Báo cáo khoa học: Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae pptx

Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus influenzae Andrew D. Cox 1 , Derek W. Hood 2 , Adele Martin 1 , Katherine M. Makepeace 2 , Mary E. Deadman 2 , Jianjun Li 1 , Jean-Robert Brisson 1 , E. Richard Moxon 2 and James C. Richards 1 1 Institute for Biological Sciences, National Research Council, Ottawa, Canada; 2 University of Oxford Department of Paediatrics, Weatherall Institute for Molecular Medicine, John Radcliffe Hospital, Oxford, UK A sialylated lacto-N-neotetraose (Sial-lNnT) structural unit was identified and structurally characterized in the lipo- polysaccharide (LPS) from the genome-sequenced strain Road (RM118) of the human pathogen Haemophilus influ- enzae grown in the presence of sialic acid. A combination of molecular genetics, MS and NMR spectroscopy techniques showed that this structural unit extended from the proximal heptose residue of the inner core region of the LPS molecule. The structure of the Sial-lNnT unit was identical to that found in meningococcal LPS, but glycoforms containing truncations of the Sial-lNnT unit, comprising fewer residues than the complete oligosaccharide component, were not detected. The finding of sialylated glycoforms that were either fully extended or absent suggests a novel biosynthetic feature for adding the terminal tetrasaccharide unit of the Sial-lNnT to the glycose acceptor at the proximal inner core heptose. Keywords: Haemophilus influenzae; LPS; mass spectrometry; NMR; sialic acid. Haemophilus influenzae remains an important cause of disease worldwide. Encapsulated strains can cause invasive, bacteraemic infections such as septicemia and strains lacking a capsule, so-called nontypeable strains (NTHi), are a common cause of otitis media and acute lower respiratory tract infections [1]. Lipopolysaccharide (LPS) is critical to the integrity and functioning of the cell wall of H. influenzae, and as a surface component is a target for host immune responses. The structure of H. influenzae LPS has been well established from several studies [2–8]. H. influenzae LPS is composed of a membrane-anchoring lipid A moiety linked to a heterogeneous core oligosaccha- ride via a phosphorylated 2-keto-3-deoxyoctulosonic acid (Kdo) residue. The core oligosaccharide of H. influenzae can be divided into inner and outer regions. The inner core consists of a L -glycero- D -manno-heptose (Hep) trisaccharide unit wherein the second heptose residue (Hep II) is substi- tuted at the 6-position by a phosphoethanolamine (PEtn) residue. Each of the Hep residues can provide a point for the addition of hexose (Hex) residues each of which can be further extended into oligosaccharide chains of the outer core. The substitution pattern and degree of extension from each Hep residue varies between and within strains. The presence of nonglycose residues, including phosphate- containing substituents and ester linked acetyl groups and glycine molecules also contribute to the structural variability of these molecules [9]. The LPS of H. influenzae lacks the O-specific side chain characteristic of enteric bacteria. Evidence from recent structural studies has confirmed the presence and defined the position of sialic acid in the LPS of H. influenzae [7,10–12]. Sialylation of LPS is a commonly observed structural modification in Neisseria spp. and is frequently found as the sialylated lacto-N-neotetraose structure (Sial-lNnT) [13,14]. Indeed in neisserial species sialylation of LPS renders the bacteria resistant to comple- ment-mediated killing by normal human serum [15]. In the gonococcus, LPS sialylation can occur only if an exogenous supply of sialic acid is available [16]. In contrast, sialylated meningococcal LPS glycoforms can be synthesized endo- genously [17]. Sialylation of H. influenzae LPS appears to depend upon the presence of an exogenous source of sialic acid or its activated form CMP-sialic acid [11,18]. Sialyla- tion of H. influenzae LPS was first indicated by alterations in the reactivity of LPS with a mAb 3F11 that is specific for the terminal lactosamine disaccharide of the lNnT group following treatment with neuraminidase [19]. Consistent with these findings, MS studies pointed to the presence of a Sial-lNnT group [20]. However, the low amounts of sialylated glycoforms present precluded definitive structural characterization of the sialylated moiety. In a recent structural study utilizing an exogenous source of sialic acid in the growth medium we identified and localized another sialylated species, namely sialyl-lactose (Sial-Lac) in H. influenzae LPS [10,11]. The genetic control of the biosynthesis of H. influenzae LPS inner and outer core oligosaccharides has been investigated extensively. Most of the genes responsible for Correspondence to A. D. Cox, Institute for Biological Sciences, National Research Council, Ottawa, ON, Canada. K1A 0R6. Fax: +44 613 952 9092, Tel.: +44 613 991 6172, E-mail: Andrew.Cox@nrc.ca Abbreviations: NTHi, nontypeable strains of Haemophilus influenzae; LPS, lipopolysaccharide; Kdo, 2-keto-3-deoxyoctulosonic acid; Hep, L -glycero- D -manno-heptose; PEtn, phosphoethanolamine; Hex, hexose; Sial-lNnT, sialylated lacto-N-neotetraose; Sial-Lac, sialyl-lactose; BHI, brain heart infusion; ES-MS, electrospray; PCho, phosphocholine. (Received 21 March 2002, revised 20 June 2002, accepted 2 July 2002) Eur. J. Biochem. 269, 4009–4019 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03090.x the biosynthesis of the glycose residues in the globotetraose- containing Road (RM118) LPS glycoforms have been recently reported [21]. Moreover, it is also known that H. influenzae has the ability to modify its LPS structure by a genetic mechanism known as phase variation [22]. We recently identified one such phase variable gene lic3A as encoding an a-2,3-sialyltransferase, which was found to be responsible for the sialylation of a lactose group in the LPS of H. influenzae strain RM118 [11]. In that study, we observed that following insertional deletion of the lic3A gene, LPS derived from the mutated strain still produced sialylated glycoforms, suggesting the presence of alternate sialylated structures. In this study we describe the identifi- cation and structural analysis of a Sial-lNnT unit in H. influenzae LPS. MATERIALS AND METHODS Bacterial strains and culture conditions The H. influenzae strain RM118 (Rd) is derived from the same source as the strain for which the complete genome has been sequenced [23]. Isogenic strains RM118 lgtC and the double mutant RM118 lgtC lic3A have been described previously [11], as have strains RM118 lic2A, RM118 lpsA and RM118 lgtF [21]. H. influenzae strains were grown at 37 °C on brain heart infusion (BHI) agar (1% w/v) supplemented with Levinthals reagent (10% v/v) and when appropriate, Neu5Ac (25 lgÆmL )1 ), CMP-Neu5Ac (50 lgÆmL )1 ), kanamycin (10 lgÆmL )1 ) or tetracycline (4 lgÆmL )1 ). Structural analysis and purification of LPS LPS was prepared for structural analysis from cells harvested after growth on batches of 20 BHI plates, supplemented with Neu5Ac. LPS was extracted by the hot phenol/water method [24] and O-deacylated as described previously [3]. MS analyses were carried out as described previously [8,25,26]. Electrospray (ES) MS was measured in the negative ion mode on a VG Quattro triple quadrupole mass spectrometer (Fisons Instruments) with an electro- spray ion source. Capillary electrophoresis (CE)-MS analysis was per- formed on a crystal Model 310 CE instrument (ATI Unicam, Boston, MA, USA) coupled to an API 3000 mass spectrometer (MDS/Sciex, Concord, Canada) via a micro- ionspray interface. A sheath solution (isopropanol/metha- nol, 2 : 1, v/v) was delivered at a flow rate of 1 lLÆmin )1 to a low dead volume tee (250 lm internal diameter; Chro- matographic Specialities, Brockville, Canada). All aqueous solutions were filtered through a 0.45-lm filter (Millipore) before use. An electrospray stainless steel needle (27 gauge) was butted against the low dead volume tee and enabled the delivery of the sheath solution to the end of the capillary column. Separation was obtained on  90 cm length of bare fused-silica capillary (192 lmo.d.· 50 lm internal diam- eter; Polymicro Technologies, Phoenix, AZ, USA) using 30 m M morpholine in deionized water (negative ion mode), pH 9.0, containing 5% methanol and 15 m M ammonium acetate/ammonium hydroxide in deionized water (positive ion mode), pH 9.0, containing 5% methanol. A voltage of 25 kV was typically applied at the injection. The outlet of the capillary was tapered to  15 lm internal diameter using a laser puller (Sutter Instruments, Novato, CA, USA). Mass spectra were acquired with dwell times of 3.0 ms per step of 1 m/z unit in full-mass scan mode. In the CE-ESMS, 30 nL sample was typically injected by using 300 mbar for a duration of 0.1 min. For CE-ESMS/MS experiments  60 nL sample was introduced using 300 mbar for 0.2min.TheMS/MSdatawereacquiredwithdwelltimes of 2.0 ms per step of 1 m/z unit. Fragment ions formed by collision activation of selected precursor ions with nitrogen in the RF-only quadrupole collision cell, were mass analysed by scanning the third quadrupole. NMRexperimentswereperformedonVarianINOVA 600 and 500 NMR spectrometers using a 5-mm triple resonance probe with Z gradient as described previously [25]. Measurements were made at 25 °C at concentrations of  2mgÆmL )1 in D 2 O, subsequent to several lyophilizations with D 2 O. For the proton chemical shift reference, the HDO resonance was set at 4.78 p.p.m. at 25 °Crelativeto the methyl resonance of external acetone at 2.225 p.p.m. All of the NMR data was acquired using Varian sequences provided with the VNMR 6.1B software. The same program was used for processing. The proton spectrum was acquired with a sweep width of 10 p.p.m., 1024 transients, water presaturation during the relaxation delay of 1.5 s and acquisition time of 1.7 s. It was processed with a low-pass digital filter for solvent suppres- sion and zero filling for a final resolution of 0.37 Hz per point. Homonuclear two-dimensional experiments, COSY (16 h), TOCSY (7 h) and NOESY (7 h) were acquired using the following parameters: water presaturation during the relaxation delay of 1.0 s, spectral width of 7 p.p.m. (COSY; 10 p.p.m.), acquisition time in t 2 of 0.15 s (COSY; 0.20 s) and 200 increments (COSY; 128) with 52 (TOCSY), 40 (NOESY) or 360 (COSY) scans per increment. The sign discrimination in F 1 was achieved by the States method. The mixing time for the NOESY and TOCSY experiments was 0.4 and 0.08 s, respectively. The phase-sensitive spectra were processedwithforwardlinearpredictionint 1 , unshifted Gaussian window functions, and zero filling to 1024*1024 complex points for a resolution of 2.9 Hz per point in both dimensions. The one-dimensional NOESY experiment was acquired with a sweep width of 16 p.p.m., water presatura- tion during the relaxation delay of 1 s, acquisition time of 1 s, a mixing time of 800 ms, selective pulse with 100 Hz bandwidth, and 2048 transients for a duration of 2 h. Using similar acquisition parameters, the one-dimensional NO- ESY-TOCSY experiment [27] was acquired with a NOE mixing time of 800 ms, spin lock time of 80 ms, and 12288 transients for a duration of 10 h. The one-dimensional TOCSY experiment was acquired with a sweep width of 16 p.p.m., water presaturation during the relaxation delay of 1 s, acquisition time of 1.7 s, mixing times of 80 ms, selective pulse with 30 Hz bandwidth, and 5120 transients for a duration of 4 h. Using similar acquisition parameters, the one-dimensional TOCSY-NOESY experiment [27] was acquired with a NOE mixing time of 800 ms, spin lock time of 80 ms, and 20480 transients for a duration of 21 h. Electrophoretic analysis of LPS LPSwaspreparedandanalysedbytricine-SDS/PAGEas described previously [10]. 4010 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002 Analytical methods and methylation analysis Sugars were determined as their alditol acetates and partially methylated alditol acetate derivatives by GLC- MS as described previously [8]. RESULTS In a previous study on LPS from H. influenzae strain RM118 lgtC we had identified a sialylated lactosyl (Sial-Lac) extension from the distal heptose residue (Hep III) of the inner core [11]. Interestingly, following mutation of the sialyltransferase gene (lic3A) responsible for addition of Neu5Ac to the lactose moiety in this strain, creating the double mutant (lgtC lic3A), sialylated glyco- forms were still observed. In SDS/PAGE analysis sialylated species of strain RM118 lgtC lic3A LPS were found as the LPS constituents with the lowest mobility, identified by virtue of an alteration in their migration pattern following treatment with neuraminidase, an enzyme that specifi- cally cleaves sialic acid residues (Fig. 1; lanes 1 and 2). H. influenzae LPS typically migrates as a complex series of bands in SDS/PAGE, each band corresponding to gain or loss of a glycose moiety due to the phase variable nature of H. influenzae LPS synthesis [3,21,22]. In LPS from RM118 lgtC lic3A it was interesting to note that the band corresponding to the sialylated LPS glycoform separated from those of fast migrating nonsialylated glycoforms by several glycose units. ES-MS analysis of the O-deacylated LPS from the RM118 lgtC lic3A double-mutant strain revealed ions with an expected composition consistent with the presence of a sialic acid residue (Fig. 2; Table 1). It has previously been established that sialic acid residues are not removed by the hydrazinolysis treatment used to O-deacylate LPS [11]. The molecular mass of the major sialylated glycoforms in the lgtC lic3A double mutant (3381.6 and 3546.0 Da) indicated the presence of an additional 527 atomic mass units, corresponding to an N-acetyl-hexosamine residue and two hexose residues over the Sial-Lac glycoform in the O-deacylated LPS from the Fig. 1. SDS/PAGE analysis of LPS from strains RM118 lgtC lic3A, RM118 lgtC, RM118 (wt), RM118 lic2A,RM118lpsA and RM118 lgtF before and after treatment with neuraminidase (as indicated by – and +, respectively). The sialylated tetrasaccharide-containing glycoforms are indicated by an asterisk. All strains were grown on media con- taining Neu5Ac except for the wild-type (wt) strain RM118. Fig. 2. Triply charged region of the ES-MS spectrum from the O-deacylated LPS obtained from strain RM118 lgtC lic3A grown on BHI plates supplemented with Neu5Ac. The structure of the inner core glycoforms is shown and ions arising from the inner core and from the addition of the sialylated tetrasaccharide are indicated. Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4011 lgtC single mutant (Table 1) [11]. The two major sialylated glycoforms observed differed by 165 atomic mass units, consistent with the gain or loss of a phosphocholine (PCho) residue. The PCho group is appended to the glucose residue at the proximal heptose residue (Hep I) in the parent strain [5] and lgtC mutant [21]. CE-MS analysis was carried out on Table 1. Negative ion ES-MS data and proposed compositions of O-deacylated LPS from H. infl uenza e strains RM118, RM118 lgtC,RM118 lgtC lic3A, RM118 lic2A and RM118 lpsA when grown on media containing sialic acid. Average mass units were used for calculation of molecular weight based on proposed composition as follows: lipid A, 953.00; Hex, 162.15; HexNAc, 203.19; Hep, 192.17; Kdo-P, 300.16; PEtn, 123.05; PCho, 165.05; Sial, 291.05. Strain [M-3H] 3– [M-2H] 2– Observed molecular ion Calculated molecular ion Relative intensity Proposed composition RM118 lgtC 853.2 1280.3 2562.6 2562.2 0.50 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 867.2 1300.9 2604.2 2604.2 0.85 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A 908.3 1362.6 2727.5 2727.3 1.00 PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 964.5 1446.6 2895.8 2895.3 0.35 Sial, PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A 1005.2 1508.3 3018.6 3018.3 0.70 Sial, PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1126.0 – 3381.0 3380.8 0.25 (Sial, 2Hex, HexNAc), 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1181.4 – 3547.2 3545.9 0.20 (Sial, 2Hex, HexNAc), PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A RM118 lgtC 867.3 1301.2 2604.7 2604.2 1.00 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A lic3A 908.3 1362.6 2727.5 2727.3 0.90 PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1126.2 – 3381.6 3380.8 0.20 (Sial, 2Hex, HexNAc), 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1181.0 – 3546.0 3545.9 0.20 (Sial, 2Hex, HexNAc), PCho, 3Hex, 3Hep, 2PEtn, Kdo-P, Lipid A RM118 812.2 – 2439.6 2439.2 0.15 3Hex, 3Hep, PEtn, Kdo-P, Lipid A 866.9 – 2603.7 2604.2 0.50 PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A 920.9 – 2765.7 2766.3 0.80 PCho, 4Hex, 3Hep, PEtn, Kdo-P, Lipid A 962.0 – 2889.0 2889.3 0.45 PCho, 4Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 964.0 – 2895.0 2895.3 1.00 Sial, PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A 989.0 – 2970.0 2969.5 0.60 PCho, 4Hex, HexNAc, 3Hep, PEtn, Kdo-P, Lipid A 1005.0 – 3018.0 3018.3 0.70 Sial, PCho, 3Hex, 3Hep, PEtn, Kdo-P, Lipid A 1030.0 – 3093.0 3092.6 0.25 PCho, 4Hex, HexNAc, 3Hep, 2PEtn, Kdo-P, Lipid A 1179.7 – 3542.1 3542.9 0.20 (Sial, 2Hex, HexNAc), 4Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1235.0 – 3708.0 3708.0 0.30 (Sial, 2Hex, HexNAc), PCho, 4Hex, 3Hep, 2PEtn, Kdo-P, Lipid A RM118 lic2A 758.0 – 2277.0 2277.4 0.40 2Hex, 3Hep, PEtn, Kdo-P, Lipid A 812.9 – 2441.7 2442.4 0.85 PCho, 2Hex, 3Hep, PEtn, Kdo-P, Lipid A 853.9 – 2564.7 2565.5 0.60 PCho, 2Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1030.9 – 3095.7 3095.6 0.30 (Sial, 2Hex, HexNAc), 2Hex, 3Hep, PEtn, Kdo-P, Lipid A 1071.9 – 3218.7 3218.7 0.90 (Sial, 2Hex, HexNAc), 2Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1085.8 – 3260.4 3260.7 0.35 (Sial, 2Hex, HexNAc), PCho, 2Hex, 3Hep, PEtn, Kdo-P, Lipid A 1126.8 – 3383.4 3383.7 0.75 (Sial, 2Hex, HexNAc), PCho, 2Hex, 3Hep, 2PEtn, Kdo-P, Lipid A RM118 lpsA 703.6 1055.9 2114.1 2115.2 0.45 Hex, 3Hep, PEtn, Kdo-P, Lipid A 744.9 1117.7 2237.3 2238.2 0.25 Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 758.7 1138.8 2279.3 2280.2 1.00 PCho, Hex, 3Hep, PEtn, Kdo-P, Lipid A 799.7 1200.4 2402.8 2403.2 0.60 PCho, Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1017.6 1526.5 3056.1 3056.4 0.20 (Sial, 2Hex, HexNAc), Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 1072.6 1609.3 3221.1 3221.5 0.40 (Sial, 2Hex, HexNAc), PCho, Hex, 3Hep, 2PEtn, Kdo-P, Lipid A 4012 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002 the O-deacylated material from the RM118 lgtC lic3A double mutant in order to obtain further information on the nature of the sialylated glycoforms. Comparison of the total ion electropherogram in negative ion mode (Fig. 3A) and selective ion scanning for m/z 290, specific for sialic acid (Fig. 3B), suggested the presence of two major populations of sialylated glycoforms consistent with the ES-MS spec- trum (Fig. 2). MS/MS analysis in positive ion mode of the doubly charged ion at m/z 1691.6 (Fig. 3C) that corre- sponds to the 3381.6 atomic mass unit glycoform produced abundant fragment ions at m/z 657 (Neu5Ac-Hex-Hex- NAc); m/z 528 (Hex-HexNAc-Hex); m/z 366 (Hex-Hex- NAc); m/z 292 (Neu5Ac) and; m/z 204 (HexNAc). This series of ions indicated the presence of a Neu5Ac-Hex- HexNAc-Hex oligosaccharide unit. In order to determine the location of this tetrasaccharide unit, the wild-type strain RM118 and a series of mutants thereof (lgtC, lic2A, lpsA) that corresponded to sequential truncations of the globoside trisaccharide extending from Hep III were examined, as well as a lgtF mutant in which there is no chain extension from Hep I [21]. In SDS/PAGE analysis of the LPS obtained from the lgtC, lic2A and lpsA mutants before and after neuraminidase treatment, the enzyme sensitive sialy- lated glycoforms showed an increased mobility. Corre- spondingly, the mobility of the major nonsialylated glycoforms increased as the length of chain from Hep III was reduced by consecutive glycose truncations (Fig. 1; lanes 3–4, 7–10), confirming that the sialylated unit was not attached via the distal heptose residue (Hep III) of the inner core LPS. This was most notable for the lpsA mutant, as the gene product of lpsA is responsible for initiation of chain extension from Hep III [21], and in this mutant background the LPS still elaborates the slower migrating sialylated glycoform. However, the lgtF mutant LPS (Fig. 1; lanes 11– 12) did not contain slower migrating sialylated glycoforms, indicating the sialylated unit to be located as an extension from the Hep I residue of the inner core. LgtF has been shown to be the glycosyltransferase required for initiation of chain extension from Hep I [21]. As expected ES-MS analysis of the O-deacylated LPS of each mutant strain (Table 1) revealed a reduction in mass of the sialylated species by a value corresponding to the removed residue. Thus the difference in mass between the O-deacylated LPS of RM118 lic2A (3383.4 Da) and RM118 lpsA (3221.1 Da) corresponds to the glucose residue (162.3 atomic mass units) that is normally attached by the lpsA gene product. In the spectra of O-deacylated LPS from the parent strain RM118 and RM118 lgtC, Sial-Lac glycoforms were observed in addition to the higher mass sialylated glycoforms. As expected Sial-Lac-containing glycoforms were not observed in the lic2A and lpsA mutants, as the required lactose acceptor is not present. It is noteworthy that di-sialylated species were not observed even in the lgtC mutant, the most appropriate background for Sial-Lac production [11]. In each of the mutant strains the higher mass sialylated glycoforms had a molecular weight consistent with the addition of a complete tetrasac- charide unit comprising Neu5Ac-Hex-HexNAc-Hex (819 atomic mass units). Closer examination of the ES-MS revealed no evidence for any glycoforms corresponding to partial addition of this sialylated unit, suggesting that this tetrasaccharide is added as a complete unit or not at all during LPS biosynthesis. The structure of the sialylated glycoforms was determined by NMR spectroscopy. To simplify the analysis the simplest structure still containing the sialylated tetrasaccharide was chosen for NMR studies, namely LPS from the lpsA mutant. The one-dimensional 1 H-NMR spectrum of the O-deacylated LPS was well resolved (Fig. 4). Characteristic signals were observed in the anomeric region for the H-1 1 H-resonances of a-configured residues at 5.76 p.p.m. (Hep II), 5.42 p.p.m. (GlcN of O-deacylated lipid A), and 5.16 p.p.m. (Hep I and Hep III). Additionally, signals were observed in the anomeric region for the H-1 1 H-resonances of b-configured residues, including the second GlcN residue of O-deacylated lipid A (4.63 p.p.m.) and the glucose residue (Glc I) at Hep I (4.54 p.p.m.) consistent with assignments previously obtained for O-deacylated LPS from the lpsA mutant that had been grown on media not containing Neu5Ac [21]. Closer examination of the b-anomeric region revealed two minor resolved signals at 4.74 and 4.45 p.p.m. due to the anomeric protons from Fig. 3. CE-ES mass spectrum of O-deacylated LPS from strain RM118 lgtC lic3A grown on BHI plates supplemented with Neu5Ac. (A) Solid line indicates the total ion electropherogram (TIE), the dotted line indicates single ion monitoring (SIM) for m/z 290 – , obtained in neg- ative ion mode with a high orifice voltage (120 V), and (B) a MS/MS experiment in positive ion mode on m/z 1691.6 2+ that corresponds to the Sial-lNnT-containing glycoform without the PCho moiety. Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4013 residues in the tetrasaccharide unit of the sialylated glyco- form. The signal at 4.74 p.p.m. produced a spin-system in a TOCSY experiment that could be assigned to N-acetyl- b- D -glucosamine (b- D -GlcNAc) by comparison to the published data (Fig. 5) [13,14]. Similarly (data not shown) the signal at 4.45 p.p.m. could be assigned to a b- D - galactose spin-system by comparison to the published data [13,14]. In the high-field region of the spectrum character- istic signals were observed for the axial and equatorial H-3 1 H-resonances of the sialic acid residue at 1.81 and 2.76 p.p.m., respectively (Fig. 4). Integration of the H-3 1 H-resonances of the sialic acid residue indicated that the sialylated glycoform was present at levels of  10% of the total LPS glycoform population. Higher percentages were anticipated from the MS analyses, but perhaps the levels of sialylated species were over-estimated in the MS studies due to the presence of the sialic acid residue in these glycoforms that would be more readily ionized in the negative ion mode. The 1 H NMR spectrum of the inner core LPS structure was assigned using COSY and TOCSY experiments (Table 2). The ring sizes and relative stereochemistries of the component monosaccharides were established from the 1 H chemical shifts and the magnitude of the coupling constants. The sequence of glycosyl residues of the inner core was determined from interresidue 1 H– 1 HNOEmeas- urements between anomeric and aglyconic protons on adjacent glycosyl residues. The NMR data was almost identical to that found previously [21] for the O-deacylated LPS of the RM118 lpsA mutant that had been grown on media not containing Neu5Ac. Under these growth conditions, the RM118 lpsA mutant does not elaborate detectable amounts of LPS containing sialylated glycoforms [21]. The structure of the oligosaccharide chain of the sialylated glycoforms was determined from a series of selective excitation NMR experiments [27]. Initially the axial resonance of the sialic acid residue at 1.81 p.p.m. was selectively irradiated in a one-dimensional NOESY Fig. 4. Anomeric region of the 1 H-NMR spectrum of the O-deacylated LPS obtained from strain RM118 lpsA grown on BHI plates supplemented with Neu5Ac. Inset, complete 1 H-NMR spectrum. The spectrum was recorded in D 2 O at pH 7.0 and 295 K. Fig. 5. Ring 1 H region of the TOCSY spectrum of the O-deacylated LPS from strain RM118 lpsA grown on BHI plates supplemented with Neu5Ac corresponding to the spin system from the residue at 4.74 p.p.m. The spectrum was recorded in D 2 O at pH 7.0 and 295 K. 4014 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002 experiment, revealing signals that by comparison to pub- lished data [13], could be assigned at 2.76 (H-3eq), 3.68 (H-4), 3.86 (H-5) and at 4.05 p.p.m. via an interresidue NOE. The latter signal was then irradiated in a one- dimensional TOCSY step following the one-dimensional NOESY step which revealed a signal at 4.56 p.p.m. that subsequently was found to correspond to the anomeric resonance of the b- D -galactose (Gal II) residue substituted by sialic acid (Fig. 6A). Selective irradiation of the anomeric resonance of the b- D -GlcNAc residue at 4.74 p.p.m. in a one-dimensional TOCSY experiment revealed the ring 1 H-signals at 3.81 (H-2), 3.73 (H-3 and H-4), and 3.59 p.p.m. (H-5). In a subsequent one-dimensional NOESY step irradiation of the H-3/H-4 signals at 3.73 p.p.m. produced signals at 4.74 and 4.56 p.p.m. corresponding to the anomeric 1 H-resonances of the b- D -GlcNAc and the b- D -galactose (Gal II) residue substi- tuting the b- D -GlcNAc, respectively (Fig. 6B). By compar- ison to the published data [13,14,28] it was clear that the chemical shifts of the b- D -GlcNAc residue were consistent with substitution at the 4-position. Thus, these experiments established the sequence of the terminal trisaccharide as a-Neu5Ac-(2–3)-b- D -Gal-(1–4)-b- D -GlcNAc. Examination of the two-dimensional TOCSY and NOESY spectra revealed characteristic signals for a 3-linked b- D -galactose (Gal I) residue at 3.60 (H-2), 3.73 (H-3), and 4.17 p.p.m. (H-4) in the spin-system arising from the anomeric resonance at 4.45 p.p.m. [14]. This inference was confirmed by a TOCSY/NOESY selective excitation experiment, where following selective irradiation of the anomeric proton at 4.45 p.p.m. in the TOCSY step, subsequent irradiation of the H-3 proton at 3.73 p.p.m. in the NOESY step revealed the anomeric proton at 4.74 p.p.m. of the b- D -GlcNAc residue, thus confirming this linkage (data not shown) and establishing the structure of the tetrasaccharide to be, a-Neu5Ac-(2–3)-b- D -Gal-(1–4)-b- D -GlcNAc-(1–3)-b- D - Gal. Due to the low intensity of the sialylated glycoforms and a high degree of overlap in the b-anomeric region of the spectrum, NMR methods were not successful in confirming the linkage position of the glucose residue at Hep I, to which the terminal tetrasaccharide unit was expected to be the attached. This is the only glucose residue present in the LPS of the RM118 lpsA mutant. Thus a methylation analysis was performed in order to determine its linkage position. Methylation analysis was carried out on the dephosphorylated O-deacylated LPS as it is known that phosphorylated residues are not readily identified following methylation analysis and a PCho residue substitutes the glucose residue in the majority of glycoforms. GLC-MS analysis of the partially methylated alditol acetates identified the expected substitution patterns for the inner core LPS, with t-Glc, t-Hep, 2-Hep and 3,4-Hep residues identified in approximately equimolar amounts. Less intense signals were also identified for 3-Gal and 4-Glc establishing the glucose residue to be 4-substituted and confirming the 3-linkages for the galactose residues of the Sial-lNnT unit (Fig. 7). This confirms that a terminal Sial-lNnT unit is linked to the Hep I residue of the LPS inner core in the sialylated glycoform. The structure of the sialylated glyco- forms of the O-deacylated LPS of H. influenzae strain RM118 and mutants causing sequential truncations from Hep III are illustrated in Fig. 8. DISCUSSION Structural analysis of H. influenzae strain RM118 lpsA LPS has revealed glycoforms containing a Sial-lNnT unit when the organism is grown on solid medium containing Neu5Ac. This structure was found to extend from the proximal heptose residue (Hep I) and is expressed in LPS glycoforms from the wild-type RM118 strain which also expresses a globoside unit (a- D -Galp-(1–4)-b- D -Galp-(1–4)-b- D -Glcp- (1–) from the distal heptose residue (Hep III). Sial-lNnT- containing glycoforms were also characterized in LPS from RM118 mutant strains with sequential truncations in the globoside unit (lgtC, lic2A,andlpsA). Sial-lNnT-containing glycoforms were not observed however, in LPS from the RM118 lgtF mutant strain. LgtF is the glucosyltransferase Table 2. 1 H NMR assignments of O-deacylated LPS from H. influenzae strain RM118 lpsA grown on media containing sialic acid. Assignments at 295 K, relative to HOD 4.78 p.p.m. ND, Not determined. H-1 H-2 H-3 H-4 H-5 NOEs Lipid A-OH a-GlcN 5.42 3.94 ND ND ND – b-GlcN 4.63 3.82 3.70 ND ND ND Inner core Hep I 5.16 4.14 4.07 ND ND 4.31 Kdo H-5 Hep II 5.76 4.27 ND ND ND 4.07 Hep I H-3 Hep III 5.16 4.06 ND ND ND 4.27 Hep II H-2 5.76 Hep II H-1 t-b-Glc (Glc I) 4.54 3.47 3.55 ND ND 4.25 Hep I H-4 4.05 Hep I H-6 Sial-lNnT unit -4)-b-Glc-(1-(Glc I) ND ND ND ND ND ND -3)-b-Gal-(1-(Gal I) 4.45 3.60 3.73 4.17 ND ND -4)-b-GlcNAc-(1- 4.74 3.81 3.73 3.74 3.59 3.73 Gal-I H-3 -3)-b-Gal-(1-(Gal II) 4.56 ND 4.05 ND ND 3.74 GlcNAc H-4 t-a-Neu5Ac 2.76 1.81 3.68 3.86 4.05 Gal-II H-3 Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4015 responsible for the initiation of oligosaccharide extension from Hep I. In the absence of the glucose acceptor the sialylated tetrasaccharide unit cannot be attached. In strain RM118, and the RM118 lgtC mutant strain, we have now identified two different sialylated species, namely Sial-Lac and sialyl-lacto-N-neotetraose (Sial-lNnT). Interestingly, glycoforms containing both sialylated oligosaccharides were not observed. It is possible that the expression of one sialylated structure sterically, or in some other way, hinders the attachment of the second sialylated group. Similarly, the high molecular weight sialylated glycoform was not observed in the wild-type RM118 strain when there was full extension of the globotetraose oligosaccharide (b- D - GalpNAc-(1–4)-a- D -Galp-(1–4)-b- D -Galp-(1–4)-b- D -Glcp- (1–) from Hep III, again perhaps indicating that steric interference of the two chains precludes coincident expres- sion of both. It is noteworthy that the majority of Sial- lNnT-containing glycoforms contain two PEtn residues. In the inner core of H. influenzae LPS, one PEtn residue is stoichiometrically present at the 6-position of the Hep II residue, and a second PEtn residue is sometimes attached to the Kdo-P moiety. The percentage of Sial-lNnT-containing glycoforms that elaborate two PEtn residues is considerably higher than the percentage of other glycoforms that elabo- rate two PEtn residues (Fig. 2), although the significance of this is unclear. Apart from the presumed steric incompat- ability of the globotetraose-containing RM118 glycoform with elaboration of the sialylated tetrasaccharide, all Sial- lNnT units are found in LPS molecules that have only the fully extended oligosaccharide at Hep III. Due to the phase- variable expression of the glycosyltransferases LgtC and Lic2A, a variety of extensions from Hep III are typically observed in the nonsialylated glycoforms [5,21]. Taken together, the correlation of fully extended structures from Hep III and the proportion of two-PEtn-containing struc- tures with the expression of the Sial-lNnT unit, would suggest that the addition of this tetrasaccharide unit utilizes a preferred LPS core structure or is perhaps a late event in the LPS biosynthesis pathway. Glycoforms in which the PCho group on Glc I is either absent or present were also detected in each of the strains investigated. The Sial-lNnT side-chain is identical to that previously observed in meningococcal and gonococcal LPS where it is found in an analogous molecular environment. It is of considerable interest that this sialylated structure has been found to increase the serum resistance of those organisms bearing this group [15]. This phenomenon is of particular importance in strains that lack a capsular structure, which in itself provides serum resistance. Recent data from our laboratory indicate that the ability of acapsular strains of H. influenzae to elaborate sialylated glycoforms can confer serum resistance in model systems. In the study of Hood et al. [11], a RM118 lgtC lic3A mutant strain that cannot synthesize Sial-Lac, but as detailed in the present study can express Sial-lNnT-containing glycoforms, was resistant to the bactericidal effect of human sera compared with the isogenic RM118 lgtC siaB strain, a strain that is unable to incorporate any sialic acid residues. It is likely that this Fig. 6. (A) One-dimensional NOESY-TOCSY spectrum using selective excitation of H-3 ax 1 H-resonance of the Neu5Ac residue in the NOESY step and of H-3 1 H-resonance of the Gal II residue in the TOCSY step and (B) one-dimensional TOCSY-NOESY spectrum using selective excitation of H-1 1 H-resonance of the GlcNAc residue in the TOCSY step and of H-3/H-4 1 H-resonance of the GlcNAc residue in the NOESY step. (A) The assignments of the 1 H-resonances of the Gal II residue are indicated. (B) The assignments of the 1 H-resonances of the GlcNAc and Gal II residues are indicated. The spectrum was recorded in D 2 O at pH 7.0 and 295 K. 4016 A. D. Cox et al. (Eur. J. Biochem. 269) Ó FEBS 2002 residual serum resistance of the RM118 lgtC lic3A strain was due to the Sial-lNnT structure. H. influenzae requires an exogenous source of sialic acid and thus the ability to sequester sialic acid from the host and to then utilize this moiety to modify LPS molecules is likely to play an important role in the virulence of H. influenzae strains. Previous studies from our laboratory had identified the sialyltransferase gene (lic3A) responsible for sialylation of the lactose moiety [11]. As shown in this study the lic3A mutant can elaborate Sial-lNnT-containing glycoforms pointing to the presence of another sialyltransferase gene in the RM118 genome that specifically adds sialic acid to the b- D -Galp-(1–4)-b- D -GlcNAcp-(1–3)-b- D -Galp-(1-trisaccha- ride. Several candidates are currently under investigation for this role and include HI0871 that is a homologue to the sialyltransferase gene lst of H. ducreyi [29] and HI1699 that is a homologue to the sialyltransferase gene lst of N. men- ingitidis [30]. This latter gene is part of a seven-gene locus, lsgA-G that when expressed in Escherichia coli results in the production of chimeric LPS that reacts with MAb 3F11 (specific for the terminal lactosamine disaccharide of lNnT) [31]. The results of the present study would suggest that the Sial-lNnT group is added to the LPS as a complete unit via a mechanism involving block addition of the terminal tetrasaccharide unit to the Glc I acceptor. This behaviour differs from the expression of the Sial-lNnT group of neisserial LPS, which involves sequential addition of monomeric units, and invariably a series of truncated Sial-lNnT structures is observed which indeed accounts for some of the different meningococcal LPS immunotypes. The presence of truncated Sial-lNnT structures is com- monly encountered due to the phase variable glyco- syltransferases of the lgt operon that synthesize the lNnT structure in meningococcal LPS [32]. Truncated structures due to incomplete biosynthesis of the Sial-lNnT unit were not observed in RM118 H. influenzae strains in this study. TheSDS/PAGEprofilesandMSdataindicatedthe presence of the Sial-lNnT structure as a complete unit and no evidence for loss of any residues in this structure was observed. This profile has also been observed in other H. influenzae strains (unpublished data). This suggests an interesting biosynthetic scheme for the attachment of this structure in H. influenzae. One explanation is that biosyn- thesis is more akin to the biosynthesis of an O-antigen, where each repeating unit of the O-antigen is often transferred as a complete unit to the growing LPS molecule. An alternative explanation could be that cooperative glycosylation reactions of a series of glycosyltransferases are required for synthesis, although such cooperativity has not been described before in H. influenzae LPS synthesis. Studies are underway in our laboratory to elucidate the genetic mechanisms involved. This study is the first to provide definitive structural evidence for the presence of a sialylated lacto-N-neotetra- ose moiety in H. influenzae LPS, a structure which is potentially of great importance to the virulence of this organism. Fig. 7. (A) GC-MS trace of partially methylated alditol acetates derived from O-deacylated, dephosphorylated LPS from strain RM118 lpsA grown on BHI plates supplemented with Neu5Ac and (B) fragmentation pattern of a 4-linked hexose residue. Fig. 8. Structural model of the sialylated tetrasaccharide-containing glycoforms of O-deacylated LPS of H. in fluenza e strain RM118. Mutant strains with truncated oligosaccharide extensions from Hep III residue are as indicated. Ó FEBS 2002 Sialylated LPS in Haemophilus influenzae (Eur. J. Biochem. 269) 4017 ACKNOWLEDGEMENTS We thank D. Krajcarski for ES-MS and S. Larocque for assistance with NMR experiments. REFERENCES 1. Foxwell, A.R., Kyd, J.M. & Cripps, A.W. (1998) Nontypable Haemophilus influenzae: Pathogenesis and prevention. Microbiol. Mol. Biol. Rev. 62, 294–308. 2. Phillips, N.J., Apicella, M.A., Griffiss, J.M. & Gibson, B.W. (1992) Structural characterization of the cell surface lipooligo- saccharides from a non-typable strain of Haemophilus influenzae. Biochemistry 31, 4515–4526. 3. Masoud, H., Moxon, E.R., Martin, A., Krajcarski, D. & Richards, J.C. 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Identification and structural characterization of a sialylated lacto-N-neotetraose structure in the lipopolysaccharide of Haemophilus in uenzae Andrew. and ions arising from the inner core and from the addition of the sialylated tetrasaccharide are indicated. Ó FEBS 2002 Sialylated LPS in Haemophilus in uenzae