycosyl composition analysis of this purifiedOS preparation consisted of Glc:Gal:L,D-Hep:D,D-Hep ina ratio of 1.5:1.5:3:1. Kdo was present but was not quantified. Compositionanalysis of the lipid A showed that it contained myristic acid, ß-hydroxymyristic acid, and glucosaminein a ratio consistent with the lipid A structure previously reported for H.influenzae (11
Helander et al., 1988). A sample of the LOS preparation was de-O-acylatedby treatment with anhydrous hydrazine and analyzed by MALDI-TOF MS.The spectrum (not shown) showed that the LOS preparation was extremelyheterogeneous consisting of a mixture of at least 25 molecules rangingin size from m/z 2600 to 3600. The OSsreleased by mild acid hydrolysis were also analyzed by MALDI-TOFMS. Again, the results (spectrum not shown) showed that the heterogeneityobserved in the de-O-acylated LOS sample was alsoobserved in the OSs in that a minimum of 17 molecules were detectedwith a the mass range between m/z 1400and 2400. The differences in masses between the various moleculesindicated that the heterogeneity was due to variation in the numberof hexosyl residues, as well as in phosphate, and PEA substituents.
Analysis of the HF-treated OSs from NTHi 9274 LOS
In order to further characterize the structures of these OSs,it was necessary to reduce the heterogeneity of the OS preparation. Thus,the phosphate and PEA substituents were removed by treatment withaqueous HF and the resulting OSs (HFOSs) were purified by Bio-GelP4 column chromatography. Two fractions, HFOSa, and HFOSb, elutedjust after the void volume of the column, the major fraction beingHFOSb. Proton NMR analysis was performed on the OS preparation priorto (Figure 1), and after (not shown) aqueousHF treatment. The NMR spectrum is quite complex due to the mixtureof OSs present. However, HF treatment resulted in the disappearanceof the PEA proton resonances at 
3.2.The spectrum also shows a complex pattern of proton resonances between 1.8 and 2.3 consistent with the H-3 protonsof Kdo and 
-acetylgroups. The spectrum of HFOSb (data not shown) also contained resonancesindicative of the H-3 methylene protons of anhydro Kdo (e.g., 4,8-anhydro Kdo)between 2.9 and 3.3 (Carlson and Krishnaiah1992). These anhydro Kdo methylene protons are exchangeable with deuterium(from D2O) and were, therefore, greatly reduced in intensitycompared with those of a normal Kdo residue. Thus, the mild acidreleased OSs and HFOSs, contain both normal, and anhydro Kdo residues.
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The HFOSs were analyzed by MALDI-TOF, MALDI-magnetic sector,and FAB-MS and the results (Table
)from all three MS methods were the same. At least seven molecularmasses were observed corresponding to compositions of Hex1-7Hep4Kdoanhydro. Ionsdue to molecules containing normal Kdo residues were also presentbut their intensities were low. The most abundant molecular ionsfound in HFOSb corresponded to molecular species of Hex2-5Hep4Kdoanhydro.These data show that HF treatment reduced the number of moleculesfrom 17 to 7, and also support the above composition results whichsuggest that all molecules in the HFOS preparation contain fourheptosyl residues with structural variation due to the number ofhexosyl residues present
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Glycosyl linkage analysis of the NTHi 9274 LOSOSs
The HFOSs were further characterized by glycosyl linkage analysis.Glycosyl linkages were determined by the preparation and GLC-MSanalysis of partially methylated alditol acetates (PMAAs). Table
shows that the PMAAs consist of terminal Glc,terminal Gal, terminal D,D-Hep, terminal L,D-Hep,6-Glc, 4-Glc, 4-D,D-Hep, 2-L,D-Hep,3,4-L,D-Hep, and a trace amount of 4-linked Gal.The 2-, and 3,4-linked heptosyl residues can be assigned as beingin the L,D-configuration based on the fact thattheir PMAAs had identical retention times to those L,D-Hep residues reported for Nisseriameningitidis NMB LOS (21Rahman et al., 1998). Both terminally linked L,D-and D,D-Hep are present. The assignment of the4-linked Hep as being in the D,D-configurationcan be made since composition analysis (described above) indicatesthat the D,D- to L,D-Hep ratiois 1:3, and the only way to achieve this ratio from the methylation valuesgiven in Table is for the 4-linkedHep to be in the D,D-configuration. Thus, as withthe composition and MS results, methylation analysis shows thatall of these NTHi 9274 OSs each contain four heptosyl residues.The methylation data also show that the three L,D-heptosylresidues exist as 3,4-linked L,D-Hep, 2-linked L,D-Hep, and terminally linked L,D-Hep.By comparison to previously reported H.influenzae LOSstructures (18Phillips et al.,1992, 1993; 13Masoud et al., 1997), the 3,4-linked L,D-Hepcorresponds to HepI, 2-linked L,D-Hep to HepII,and terminally linked L,D-Hep to HepIII. However,the fact that the levels of 2-linked L,D-Hep andterminally linked L,D-Hep are larger and smaller,respectively, than the level of 3,4-linked L,D-Hepsuggests that, for some of the LOS molecules, HepIII is glycosylatedat O-2. This substitution pattern for the L,D-heptosylresidues is consistent with that previously reported for H.influenzae LOSs(18Phillips et al., 1992,1993; 13Masoud et al., 1997).Thus, a comparison of these methylation data with the previouslypublished structures for H.influenzae LOSs implies thatthe three L,D-heptosyl residues in NTHi 9274 LOSpreparation have the same structural arrangement as thatpreviously published, and that the fourth D,D-Hep(HepIV), unique to NTHi 9274 LOS, is part of an OS which is attachedto O-4 of HepI (discussed further below). The presence of such a D,D-Hep has not been previously reported in H.influenzae LOSs,however, it has been reported in the LOSs from Haemophilusducreyi (14Melaugh et al.,1994, 1996; 23Schweda et al., 1995a).
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The procedure and columns used for GLC-MS analysis did not allowthe detection of phosphorylated glycosyl residues. However, comparisonof the glycosyl linkages of the OSs with those of the HFOSs showsthat the former give reduced levels of 2-linked L,D-Hep(HepII, or glycosylated HepIII), terminal L,D-Hep(HepIII) and terminal D,D-Hep (HepIV) (data not shown).This indicates that these residues contain phosphate or PEA substituents.The locations of these substituents were determined by methylationof the OSs, followed by HF treatment and ethylation. Preparationand analysis of the partially methylated/ethylated heptitolacetates gave the following results: 1,2,5-triacetyl-6-ethyl-3,4,7-trimethylheptitol,1,2,5-triacetyl-7-ethyl-3,4,6-trimethyl, 1,5-diacetyl-2-ethyl-3,4,6,7-tetramethylheptitol,1,5-diacetyl-4-ethyl-2,3,6,7-tetramethylheptitol, 1,5-diacetyl-6-ethyl-2,3,4,7-tetramethylheptitol,and 1,5-dia-cetyl-7-ethyl-2,3,4,6-tetramethylheptitol. These resultsindicate that the 2-linked heptosyl residues (HepII, or HepIII glycosylatedat O-2) are substituted with phosphate or PEA substituents at O-6or O-7, and that the terminally linked HepIII and HepIV can each besubstituted at the O-2, O-4, O-6, or O-7 positions. For the determinationof the Kdo linkage, the intact LOS molecules were permethylated,followed by methanolysis and analyzed by GLC-MS. The results demonstratedthat the LOS has only one type of Kdo residue namely 5-linked Kdo.This result is the same as the Kdo linkage in the previously reported H.influenzae LOS (18
Phillips et al., 1992, 1993; 13Masoud et al., 1997) structures.
Tandem MS/MS analysis of the permethylatedHFOS from NTHi 9274 LOS
The HFOS samples were permethylated and major molecular ionswere analyzed by tandem MS/MS in order to determine thepossible glycosyl sequences of some of the LOS molecules presentin this strain. The permethylated HFOSs were first characterizedby FAB-MS and ESI-MS analyses in the positive mode. The FAB-MS spectrumis shown in Figure 2. The observed m/z valuesfor molecular ions of the permethylated HFOSs are shown in Table and compared with calculated values. Forboth FAB-MS and ESI-MS, four series of ammoniated and/orsodiated molecular ions were found; two major series of higher intensities,and two minor series. One major ion series corresponded to OSs havingions of m/z values of 1695.0, 1899.0,2103.2, and 2308 due to molecules of compositions givenin Table ; each molecule having ananhydro Kdo at its reducing end (Figure 2,structure b of Scheme B). The second major seriesof ions was due to OSs having m/z valuesof 1723.0, 1927.1, 2131.4, and 2336.5 and having the compositionsgiven in Table with a modified Kdomoiety at their reducing ends. While the structure of this modifiedKdo residue is not known with certainty, the sequential methylation procedureused may have resulted in either structure a (Figure 2 Scheme A) or structure d (Figure 2, Scheme B). A third minor series of ions indicatedmolecules which may have a modified Kdo of structure c (Figure 2, Scheme B). Each of these series of ions isdue to five different molecules, HFOS1 to HFOS5 corresponding topermethylated Hex1-5Hep4Kdo1 anddiffering only in the type of Kdo modification. A fourth minor seriesof ions of m/z 1465, 1669, and 1873 consistingof Hex1-3Hep4 and a modified Kdowhich can not be accounted for by the schemes shown in Figure 2. Two other expected ions, those for permethylatedHFOS6 and HFOS7 (see Table 
), werenot observed since they had masses above the maximum detectable ionof m/z 2400.
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Positive ion tandem mass spectrometry (MS/MS) was performed onthree major molecular ions of the permethylated HFOSs, i.e.HFOS2, HFOS3, and HFOS4. The MS/MS spectrum of the parent [M + NH4]+ ionat m/z 1723 (Figure 3),HSOS2, shows X-type ions resulting from the cleavage of the C1-C2and C5-O5 bonds with charge retention on the resulting reducingend fragment. These fragment ions are consistent with two branchingOSs attached to the 3,4-linked L,D-Hep (HepI).One is a Hep-Hex- disaccharide branch defined by the ions m/z 1489 and1285. The second is a Hex-Hep-Hep- trisaccharide branch definedby ions m/z 1533, 1285, and 1037. ThisMS/MS spectrum also shows the presence some of the oxoniumB1 ions resulting from the cleavage of the glycosidic bond at theheptosyl and hexosyl residues. These ions are m/z 263and 219 for terminal heptosyl and hexosyl residues, respectively,and gave secondary fragment ions, respectively, at m/z at199 and 187 by elimination of one and two molecules of methanol,respectively. The mechanism of this type of elimination to forma conjugate diene has been described previously (28
Viseux et al., 1997). Similarly, the B2 oxoniumions of either the Hex-Hep or Hep-Hex disaccharide fragments at m/z 467 (i.e., from either of the nonreducingtermini) each gave a secondary fragment ion at m/z 435by elimination of methanol. It is also possible, as reported previously(28Viseux et al., 1997),for the diene ion of m/z 435 to form fragmentions either at m/z 199 due to eliminationof a hexosyl residue, or at m/z 155 dueto elimination of a heptosyl residue. The Hex-Hep-Hep branch isconsistent with a Glc-12-L,D-Hep-12-L,D-Hep- trisaccharide(i.e., Glc-HepIII-HepII-) attached to O3 of the 3,4-linked L,D-heptosylresidue (HepI), a structural feature of other H.influenzae LOSs(18Phillips et al., 1992,1993; 13Masoud et al., 1997).The remaining heptosyl residue would be the HepIV of the Hep-Hex-disaccharide that is attached to O4 of HepI. This disaccharide maybe the same as the D,D-Hep-16-Glc-disaccharide component reported for H.ducreyi (14Melaugh et al., 1994,1996; 23Schweda et al., 1995a).The presence of such a disaccharide is supported by the observationof 6-linked Glc in the NTHi 9274 LOS (Table ). These mass fragmentation data are consistentwith the structure shown in Figure 3.
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The MS/MS spectrum of the parent [M + NH4]+ ionat m/z 1927.1, which corresponds to anoctasaccharide, HSOS3, is shown in Figure 4.In this spectrum, a series of X-type fragment ions at m/z 1737,1489 and 1285 define a Hex-Hep-Hex trisaccharide branch,and a very minor fragment ion at m/z 1241 suggest a secondHex-Hep-Hep- trisaccharide branch (i.e., m/z 1737,1489, and 1241). Like the previous OS fragmentation (Figure 3), this MS/MS spectrum shows B1 andB2 oxonium ions from the terminal ends of both branches which givethe secondary ion fragments as shown in Figure 4.These results are consistent with this m/z 1927.1OS having the same structure as that of the m/z 1723OS but with an additional hexosyl residue attached to HepIV.
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The MS/MS spectrum (Figure 5)of the parent [M + NH4]+ ionat m/z 2131 for HFOS4 produced fragmentions in a similar manner to that of HFOS2 and HFOS3, and indicatethat HFOS4 is a nonasaccharide (Figure 5).Using the same rationale as described for HFOS3, we conclude thatHFOS4 has the same structure as HFOS3 but with a Hex-Hex disaccharide attachedto HepIV.
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Discussion |
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In this report it has been shown that the NTHi 9274 LOS preparationcan contain at least 25 different structures which vary from oneanother in the degree and position of phosphate and PEA substitution,and in the number of glycosyl residues present in two branchingoligosaccharides. A comparison of the NTHi 9274 LOS structures withthose reported for other H.influenzae strains isshown in Figure 6.
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The boxed L,D-Hep3Kdo region (i.e.,HepIII-HepII-HepI-Kdo) has been reported for other H.influenzae LOSs(18
Phillips et al., 1992,1993; 13Masoud et al., 1997)and represents a type of "inner core" structurefor these LOSs. The data presented above show that the NTHi 9274LOS has extended this "inner core" by adding a D,D-Hep-Hex disaccharide to O4 of HepI as shownin Figure 6B. As stated above, a similarstructural feature, namely D,D-Hep-16-Glc,has been reported for H. ducreyi LOSs(14Melaugh et al., 1994,1996; 23Schweda et al., 1995a),but not for H.influenzae LOSs. Since the NTHi 9274 LOSalso contains 6-linked Glc (which is not reported in other H.influenzae LOSs),it is likely that the D,D-Hep-Hex region of NTHi9274 LOS is, in fact, D,D-Hep-16-Glc.
In addition to the uniqueness of the D,D-Hep-Glcinner core extension in NTHi 9274 LOSs, there are other significant featuresof the various oligosaccharide substitutions to this extended innercore. In the case of other reported H.influenzae LOSs,whenever an R2 oligosaccharide is present it is attached toO3 of the 2-linked L,D-heptosyl residue (HepII).The absence of any 2,3-linked L,D-Hep togetherwith the MS/MS data show that none of the various NTHi9274 LOS molecules have an R2 oligosaccharide substitution.The absence of an R2 oligosaccharide has also been reportedfor H.influenzae RM.118–28 (22Risberg et al., 1997), H.influenzae AH1-3 (23Schweda et al., 1995a),and NTHi 2019 LOSs (18Phillips et al., 1992).
Structural variation of H.influenzae LOSs isreported to be due to the various OSs attached to this "innercore" structure at HepI, HepII, or HepIII (i.e., R1,R2, and R3 of Figure 6).These OSs are reported to consist of a single glucosyl residue,a phosphocholine substituted glucosyl residue, lactosyl or diglucosyl disaccharides,the p
epitope (-Gal-14-ß-Gal-14-ß-Glc-), truncatedlacto-N-neotetraose, and sialylated versions ofthe lacto-N-neotetraose (18Phillips et al., 1992, 1993, 1996; 13Masoud et al., 1997; 22Risberg et al., 1997). The fact that NTHi 9274 OSslack GlcNAc indicates that none of the variant LOS molecules fromthis strain contains lacto-N-neotetraose or its sialylatedversion. Also, the absence of a 4-linked galactosyl residue rulesout the presence of the p epitope structurein these various LOS molecules.
While the more extended oligosaccharide structures such as lacto-N-neotetraose and the p epitopeare not present in NTHi 9274 LOS, the data suggest that the NTHi9274 LOS molecules contain many of the more truncatedstructural features reported to be present in other H.influenzae LOSs.The presence of terminally and 4-linked Glc indicates that someof the some of the NTHi 9274 LOS molecules have the frequently reported Glc-14-Glc- disaccharide structural featureas the R1 oligosaccharide. Likewise, the presenceof terminally linked Gal and 4-linked Glc may indicate that someof these NTHi 9274 LOS molecules could contain a lactosyl substitutionat the R1 position; however, it is also possible thatthe terminally linked Gal is totally accounted for by the hexosylresidue that is linked to HepIII, i.e., R3. It is clearfrom the MS/MS data that when NTHi 9274 LOS molecules containR3, it exists as a single hexosyl residue. This is alsotrue for other H.influenzae LOSs in which the HepIIIis reported to be glycosylated at O2 by Gal (13Masoud et al., 1997), or by Glc (22Risberg et al., 1997). Again, the presence ofterminally linked Gal and Glc supports the possibility that therecould be versions of the NTHi 9274 LOS in which R3 iseither Glc or Gal. The various possible R1 and R3 OSsfor NTHi 9274 LOS molecules are summarized in Table . These structures are consistent with thelinkages reported in Table , and withthe MS/MS spectra shown in Figures 3–5. The presence of significant levels of bothterminally linked D,D-Hep and terminally linked L,D-Hep suggest that there are a significant numberof molecules without R1 or R3 OSs which maynot be fully accounted for by the MS data. These molecules may behave been lost during purification of the HFOSs. Purification oflarger amounts of OSs is in progress in order to obtain more completesequence information on these variable OSs.
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Finally, our data also indicate that NTHi 9274 LOSs vary greatlyin phosphate and PEA substitution. A small portion of the variousLOSs may not have phosphate or PEA substituents while the vast majorityhave phosphate or PEA groups on the 2-linked L,D-heptosylresidues (HepII, or HepIII when glycosylated at O-2) at O-6 or O-7,and on the terminally linked D,D-Hep (HepIV) or L,D-Hep (HepIII) at O-2, O-4, O-6, or O-7 positions.
Taken together, the hexosyl, phosphate, and PEA variation revealthat the NTHi 9274 LOS preparation is extremely heterogeneous.The presence of numerous LOS molecules containing various truncatedoligosaccharides that are common to many H.influenzae LOSsmay explain why antibodies to NTHi 9274 LOS react with the majorityof NTHi clinical isolates and show bactericidal activity to theseisolates (9Gu et al., 1996b).The exact glycosyl sequence of the R1 OSs of the NTHi9274 LOSs is under further investigation.
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Material and methods |
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Bacterial strains, condition of growth, and LOSpreparation
NTHi strain 9274, isolated from middle ear fluid removed from apatient with otitis media, was provided by M.A.Apicella, Universityof Iowa. The strain was grown on chocolate agar at 37°Cunder 5% CO2 for 8 h and transferred to 200ml of 3% brain heart infusion medium (Difco Laboratories,Detroit, MI) containing NAD (5 µg/ml)and hemin (2 µg/ml) (SigmaChemical Co., St. Louis) in a 500 ml bottle. The bacteria in thebottle were incubated overnight at 150 r.p.m. in an incubator shaker (modelG-25; New Brunswick Scientific Co., Edison, NJ) at 37°C.The culture was transferred to five 2.8 l baffled Fernbach flasks,each of which contained 1.4 l of the same medium. The flasks wereshaken at 140 r.p.m. and maintained at 37°Cfor 24 h. Each culture was centrifuged at 15,000 x g at 4°C for 30 minto separate the bacterial cell from the supernatant. LOS was prepared fromthe bacterial cells by modified phenol-water extraction method (7
Gu et al., 1995). The proteinand nucleic acid contents of both purified LOSs were each less than1% (25Smith et al.,1985).
Preparation of LOS OSs
The LOS preparation (20 mg) from NTHi strain 9274 was hydrolyzedin aqueous 1% acetic acid (10 ml) for 2 h at 100°C. Thehydrolyzate was centrifuged at 10,000 x g for 20 min, and the supernatant was collected.The pellet was washed once with 5 ml of water and collected by centrifugation.The wash water was added to the original supernatant, and the remaining lipidA was extracted with diethyl ether (three times, 5 ml volumes eachtime). The aqueous phase containing the OSs was lyophilized. Thelyophilized OSs were dissolved in 0.5 ml of water, filtered withMicrofilterfuge tubes containing 0.45 µmpore size Nylon-66 membrane filters, applied to a Bio-Gel P-4 column (70 x 1.6 cm), and eluted with deionizedwater containing 1% 1-butanol. Fractions were assayed forcarbohydrate by the phenol-sulfuric acid assay, and the OS peakswere pooled and lyophilized.
De-O-acylation of LOS
The LOS sample of NTHi 9274 was de-O-acylatedaccording to the procedure of Helander et al. (11Helander et al., 1988). Approximately8 mg of LOS were incubated with 1 ml of anhydrous hydrazine for20 min at 37°C. The solution was cooledto –20°C, and 5 ml of chilledacetone were added drop-wise to precipitate the de-O-acylatedLOS. The sample was then centrifuged at 12,000 x g for 20 min at 4°C.The supernatant was removed, and the pellet was washed again withcold acetone and centrifuged. The precipitated de-O-acylatedLOS was then resuspended in 1 ml of water and lyophilized.
Treatment of LOS and OS with aqueous HF
The LOS and OS samples (8 mg) were each placed in 1.5 ml polypropylenetubes. The samples were treated with cold aqueous 48% hydrogenfluoride (HF) (100 µl) and kept for24 h at 4°C (12Kenne et al., 1993). The HF was removed by flushingunder a stream of air followed by addition of diethyl ether (600 µl) and drying with a stream of air.This step was repeated three times. The dry pellet was dissolvedin water and lyophilized. The lyophilized dephosphorylated sampleswere dissolved in 0.5 ml of water, and filtered with Microfilterfugetubes containing 0.45 µm pore size Nylon66 membrane filters. The HF-treated OS sample (HFOS) was then appliedto a Bio-Gel P-4 column (70 x 1.6 cm),and eluted with water containing 1% 1-butanol. Fractionswere assayed for carbohydrate by the phenol-sulfuric acid assay,and the OS peaks were pooled and lyophilized.
Glycosyl composition analyses
Glycosyl composition analysis of LOS and OS samples (0.5 mg each)was performed by hydrolysis in 2 M trifluoroaceticacid (0.5 ml) in a closed vial at 120°Cfor 3 h. The glycoses in the hydrolyzate were reduced with NaBH4,acetylated, and analyzed by gas liquid chromatography (GLC) andby combined GLC-mass spectrometry (MS) (31Yorket al., 1985). For the determination ofKdo, the LOS sample (0.5 mg) was dried in vacuum, and methanolyzedin 1 ml of methanolic 2 M HCl at 80°Cfor 4 h. The released methyl glycoside residues were trimethylsilylated, andanalyzed by GLC-MS (31York et al.,1985). The absolute configurations of the glycoses presentin the OS from NTHi were determined by GLC-MS analysis of the trimethylsilylated (S/R)-2-butylglycosides (6Gerwig et al.,1979). GLC and GLC-MS analyses were performed on fusedsilica capillary columns (length, 30 m; inner diameter, 0.32 mm)with helium as the carrier. A DB-5 column (J & W Scientific)was used for aminoglycosyl alditol acetate derivatives, and an SP2330column (Supelco, Bellefonte, PA) was used for the neutral glycosylalditol acetate derivatives. The DB-5 column was also used for theanalysis of trimethylsilyl methyl or 2-butyl glycosides. GLC equipment consistedof HP5890 gas chromatograph equipped with a flame ionization detector(Hewlett-Packard). GLC-MS (EI) was performed using a Hewlett-Packard5970 MSD.
Glycosyl linkage analyses
Glycosyl linkage analyses were carried out using a modified NaOH-method(Ciucanu and Kerek 1984). Samples (1 mg each) were dissolved indimethylsulfoxide (100 µl), powdered NaOH(100 mg) was added, and the reaction mixture was stirred rapidlyfor 30 min. Methylation was performed by the sequential additionsof methyl iodide (40, 40, and 80 µl)at 10 min intervals. After an additional 20 min stirring,the methylated glycans were recovered in the organic phase afteraddition of chloroform (0.5 ml) and 1 M sodiumthiosulfate (1 ml). The extraction with chloroform was repeatedtwo more times, the organic phases were combined and dried witha stream of nitrogen. The permethylated product residue was suspendedin deionized water and was further purified by reverse-phase chromatographyon a Sep-Pak C18 cartridge as previously described (29Waeghe et al., 1983). Methylated glycans were hydrolysedwith 2 M trifluoroacetic acid (120°C,3 h), reduced with NaBH4 or NaB(2H)4,acetylated, and analyzed by GLC and GLC-MS (31Yorket al., 1985). For the determination ofKdo linkages, the permethylated LOS was methanolized in 1 ml of methanolic2 N HCl at 80°C for4 h, and the released methyl glycoside residues were acetylatedand analyzed by GLC-MS (5Edebrink et al., 1994).
Lipid A purification
Lipid A was released from the LPS (10 mg) by hydrolysis in aqueous1% acetic acid (5 ml) at 100°Cfor 2 h. The precipitated lipid A was collected by centrifugation(10,000 x g). The precipitatewas resuspended with water (2 ml) and partitioned into chloroform(2 ml). The chloroform layer containing purified lipid was concentratedto dryness.
Fatty acid analysis
Total fatty acids were released by methanolysis of lipid A with methanolic M HCl at 80°Cfor 16 h, and were trimethylsilylated. The resulting fattyacid methyl esters were analyzed by GLC-MS (31Yorket al., 1985). Ester and amide-linked fatty acidswere distinguished by preferential release of the ester-linked fattyacids using anhydrous sodium methoxide (30Wollenweberand Rietschel, 1990).
Mass spectrometry
Fast atom bombardment mass spectrometry (FAB-MS) was performedin the positive mode with a JEOL (Tokyo, Japan) SX/SX 102Atandem mass spectrometer, which was operated at 10 kV acceleratingpotential. Ions were produced by FAB with xenon, using a JEOL FABgun operated at 6 kV in a conventional FAB ion source. Spectra acquiredfor the first MS are averaged profile data of three scans as recordedby a JEOL XMS data system. These spectra were acquired from 200to 2000 m/z at a rate that would scanthe mass range from 0 to 2500 in 1 min. A filtering rate of 100Hz was used in acquiring these spectra. The samples were dissolvedin water, and 1 µl aliquots were mixedwith an equal volume of the FAB matrix, thioglycerol, on the probetip. The MS-MS spectrum was obtained by using a linked scan (B/Econstant) at a rate that would scan the mass range from 5 to 2400in 1 min with a filtering of 300 Hz.
Matrix-assisted laser desorption ionization time-of-flight massspectrometry (MALDITOF-MS) was performed on an LDI 1700XP spectrometer(Linear Scientific, Reno, NV) The instrument was operated at anaccelerating voltage of 30 kV and an extractor voltage of 9 kV.The pressure was 
2.1 x 10–6 torr. Thesample was ionized with a nitrogen laser (
= 337nm) with a pulse width of 3 ns and a 4 to 7.5 µJpulse. The samples were dissolved in water, and the matrix (2,4-dihydroxybenzoic acid)-samplemixture (1–2 µl) was appliedto the probe and quickly dried under vacuum. The sample solutionwas serially dried with matrix to obtain optimal sensitivity. Themass spectra were recorded from m/z 400to 10,000. The spectra are the average of 100–250 acquisitions.A mixture of maltooligosaccharides was used as the calibrationstandard.
The MALDI magnetic sector experiment was performed on the firsttwo sectors of a JEOL (Tokyo, Japan) SX/SX102A tandem four-sectormass spectrometer with a point detector located at the end of MS1.In the MALDI configuration, the FAB gun is replaced by a JEOL MALDIkit (JUS-MALDI, JEOL USA Inc., Peabody, MA), which includes a nitrogen laserof 337 nm wavelength, 3 ns pulse width, and 250 µJmaximum pulse energy. This laser was operated at a pulse rate of 20Hz. MS1 was operated at 10 kV, full accelerating voltage, for allexperiments except the survey scan of the maltooligosaccharidemixture was used as the calibration standard, where the acceleratingvoltage was lowered to 6 kV to enable the magnetic field to scanup to 4000 m/z units. The spectra wereacquired at a rate that would scan the mass range 0–2400 in1 min with 300 Hz filtering. Spectra are averaged profile data asrecorded by a JEOL complement data system.
Electrospray ionization mass spectrometry (ESI-MS) was performedwith a SCIEX API-III mass analyzer operated in the positive ionmode with an orifice potential of 50 V. Spectra are the accumulationof 10–15 scans collected over the mass range of 400–2000.The sample was dissolved in methanol at a final concentration of2 µg/µl,and pumped into the mass spectrometer at a rate of 3 µl/min.
Nuclear magnetic resonance spectroscopy (NMR)
Samples were exchanged several times with D2O, dissolvedin D2O, and analyzed at 298 K, using a Bruker AM500 spectrometer. Chemicalshifts were measured relative to sodium 3-trimethylsilylpropionate-2,2,3,3-d4 ( 0.00).
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This work was funded in part by the Department of Energy GrantDE-FG09-93ER20097 to the CCRC, and by an NDCD contract (263-MD-540088)to the CCRC.
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a Towhom correspondence should be addressed at: Complex Carbohydrate ResearchCenter, The University of Georgia, 220 Riverbend Road, Athens, GA 30602
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