Báo cáo Y học: Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd–/b+ of Haemophilus influenzae docx

Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd – /b + of Haemophilus influenzae Sven Mu¨ ller-Loennies, Lore Brade and Helmut Brade Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany From the lipopolysaccharide of the deep rough mutant I-69 Rd – /b + of Haemophilus influenzae two oligosaccharides were obtained after de-O-acylation and separation by high-performance anion exchange chromatography. Their chemical structures w ere determined by one- a nd two-dimensional 1 H-, 13 C- and 31 P-NMR spectroscopy as aKdo-4P -(2 fi 6)-bGlcN-4P-(1 fi 6)- aGlcN-1P and aKdo-5P-(2 fi 6)- bGlcN-4P-(1 fi 6)-aGlcN-1P. The spe- cificity of mAbs S42-21 and S42-16 specific for Kdo-4P or Kdo-5P, r e spectively [Rozalski, A., Brade L., Kosma P., Moxon R., Kusumoto S., & B rade H. (1997). Mol. Micro- biol. 23, 569–577] was confirmed with neoglycoconjugates obtained by conjugation of the isolated oligosaccharides to BSA. In addition, a mAb S42-10-8 with unknown epitope specificity could be assigned using the neoglycoconjugates described herein. This mAb binds to an epitope composed of the b isphosphorylated glucosamine b ackbone of lipid A a nd Kdo-4P, w hereby the latter determines t he specificity strictly by the position of the phosphate group. Keywords: carbohydrate antibody; Kdo-phosphate; neoglycoconjugate; serology; sugar phosphate. Haemophilus influenzae normally colonizes the human nasopharynx but may cause severe infections, in particular meningitis, in children. A m ajor virulence factor of this human p athogen is the type b capsule, an acidic polysac- charide composed of ribose, ribitol a nd phosphate and which i s the basis of an e ffec tive conjugate vaccine [1]. Among other viru lence factors i s the lipopolysacchari de (LPS) in which we are interested for various reasons: (a) LPS is an essential component of the outer membrane in all Gram-negative bacteria; (b) LPS is the endotoxin of Gram- negative bacteria; (c) LPS is a major surface antigen leading to the induction of protective a ntibodies; and (d) the understanding of the biosynthesis of LPS may allow the distinct blockage of e ssential steps as a new strategy for the development of antibiotics [2,3]. The smallest LPS structure which still allows the bacter- ium t o survive was f ound in the mutant strain I-69 Rd – /b + of H. influenzae (referredtohereasI-69)wherea single phosphorylated 3-deoxy- D -manno-oct-2-ulopyrano- sonic acid (Kdo) residue is linked to the lipid A moiety. Helander et al. h ave shown t hat t he I-69 LPS was composed of two molecular s pecies with Kdo phosphorylated at either position 4 or 5 [4]. The Kdo transferase of I-69 has been cloned and characterized and the phosphokinase adding the phospho- ryl group to position 4 of the Kdo residue has also been cloned [5,6]. Coexpression of both e nzymes in an Escheri- chia coli strain lacking its own Kdo transferase led to the synthesis o f an LPS which contained exclusively Kdo-4P [7]. For this study mAbs were useful to identify the secondary gene products. We have reported earlier on mAb recogni- zing either t h e 4- or 5-phosphorylated Kdo which was chemically synthesized and c onjugated to BSA [8]. In addition, we found mAb S 42-10-8 w hich was specific f or the I-69 LPS but did not react with Kdo-4P or Kdo-5P alone. Therefore, this antibody was assumed to recognize an epitope requiring, i n addition to a phosphorylated Kdo residue, the phosphorylated lipid A backbone. As the LPS species containing the Kdo-4P or Kdo-5 P could not be separated at t hat time and were not yet chemically synthesized, the s pecificity of this m Ab has not yet been elucidated. Here, we report on: (a) t he successful separation of the deacylated carboh ydrate backbone of I-69 LPS into two pure oligosaccharides containing either Kdo-4P or Kdo-5P; (b) the structural analysis of both oligosaccharides by NMR; and (c) t he characterization of a n ew mAb recognizing a phosphorylated carbohydrate epitope. MATERIALS AND METHODS Bacteria and bacterial LPS H. influenzae I-69 Rd – /b + was cultivated a s d escribed previously [9]. Bacteria were washed with ethanol, acetone (twice), and ether, and dried. LPS was extracted from dry bacteria by the phenol/chloroform/petroleum ether method [10] in a yield of 4.4% of dry bacteria. De-O-acylated LPS was prepared after hydrazine treatment of LPS for 30 min at 37 °C (yield: 81% based on the glucosamine content), and deacylated LPS ( LPS deac ) w as ob tained by hydrolysis of de-O-acylated LPS in 4 M KOHasreported[11].LPS deac was further purified by preparative high performance anion exchange chromatography(HPAEC) u sing water as e luent A Correspondence to H. Brade, Research Center Borstel, Center for Medicine and Biosciences, Parkallee 22, D-2 3845 Bor stel, Ge rmany. Fax: + 49 4537 188419, Tel.: + 49 4537 188474, E-mail: hbrade@ fz-borstel.de Abbreviations: HPAEC, high performance anion exchange chroma- tography;Kdo,3-deoxy- D -manno-oct-2-ulopyranosonic acid; LPS, lipopolysaccharide; LPS deac , deacylated LPS. Note:S.Mu ¨ ller-Loennies and L. Brade contibuted equally to this work. (Received 8 August 2001, revised 2 1 December 2001, accepted 3 January 2002) Eur. J. Biochem. 269, 1237–1242 (2002) Ó FEBS 2002 and 1 M ammonium acetate as eluent B and a gradient of 1% to 99% over 80 min. Desalting was achieved by gel filtration on a column of 100 · 1.5 cm Sephadex G10 in pyridine/acetic acid/water (4 : 10 : 10 00, v/v/v) at a flow rate of 1 mLÆmin )1 . Fractions 1 and 2 were obtained in pure form in yields of 21.6 and 9.5%, respectively, based on the glucosamine content. NMR spectroscopy The deacylated LPS from H. influenzae I-69 was investi- gated by one-dimensional 1 H-NMR- and 13 C-NMR and spectroscopy at 600 and 150 MHz, r espectively, on a Bruker DRX 600 A vance spectrometer; 31 P-NMR spectra were recorded on a Bruker D PX 360 Avance spectrometer a t 145 MHz. All spectra were recorded on a 0.5-mL solution of 5 mg s ample i n D 2 O. As reference served acetone 2.225 p.p.m. ( 1 H), d ioxane 67.4 p.p.m. ( 13 C) and 85% phosphoric acid 0 p.p.m. ( 31 P). All spectra were run at a temperature of 300 K. For 31 P measurements the pD was adjusted to pD 2. Other measurements were performed at pD 6 due to the a cid labile nature of the Kdo-linkage. Two-dimensional homonuclear 1 H, 1 H-DQF-COSY was recorded over a sp ectral width of 7.5 p.p.m. in both dimensions recording 512 experimen ts of 32 s cans. Four thousand data points were recorded in F2. Zero-filling was applied in F1 to 1000 data points. Heteronuclear 1 H, 13 C-NMR correlation spectroscopy was recorded as HMQC. Two thousand data points were recorded in F2 over a spectral width of 10 p.p.m. and 256 experiments consisting of 24 scans per increment. Phase cycling w as performed using States-TPPI. Prior to Fourier transfor- mation zero-filling was applied in F1 to 512 data points. 31 P-NMR spectroscopy was recorded with continuous wave decoupling during acquisition. A total of 32 scans was recorded. For 1 H, 31 P-NMR COSY a HMQC experiment was recorded consisting of 256 experiments and 32 scans each. Two thousand data points were collected ove r a spectral w idth of 10 p.p.m. in F2 and zero filling was applied in F1 to yield 512 data points. The spectral width w as 10 p.p.m. in F1. Neoglycoconjugates The amino groups of the glucosamine residues in LPS deac and in the oligosaccharides obtained from LPS deac were activated with glutardialdehyde and conjugated to BSA as described [12]. The amount of ligand present in the conjugates was d etermined by measuring the amount of protein (Bradford assay, Bio-Rad) and glucosamine (Table 1). MAbs Monoclonal antibodies S 42-16, S42-21 and S42-10-8 were obtained after immunization and selection as described [8]. Culture s upernatants were p repared in at least 100 mL quantities and antibodies were purified on protein G-Sepharose (Pharmacia/LKB) according to the supplier’s instructions. Purification was a scertained by SDS/PAGE and protein concentrations were determined by the bicin- choninic acid assay (Pierce). Serology For ELISA, neoglycoconjugates were coated onto Maxi- Sorp microtiter plates (U-bottom, Nunc). Antigen solutions were adjusted to equimolar concentrations based on the amount of ligand present in the respective glycoconjugate. Unless stated otherwise, 50 lL volumes were u sed. Micro- titer plates w ere coated with the respective antigen solution in 50 m M carbonate buffer pH 9 .2 at 4 °C overnight. Plates were washed twice with distilled wate r; further washing was carried out in NaCl/P i supplemented with 0.05% Tween 20 (Bio-Rad) and 0.01% thimerosal (NaCl/P i /Tween-T). Pla tes were then blocked w ith NaCl/P i /Tween-T supplemented with 2.5% casein (NaCl/P i /Tween-TC) f or 1 h a t 37 °Cona rocking platform f ollowed by t wo washes. A ppropriate antibody dilutions in NaCl/P i /Tween-TC supplemented with 5% BSA were added and incubated for 1 h at 37 °C. After washing, peroxidase-conjugated goat anti-(mouse IgG) Ig (heavy and light chain specific; Dianova) was added (diluted 1 : 1000) and incubation was continued for 1 h at 37 °C. After three washes in NaCl/P i /Tween-T, the plates were wash ed in substrate buffer ( 0.1 M sodium citrate, pH 4.5). Substrate solution was freshly prepared and was composed of azino-di-3-ethylbenzthiazolinsulfonic acid (1 mg) dissolved in substrate buffer ( 1 mL) with sonication in an ultrasound water bath for 3 min followed by the addition of hydrogen peroxide (25 lLofa0.1%solution). After 3 0 min at 3 7 °C, the reaction w as stopped b y t he addition of 2% aqueous oxalic acid and the plates were read with a microplate reader (Dynatech MR 700) at 405 nm. For ELISA using LPS as a solid-phase antigen another protocol was used. Polyvinyl microtiter plates (Falcon 3911) were coated with various amounts of LPS dissolved in NaCl/ P i (10 m M pH 7.3, 0.9% NaCl, 50 lL) at 4 °C overnight or at 37 °C for 1 h. All following steps were performed at 37 °C with gentle agitation and all washing steps were performed four times. Coated plates were washed in NaCl/P i , blocked for 1 h with blocking b uffer (2.5% casein in NaCl/P i )and then incubated f or 1 h with mAb diluted in blocking buffer (50 lL). Plates were washed in NaCl/P i and incubated for Table 1. Oligosaccharides and neoglycoconjugates used in this study. For derivatization procedures see Materials and methods. Molar ratio o f ligand to protein given in parentheses. Chemical structure Abbreviation Amount of ligand (nmolÆmg )1 ) aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P Kdo4PGlcN 2 P 2 aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P Kdo5PGlcN 2 P 2 aKdo-4/5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA LPS deac -BSA 33 (2.4) aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA Kdo4P-GlcN 2 P 2 -BSA 16 (1.1) aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P-BSA Kdo5P-GlcN 2 P 2 -BSA 15 (1.0) 1238 S. Mu ¨ ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002 1 h with peroxidase-conjugated goat anti-(mouse IgG) Ig or goat anti-(rabbit IgG) Ig (heavy and light chain specific, Dianova; diluted 1 : 1000 in blocking buffer, 50 lL). Further development of the reaction was as described a bove. All tests were set up in quadruplicate. Confidence v alues of the means were less than 10%. RESULTS Isolation and structural analysis of the phosphorylated carbohydrate backbone of I-69 LPS The LPS of H. influenzae I-69 was s uccessively de-O-acylated and de-N-acylated with hydrazine and potassium hyd rox- ide, respective ly, leading to two major products as revealed by HPAEC (Fig. 1). The two peaks, compounds 1 and 2, could be separated from each other b y preparative HPAEC with yields of 11.6 mg (21.6% of LPS) and 5.1 mg (9.5% of LPS) for Kdo-4P-GlcN 2 -P 2 and K do-5P-GlcN 2 -P 2 , respectively. Both compounds were identified by one- and two- dimensional NMR spectroscopy. Spectra of both contained characteristic signals o f a single a-Kdo-residue, one b-linked GlcN and one a-configured GlcN [7]. In addition, three phosphate-residues were identified by 31 P-NMR spectro- scopy (Fig. 2). With respect to the carbohydrate and phosphate composition the two compounds were identical and was reflected by almost identical one-dimensional 1 H-NMR spectra (Fig. 3, Table 2). As expected the com- pounds differed in their phosphate substitution (Fig. 3, Table 4). Both compounds contained one glycosidic phos- phate linked to the a-GlcN (A) of the lipid A backbone leading to a splitting of the signal of its anomeric proton and another phosphate linked to the 4-position of the b-config- ured GlcN (B). The far downfield position of the chemical shifts of proton H-4 and carbon C-4 of the Kdo-residue (C) of compound 1 and the downfield shift to t he same frequencies of proton H-5 and carbon C-5 of the Kdo-resi- due (C) of compound 2 identified compound 1 as Kdo-4P- GlcN 2 -P 2 and compound 2 as Kdo-5P-GlcN 2 -P 2 (Tables 2 –4). The correct position o f p hosphates was finally determined by 1 H, 31 P-HMQC spectroscopy. Serology Both oligosaccharides were activated with glutardialdehyde and conjugated to BSA as described [ 12]. Chemical analyses indicated a molar ratio of protein to ligand of 1 : 1.1 and 1 : 1.0 f or Kdo-4P-GlcN 2 -P 2 -BSA and Kdo-5P-GlcN 2 - P 2 -BSA, r espectively. Both neoglycoconjugates w ere used in ELISA to determine the epitope specificities of mAb. LPS and LPS deac -BSA were used for comparison, whereby the latter contained a mixture of 4- and 5-phosp horylated Kdo in the ratio as it occurs in natural LPS. Clone S42-16 and S42-21 were confirmed to be specific for Kdo-5P and Kdo-4P, respectively. As seen in Fig. 4B clone S42-16 bound over a wide range of antigen coating concentrations (10–0.08 pmol per well) to Kdo-5P-GlcN 2 -P 2 -BSA at antibody concentrations as low as 1 ngÆmL )1 . No binding of this antibody was observed with Kdo-4P-GlcN 2 -P 2 -BSA even at highest antigen concentration (10 pmol per w ell) and antibody concentration ( 10 lgÆmL )1 ) (Fig. 4A). The mAb S42-21 bound only to Kdo-4P-GlcN 2 -P 2 -BSA (Fig. 4 C) but not to Kdo-5 P-GlcN 2 -P 2 -BSA (Fig. 4D) The affinity of mAb S42-21 was approximately 200 times lower t han t hat o f m Ab S42-16 for the homologous epitope. Fig. 1. HPAEC chromatogram of deacylated LPS from H. influenzae I-69. Shown is the analytical separatio n of the crude mixture (A) and the a nalytical chromatography of the isolated species (B a nd C). Peaks 1and2representaKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P, respectively. Fig. 2. 31 P-NMR spectrum of aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)- aGlcN-1P (top) and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (bottom). Ó FEBS 2002 Structure and antigenicity of H. influenzae LPS (Eur. J. Biochem. 269) 1239 The generation of clone S42-10-8 has been reported previously [8] but its e pitope specificity c ould not be determined so far. Binding of this antibody was tested i n ELISA using various concentrations of Kdo-4P-GlcN 2 - P 2 -BSA and Kdo-5P-GlcN 2 -P 2 -BSA, LPS or LPS deac - BSA. Fig. 3. 1 H-NMR spectr a of aKdo-4P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (top) and aKdo-5P-(2 fi 6)-bGlcN-4P-(1 fi 6)-aGlcN-1P (bottom). The asterisk indicates signals o f tryethylamine. Table 2 . 1 H-NMR chemical s hift data of compounds 1 and 2. NR, not resolved. Compound Residue 1 H-Chemical shift (p.p.m.) and coupling constants (Hz) for proton H-1 H-2 H-3ax H-3eq H-4 H-5 H-6a H-6b H-7 H-8a H-8b 1Afi6aGlcN1P 5.659 3.380 3.873 3.448 4.123 3.852 4.248 4; 8 a 10 10 10 12; 9 4 B fi 6bGlcN4P 4.908 3.072 3.859 3.859 3.740 3.495 3.698 810 CaKdo4P 1.925 2.149 4.514 4.179 3.766 3.926 3.918 3.652 )12; 12 6 6 9 12; NR 6 2Afi6aGlcN1P 5.654 3.364 3.889 3.426 4.124 3.861 4.229 4; 7 a 11 10 10 12; 9 4 B fi 6bGlcN4P 4.902 3.074 3.854 3.840 3.727 3.471 3.714 81010 CaKdo5P 1.919 )12; 12 2.142 5 4.141 4.507 3.736 9 3.907 3.941 13; NR 3.649 a3 J H-1,P . 1240 S. Mu ¨ ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002 As seen in Fig. 4E, mAb S42-10-8 bound to Kdo-4P- GlcN 2 -P 2 -BSA and with comparable a ffinity to LPS (Fig. 5A) or LPS deac -BSA (Fig. 5B) as solid phase antigen. No binding was observed with Kdo-5P-GlcN 2 -P 2 -BSA (Fig. 4F). The data show, together with those published earlier [8], that mAb S42-10-8 binds to a complex epitope composed of Kdo-4P linked to t he bisphosphorylated glucosamine backbone of the LPS of H. influenzae I-69. Although, Kdo-4P alone is not bound to the antibody, the position of the phosphate group strictly determines the specificity of the e pitope as no binding was observed with antigens containing Kdo-5 P instead o f Kdo-4P or with antigens containing nonphosphorylated Kdo. DISCUSSION Kdo is a common constituent of LPS and its presence is essential for the survival of Gram-negative bacteria. A c- cording t o our present knowledge of the Kdo-lipid A r egion one Kdo residue is linked to position 6¢ of the glucosamine disaccharide backbone of lipid A and is substituted at position 5 by another sugar and at position 4 by another sugar or phosphate [13]. The LPS of the deep rough mutant I-69 of H. influenzae is unique in being composed of only o ne Table 4. 31 P-NMR chemical shifts of compounds 1 and 2. Residue 31 P-Chemical shift (p.p.m.) for compound 12 A)1.41 )1.64 B 0.58 )0.01 C 0.68 1.65 Table 3. 13 C-NMR chemical shift data of compounds 1 and 2. ND, not determined. Compound Residue 13 C-Chemical shift ( p.p.m.) of carbon C-1 C-2 C-3 C-4 C-5 C-6 C-7 C-8 1Afi6aGlcN1P 91.10 54.45 69.76 70.07 72.69 69.36 B fi 6bGlcN4P 99.14 55.74 72.08 74.44 74.20 62.40 C aKdo4P ND ND 33.77 70.98 65.88 71.68 69.77 63.47 2Afi6aGlcN1P 91.16 54.45 69.61 70.12 72.68 69.20 B fi 6bGlcN4P 99.02 55.76 72.02 74.52 74.09 62.60 C aKdo5P ND ND 34.92 65.99 71.00 71.64 69.65 63.07 Fig. 4. Binding curves of mAbs S42-16 (A and B), S42-21 (C and D), and S42-10-8 (E a nd F) to Kdo4P-GlcN 2 P 2 -BSA (A, C and E) and Kdo5P-GlcN 2 P 2 -BSA (B, D and F). ELISA plates were coated with 200 (d), 100 (m), 50 (j)25(r), 12.5 (s), 6.3 (n), 3.2 (h)and1.6(e) pmol ligandÆml )1 and reacted with the mAb c oncen tratio ns indicated on the abscissa. Values are the mean of quadruplicates with confidence values not exceeding 10 %. Fig. 5. Binding curve of mAb S42-10-8. The ligands were I-69 LPS (A) and LPS deac -BSA (B). The coating concentrat ions used were 400 (d), 200 (m), 100 (j)50(r), 25 (s), 12.5 (n), 6.3 (h)and3.2(e) pmolÆml )1 for LPS deac -BSA. D ue to the poor c oating effi ciency of LPS 2000 (d), 1000 (m), 500 (j)250(r), 125 (s), 63 (n), 32 (h)and16 (e)pmolÆml )1 were used for the immobilization of LPS. Both were reacted with mAb concentrations indicated o n the abs cissa. Values are the mean of quadruplicates with confidence values not exceeding 10%. Ó FEBS 2002 Structure and antigenicity of H. influenzae LPS (Eur. J. Biochem. 269) 1241 phosphorylated Kdo residue in addition to lipid A whereby the Kdo is phosphorylated either at position 4 or 5. There was some uncertainty in the beginning whether the Kdo-5P was the result of phosphate migration [4], however, when mAbs specific for the 4- or 5-P became available it could b e shown that both antibodies bound to native bacteria [8]. The final proof that both phosphates are made by the bacterium was provided recently when we coexpressed the monofunc- tional Kdo transferase and a phosphokinase of H. influenzae in E. coli resulting in L PS whic h c ontained exclusively Kdo- 4P [7]. As the LPS obtained from this recombinant strain was deacylated by the same pro tocol as used in this study it is apparent that the appearance of the 5 P is not the result of phosphate migration. Therefore, we conclude that H. influenzae possesses two independent phosphokinases attaching phosphate to position 4 or 5 whereby the 5 -kinase has not yet been identified. With the results presented here the complete structures of the phosphorylated carbohydrate backbones of both LPS species made by H. influenzae I-69 are uniquivocally established and we have presented a protocol for p reparing these two oligosaccharides in sufficient quantities. We have performed this s tudy not only t o d efinitely identify the two differently phosphorylated LPS species bu t also to learn more about the recognition of charged carbohydrate epitopes b y antibodies. W e are interested in this aspect to better understand protein–carbohydrate inter- actions in general and the b inding of antibodies against bacterial LPS in particular, as some of them are able to neutralize the endotoxic activities of LPS which are embed- ded i n t he phosphorylated lipid A moiety [ 14]. W e h ave already characterized antibodies against the isolated lipid A moiety [15] or against Kdo [16] or Kdo- P [8]. In this context mAb S42-10-8 against I-69 LPS was of specific interest f or us as it binds to an epitope composed of Kdo-P and lipid A; however, its detailed epitope specificity could not be inves- tigated so far due to the lack o f a ppropriately defined antigens. The successful separation of these o ligosaccharides described here together w ith a previously described conju- gation protocol [14] allowed the characterization of the epitope specificity of mAb S42-10-8. The binding data obtained i n ELISA unequivocally proved that this m Ab recognizes the trisaccharide aKdo-4P-(2–6)-bGlcN-4P- (1–6)-aGlcN-1P; it does not bind to Kdo, Kdo-4P,Kdo- 5Por aKdo-(2–4)-aKdo-(2–6)-bGlcN-4P-(1–6)-aGlcN-1P. The availability of both oligosaccharides as free ligands and as neoglycoconjugates now enables us to investigate further this antibody by NMR and crystallography. ACKNOWLEDGEMENTS We thank R . Moxon (Oxford, UK) for strain I-69 and V. Susott and S. Cohrs for technical assistance. This w ork was supported by the Deutsche Forschungsgemeinschaft (grant SFB470/C1 to L. B.). REFERENCES 1. Moxon, E.R. (1992) Session III: Pathogenesis of invasive Hae- mophilus influenzae disease. Molecular basis of invasive Haemo- philus influenzae type b disease. J. Infect. Dis. 165, S77–S81. 2. Raetz, C.R.H. (1990) Biochemistry of endotoxins. Ann. Rev. Biochem. 59, 129–170. 3. Rietschel, E.T., Kirikae, T., Schade, F.U., Mamat, U., Schmidt, G., Loppno w, H., Ulmer, A.J., Za ¨ hringer, U., Seydel, U., Di Padova, F., Schreier, M. & Brade, H. (1994) Bacterial endotoxin: Molecular relationships of structure to activity and func tion. FASEB J. 8, 217–225. 4. Helander, I., Li ndner, B., Brade, H., Altmann, K ., Lindberg, A.A., Rietschel, E.Th & Za ¨ hringer, U. (1988) Chemical s tructure of the lipopolysaccharide of Haemophilus influenzae strain I-69 Rd – /b + . Eur. J. Biochem. 177 , 483–492. 5. White, K.A., Kalashov, I.A., Cotter, R.J . & Raetz, C.R.H. (1997) A mono-functional 3-deoxy- D -manno-octulosonic acid (K do ) transferase and a Kdo kinase in extracts of Haemophilus influen- zae. J. Biol. C hem. 272, 16555–16563. 6. White, K.A., Lin, S., Cotter, R.J. & Raetz, C.R.H. ( 1999) A Haemophilus influenzae gene that encodes a membrane bo und 3-deoxy- D -manno-octulosonic Acid (Kdo) kinase. J. Biol. Chem. 274, 31391–31400. 7. Brabetz, W., Mu ¨ ller-Loennies, S. & Brade, H. (2000) 3-Deoxy- D -manno-oct-2-ulosonic acid (Kdo) transferase (WaaA) and kdo kinase (KdkA) of Haemophilus influenzae are both required to complement a w aaA knockout mutation of Es cherichia coli. J. Biol. Chem. 275, 34954–34962. 8. Rozalski, A., Brade, L., K osma, P., Moxon, R., Kusumoto, S . & Brade, H. (1 997) Chara cterization of monoclonal antibodies recognizing three distinct, phosphorylated carbohydrate epitopes in the lipopolysaccharide o f the deep rough mutant I-69 Rd-/b+ of Haemophilus influenzae. Mol. Microbiol. 23, 569–577. 9. Zamze, S.E ., Ferguson, M.A.J., Moxon, E.R., Dwek, R.A. & Rademacher, T.W. (1987) Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influen zae lipopolysaccharide. Biochem. J. 245, 583–587. 10. Galanos, C., Lu ¨ deritz, O. & Westphal, O. (1969) A new method for the extraction of R. lipopolysaccharides. Eur. J. Biochem. 9, 245–249. 11. Holst, O., Broer, W., Thomas-Oates, J.E., Mamat, U. & Brade, H. (1993) Structural analysis of two oligosaccharide biphosphates isolated from the lipopolysaccharide of a recombinant strain of Escherichia coli F515 ( Re chemotype) expressing t he genus-specific epitope of Chlamydia lipopolysaccharide. Eur. J. Biochem. 214, 703–710. 12. Brade, L., Holst, O. & Brade, H. (1993) An artificial glycoconju- gate containing the bisph osphorylated glucosamine disaccharide backbone of lipid A b inds lipid A m onoclonal antibodies. Infect. Immun. 61, 4514–4517. 13. Holst, O. (1999) Chemical structure o f the core region of lipo- polysaccharides. In EndotoxininHealthandDisease.(Brade, H., Opal, S.M., Vogel, S.N. & Morrison, D.C., eds), pp. 115–154. Marcel Dekker Inc., New York. 14. Di Padova, F.E., Brade, H., Barclay, G.R., Poxton, I.R., Liehl, E., Schuetze, E., Kocher, H.P., Ramsay, G ., Schreier, M .H., McClelland, D.B.L. & R ietschel, E.T. (1993) A broadly cross- protective monoclonal antibody binding to Escherichia coli and Salmonella lipopolysaccharide. Infect. Immun. 61, 3863–3872. 15. Kuhn, H M., Brade, L., Appelmelk, B.J., Kusumoto, S., Riets- chel, E.T. & Brad e, H. (1992) Ch aracterization of the epitope specificity of murine monoclonal antibodies directed against lipid A. Infect. Immun. 60 , 2201–2210. 16. Brade, H., Brabetz, W., Brade, L., Holst, O., Lo ¨ bau, S., Lucakova, M., Mamat, U., Rozalski, A., Zych, K. & Kosma, P. (1997) Chl amydial lipopolysaccharide. J. Endotoxin Res. 4, 67–84. 1242 S. Mu ¨ ller-Loennies et al. (Eur. J. Biochem. 269) Ó FEBS 2002 . Chemical structure and immunoreactivity of the lipopolysaccharide of the deep rough mutant I-69 Rd – /b + of Haemophilus influenzae Sven Mu¨ ller-Loennies, Lore Brade and Helmut Brade Research. Medicine and Biosciences, Borstel, Germany From the lipopolysaccharide of the deep rough mutant I-69 Rd – /b + of Haemophilus influenzae two oligosaccharides were obtained after de-O-acylation and. leading to the induction of protective a ntibodies; and (d) the understanding of the biosynthesis of LPS may allow the distinct blockage of e ssential steps as a new strategy for the development of antibiotics