Eur J Biochem 269, 560±572 (2002) Ó FEBS 2002 N-Methylation in polylegionaminic acid is associated with the phase-variable epitope of Legionella pneumophila serogroup lipopolysaccharide Identi®cation of 5-(N,N-dimethylacetimidoyl)amino- and 5-acetimidoyl (N-methyl)amino-7-acetamido-3,5,7,9-tetradeoxynon-2-ulosonic acid in the O-chain polysaccharide Oliver Kooistra1, Edeltraud Luneberg2, Yuriy A Knirel1,3, Matthias Frosch2 and Ulrich Zahringer1 È È Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany; 2Institute for Hygiene and Microbiology, University of WuÈrzburg, Germany; 3N D Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia Previously, a phase-variable epitope was detected in the virulent wild-type strain RC1 of Legionella pneumophila serogroup subgroup OLDA using a lipopolysaccharidespeci®c monoclonal antibody, mAb 2625 [Luneberg, E., È Zahringer, U., Knirel, Y A., Steinmann, D., Hartmann, M., È Steinmetz, I., Rohde, M., Kohl, J & Frosch, M (1998) J Exp Med 188, 49±60] In the present study, an isogenic mutant strain, termed 5215, was constructed by deletion of genes involved in the biosynthesis of the mAb 2625 epitope Mutant 5215 was as virulent as the parental wild-type RC1 but did not bind mAb 2625 The two strains showed no di€erence in the core oligosaccharide and lipid A but in the O-chain polysaccharide structure, which is a homopolymer of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (a derivative of legionaminic acid) NMR spectroscopic studies revealed a hitherto unknown modi®cation of bacterial polysaccharides in the wild-type strain, namely N-methylation of the 5-acetimidoylamino group on a single legionaminic acid residue that is located, most likely, proximal to the core oligosaccharide Two major N-methylated substituents, the (N,Ndimethylacetimidoyl)amino and acetimidoyl(N-methyl) amino groups, could be allocated to the long- and middlechain O-polysaccharide species, respectively N-Methylation of legionaminic acid that was absent from the isogenic mutant 5215 and from the spontaneous phase variant 811, correlated with the presence of the mAb 2625 epitope Legionella pneumophila is a facultative intracellular parasite and the cause of legionellosis, a pneumonia with sometimes fatal progression [1] The reservoirs of legionellae are natural or man-made water systems and their natural hosts are various amoebae species [2] In the human lung L pneumophila invades and replicates within alveolar macrophages [3] The serogroup-speci®c antigens of the Gram-negative legionellae reside in the lipopolysaccharide (LPS) of the outer membrane [4,5] The chemical structure of L pneumophila serogroup (Sg) LPS has been extensively studied [6±12] (Fig 1) The O-chain polysaccharide (OPS) of the LPS is an a-(2 ® 4)-linked homopolymer of the 5-N-acetimidoyl7-N-acetyl derivative of 5,7-diamino-3,5,7,9-tetradeoxynon2-ulosonic acid, termed legionaminic acid [7] Initially, the D-glycero-L-galacto con®guration was ascribed to legionaminic acid [7], but this was later revised ®rst to the L-glycero -D-galacto con®guration [13,14] and, ®nally, to the D-glycero-D-galacto con®guration [15,16] Similarities in the biosynthesis pathway of legionaminic acid and neuraminic acid (5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2ulosonic acid) have been described previously [17] In strains belonging to the Pontiac group, e.g Philadelphia [5,18], polylegionaminic acid is quantitatively 8-O-acetylated [7,19], but in other Sg strains of the non-Pontiac group, including those of subgroup OLDA [5,18], it is only speci®cally 8-O-acetylated at a few legionaminic acid residues [12] In the Pontiac group, the 8-O-acetyl has been identi®ed as a part of the epitope of LPS-speci®c monoclonal antibodies mAb and mAb 3/1 [18,19], and the 8-Oacetyl transferase-encoding gene lag-1 has been described previously [20] In L pneumophila Sg LPS, the OPS is Correspondence to U Zahringer, Forschungszentrum Borstel, È Zentrum fur Medizin und Biowissenschaften, Parkallee 22, D-23845 È Borstel, Germany Fax: + 49 4537 188612, Tel.: + 49 4537 188462, E-mail: uzaehr@fz-borstel.de Abbreviations: LPS, lipopolysaccharide; OPS, O-chain polysaccharide; PS, polysaccharide; Sg, serogroup; GPC, gel-permeation chromatography; HMBC, heteronuclear multiple-bond correlation; DEPT, distortionless enhancement by polarization transfer; Kdo, 3-deoxyD-manno-oct-2-ulosonic acid; Rha, L-rhamnose; BYCE, bu€ered charcoal yeast extract Note: part of this study was presented at the 20th International Carbohydrate Symposium, Hamburg, Germany, August 13±September 1, 2000 (Received August 2001, revised 13 November 2001, accepted 16 November 2001) Keywords: N-methylation; lipopolysaccharide; O-chain polysaccharide; phase variation; Legionella pneumophila Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur J Biochem 269) 561 Fig Schematic representation of the L pneumophila serogroup lipopolysaccharide structure Adapted from [6±12,15,16,20] Sugar abbreviations: GlcN3N, 2,3-diamino-2,3-dideoxy-D-glucose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; Man, D-mannose; GlcNAc, 2-acetamido-2-deoxy-Dglucose (N-acetyl-D-glucosamine); QuiNAc, 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine); Rha, L-rhamnose; Leg and 4e-Leg, derivatives of legionaminic and 4-epilegionaminic acid, respectively OAc stands for O-acetyl group linked to a terminal nonreducing L-rhamnose residue (RhaII) of the core oligosaccharide [9,10] (Fig 1) The LPS core oligosaccharide lacks heptose and phosphate, contains abundant 6-deoxy sugars and N-acetylated amino sugars, and is highly O-acetylated [8±10,12] Lipid A of L pneumophila Sg consists of unusual long-chain and branched fatty acids [6,8], which may account for its low endotoxic potential [21±23] Recently, phase-variable expression of an LPS epitope of L pneumophila has been reported [24] The LPS phasevariant strain 811 was isolated from the virulent wild-type strain RC1 (Sg 1, subgroup OLDA), and found to be avirulent and serum sensitive [24] The altered LPS phenotype could be distinguished with the aid of the LPS-speci®c monoclonal antibody mAb 2625, which bound to wild-type RC1 but not to phase variant 811 Revertants from strain 811 retain all phenotypic characteristics of the wild-type [24] We have recently shown that phase variation in L pneumophila depends on chromosomal excision and insertion of an unstable 29-kb genetic element of presumably phage origin [25] The 29-kb sequence is located in a de®ned and conserved site within the chromosome of the virulent wild-type strain RC1 In contrast, in the phasevariant strain 811, the 29-kb element is excised from the chromosome and replicates as a high copy plasmid resulting in the avirulent phenotype [25] In another study, we characterized a 32-kb gene locus responsible for LPS biosynthesis in L pneumophila [17] Complementation of the spontaneous stable LPS mutant strain 137 that did not bind mAb 2625 was achieved with a gene from the avirulent parental wild-type strain 5097 The gene, designated ORF 8, was able to restore mAb 2625 binding and exhibited homologies to bacterial methyl transferase genes [17] Until now, the structure of the mAb 2625 epitope was not known, because in previous studies no structural difference could be detected between the LPS of wild-type RC1 and phase variant 811 [24] or between the LPS of the avirulent wild-type 5097 and the corresponding mutant 137 [17] As the mAb 2625 LPS epitope was found to be associated with alteration in virulence and serum resistance upon phase variation [24], we were interested in the elucidation of its chemical structure The aim of this study was to identify the mAb 2625 epitope and to correlate it to virulence For this purpose, the isogenic mutant 5215 was generated by deletion of the LPS biosynthesis operon ORF 8±12 in the virulent wild-type RC1 With the aid of this genetically de®ned mutant that did not bind mAb 2625, we were able to correlate the mAb 2625 epitope to N-methylation of the 5-acetimidoylamino group on legionaminic acid, a modi®cation that has not been found to date in bacterial polysaccharides Studies of binding of mAb 2625 to the N-methylated legionaminic acid derivatives is described in the accompanying paper [26] MATERIALS AND METHODS Bacterial strains and cultivation L pneumophila wild-type strain RC1 (Sg 1, subgroup OLDA) is a virulent patient isolate that binds mAb 2625 [17,24] The unstable phase-variant strain 811 derived from strain RC1 is avirulent and does not bind mAb 2625 [24] Phase variation is mediated by chromosomal insertion and excision of a 29-kb genetic element of presumably phage origin It is presumed that a regulatory factor is affected upon phase variation leading to numerous phenotypic alterations [25] L pneumophila wild-type strain 5097 (Sg 1, subgroup OLDA) was obtained from the American Type Culture Collection, Rockville, MD (ATCC 43109) Strain 5097 is not virulent (presumably due to long-term passage on arti®cial media) and binds mAb 2625 The spontaneous stable mutant strain 137 that does not bind mAb 2625 was derived from strain 5097 [17] A point mutation in ORF resulting in a frame shift with presumably polar effects is responsible for loss of the mAb 2625 epitope in mutant strain 137 [17] All strains were grown on buffered charcoal yeast extract (BCYE) agar with a-growth supplement (Merck) Construction of the L pneumophila ORF 8±12 mutant strain 5215 from wild-type strain RC1 For deletion of the ORF 8±12 operon, a 7150 bp HpaI±SpeI restriction fragment from pEL28 (covering position 456 of ORF to position 710 of ORF 13) was ligated into the HincII site of pBC From the resulting plasmid pMH546 562 O Kooistra et al (Eur J Biochem 269) Ó FEBS 2002 Fig Schematic depiction of plasmid constructs employed for generation of the ORF 8±12 mutant 5215 For detailed description see Materials and methods On top of the plasmids a schematic diagram of the 32-kb gene locus of L pneumophila wild-type RC1 required for LPS biosynthesis is depicted (adopted from [17]) The direction of the operons is indicated by arrowheads and designation of selected ORFs are shown above gene blocks (Fig 2) a 3-kb NsiI fragment (covering position 275 of ORF to position 399 of ORF 11) was deleted and a kanamycin resistance cassette was inserted inverse to the direction of transcription of ORFs 8±12 The kanamycin cassette was a 1282 bp EcoRI restriction fragment from plasmid pUC4K (Pharmacia) Orientation of the insertion was con®rmed by sequence analysis The resulting construct was termed pMH549 (Fig 2) The entire 5.5-kb insert of pHM549 was excised with NotI±ApaI (restriction sites of the multiple cloning site of pBC) and ligated into the EcoRV site of plasmid vector pLAW344 [27], resulting in plasmid pMH12 (Fig 2) pLAW344 harbours the sacB gene and allows counter-selection for homologous recombination pMH12 was introduced into L pneumophila strain RC1 by electroporation and bacteria were plated on BYCE agar supplemented with kanamycin (50 lgámL)1) Grown transformants were harvested, suspended in sterile distilled water, and plated on BCYE agar supplemented with kanamycin and 5% sucrose for selection of homologous recombination by double cross-over Homologous recombination was con®rmed by Southern blot and PCR analysis in the resulting mutant strain, termed 5215 Isolation and fractionation of LPS LPS from wild-type RC1, mutant 5215, and phase variant 811 was extracted from enzyme-digested cells by a modi®ed phenol/chloroform/petroleum ether procedure as described previously [6,7] The LPS yields from dry cells were 9, 16, and 6% (w/w), respectively Fractionation of the intact LPS by OPS chain-length was achieved by gel-permeation chromatography (GPC) in 10 mM Tris buffer (pH 8.0) containing mM EDTA, 0.25% (w/w) sodium deoxycholate, 0.2 M NaCl, and 0.02% (w/w) NaN3 as described previously [28] Brie¯y, mg LPS were dissolved under ultrasonication in 3.5 mL mM EDTA and 2% (w/w) sodium deoxycholate Non-soluble material was removed by centrifugation and discarded The sample was applied to a column (2.5 ´ 120 cm; Bio-Rad) of Sephacryl S-200 HR (Pharmacia) using the buffer mentioned above at a pump rate of 10 mLáh)1 and a differential refractometer for monitoring (Knauer) Fractions of  mL were collected SDS/PAGE and Western blot SDS/PAGE was carried out in 14% polyacrylamide gels using a Mini-Protean II system (Bio-Rad) LPS bands were visualized by the silver-staining technique described elsewhere [29] For extracted LPS samples, 100 lL stocksolution (2 mgámL)1) was mixed with 100 lL sample solution [30] and incubated at 95 °C for Then, 0.75 lL (0.75 lg LPS) of this solution was applied per lane For fractionated LPS, mL of each fraction was dried in an evaporation centrifuge, dissolved in 100 lL water, mixed with 100 lL sample solution [30], and incubated at 95 °C for The sample volume applied per lane varied from 0.25 to 2.5 lL, depending on the refraction index of the corresponding fraction in GPC For Western blot analysis of extracted LPS samples, lg LPS per lane were applied to 12.5% polyacrylamide gels The sample volume of fractionated LPS applied per lane in Western blot analysis varied from 0.75 to 7.5 lL and was three times as high as that in silver-stained SDS/PAGE Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur J Biochem 269) 563 Western blotting onto nitrocellulose ®lter membranes was carried out as described previously [31] Immunostaining was performed subsequently with mAb 2625 [24] or mAb LPS-1 (Progen) and alkaline phosphatase-conjugated goat anti-(mouse Ig) Ig (Dianova) [24] Chemical analysis GLC was performed with a Hewlett-Packard Model 5890 Series II instrument equipped with a 30-m capillary column of SPB-5 (Supelco) using a temperature gradient 150 ® 320 °C at °Cámin)1 Monosaccharides were analysed by GLC as the alditol acetates after hydrolysis with 0.1 M HCl for 48 h at 100 °C for neutral sugars [32] or with 10 M HCl for 30 at 80 °C for amino sugars [33] Following hydrolysis, sugars were dried, reduced with NaBH4, and acetylated with acetic anhydride in pyridine (30 min, 85 °C) Total hexosamine was determined by the Morgan±Elson reaction after acid hydrolysis (4 M HCl, 16 h, 100 °C) [34] 3-Deoxy-D-manno-oct-2-ulosonic acid (Kdo) was determined by the thiobarbituric acid assay according to the modi®ed method [35] Quanti®cation of total phosphate was carried out by the ascorbic acid method [36] Fatty acids of the lipid A portion were analysed by GLC and combined GLC-MS as the methyl esters after methanolysis (2 M HCl/MeOH, 24 h, 120 °C) and trimethylsilylation with N,O-bis-(trimethylsilyl)-tri¯uoroacetamide as described previously [6,37] Preparation, modi®cation, and fractionation of the OPS LPS each of wild-type RC1, mutant 5215, and phase variant 811 (750, 300, and 450 mg, respectively) was degraded at 100 °C for h with 0.1 M NaOAc/HOAc buffer (pH 4.4, 10 mgámL)1 LPS), and the resultant lipid A precipitate was removed by centrifugation (5000 g, 30 min) The supernatant was lyophilized and desalted by GPC on a column (2.5 ´ 50 cm; Bio-Rad) of Sephadex G-50 (S) (Pharmacia) using 50 mM pyridinium/acetate buffer (pH 4.3) and monitoring with a differential refractometer (Knauer) to give the corresponding polysaccharide (PS) portion PS was de-O-acetylated with 20% (v/v) aqueous NH4OH at 37 °C for 16 h (10 mgámL)1 PS); the following lyophilization and desalting as described above yielded PSNH4OH To obtain PSNH4OH devoid of core sugars, PSNH4OH was treated with 48% (v/v) aqueous hydro¯uoric acid (HF) at °C for days [38] (10 mgámL)1 PSNH4OH), the reagent was removed under a stream of nitrogen, and the resultant PSNH4OH/HF was desalted as above PSNH4OH/HF was fractionated by tandem GPC on two directly connected columns (2.5 ´ 120 cm each; Bio-Rad) of Toyopearl TSK HW-50 (S) (Supelco) and Fractogel TSK HW-40 (S) (Merck) Pyridinium/acetate (50 mM) buffer (pH 4.3) containing 10% (v/v) acetonitrile was used for elution at a pump rate of 15 mLáh)1 and a differential refractometer (Knauer) for monitoring Fractions of  mL were collected, appropriately combined into six pools, and lyophilized NMR spectroscopy H NMR, 2D 1H,1H NMR (COSY and NOESY with mixing time 300 ms), and H-detected 1H,13C NMR (HMQC and HMBC) spectra were recorded with a Bruker Avance DRX-600 spectrometer 13C NMR and DEPT-135 NMR spectra were recorded with a Bruker Avance DPX-360 spectrometer Standard Bruker software was used to acquire and process the NMR data Samples were lyophilized three times from 2H2O and measured in 2H2O (2H, 99.996%; Cambridge Isotope Laboratories) at 27 °C Chemical shifts were referenced to external acetone (dH 2.225 p.p.m.; dC 31.45 p.p.m.) RESULTS Construction and characterization of the ORF 8±12 mutant strain 5215 from wild-type strain RC1 Genes involved in formation of the LPS epitope bound by mAb 2625 were identi®ed on a LPS biosynthesis gene cluster and assigned to the ORF 8±12 operon [17] ORF showed homologies to bacterial methyl transferases [39,40], ORF exhibited sequence similarities to Neu5Ac condensing enzymes (e.g SiaC of Neisseria meningitidis [41]), and ORF 11 showed vague homologies to plant methyl transferases [42] The proteins encoded by the remaining ORFs 10 and 12 showed no homologies to known aminoacid sequences The only ORF 8±12 mutant accessible was mutant 137 derived from the avirulent wild-type 5097 Therefore, the isogenic ORF 8±12 deletion mutant 5215 was constructed from wild-type RC1 by homologous recombination (Fig 2) in order to compare the LPS structures of wild-type RC1, the genetically de®ned mutant 5215, and the phase variant 811 Unexpectedly, virulence and serum resistance were not affected in mutant 5215, when compared to the parental wild-type RC1 (data not shown), which revealed that the mAb 2625 LPS epitope itself is not associated with virulence of L pneumophila strain RC1 Characterization of LPS from wild-type RC1 and mutant 5215 In silver-stained SDS/PAGE (Fig 3A), LPS from wild-type RC1 and mutant 5215 displayed no difference in banding pattern Both LPS showed a characteristic bimodal distribution by OPS chain-length In Western blot analysis, LPS from mutant 5215 did not bind to mAb 2625 (Fig 3B) but bound to mAb LPS-1 in a different manner compared to LPS from wild-type RC1 (Fig 3C) MAb LPS-1 is known to recognize speci®cally a conserved epitope located in the outer core region of the LPS from L pneumophila Sg strains [12,24,43] Chemical analyses of both wild-type RC1 and mutant 5215 LPS revealed L-rhamnose, D-mannose, Kdo, and amino sugars, i.e total hexosamine according to the Morgan±Elson reaction after acid hydrolysis (total of 2-amino-2-deoxy-D2,3-diamino-2,3-dideoxy-D-glucose, glucose, and 2-amino-2,6-dideoxy-D-glucose), in the molar ratios of  : : : According to GLC data, the ratio of 2-amino-2-deoxy-D-glucose and 2-amino-2,6-dideoxy-Dglucose was the same in the LPS from wild-type RC1 and mutant 5215 Comprehensive analysis by NMR spectroscopy and MALDI-TOF MS revealed no difference in composition and structure of the core oligosaccharides isolated from wild-type RC1 and mutant 5215 [12] NMR spectroscopy revealed that the lipid A backbone is a 564 O Kooistra et al (Eur J Biochem 269) Ó FEBS 2002 Fig Silver-stained SDS/PAGE (A) and Western blot with mAb 2625 (B) and mAb LPS-1 (C) of L pneumophila LPS from wild-type RC1 (lane 1) and mutant 5215 (lane 2) In A, 0.75 lg LPS per lane was applied, and lg LPS in B and C 1,4Â-bisphosphorylated (1 đ 6)-linked disaccharide of 2,3-diamino-2,3-dideoxy-D-glucose in both wild-type RC1 and mutant 5215 [44] Fatty acid composition of lipid A of both strains was almost identical as well [44] Investigations of the core oligosaccharide [12] and the lipid A backbone [44] of the LPS from phase variant 811 revealed that both were unchanged compared to wild-type RC1 However, lipid A of phase variant 811 contained 3-hydroxylated fatty acids of different chain-length compared to lipid A of wild-type RC1 [44] Fig Fractionation of LPS from L pneumophila wild-type RC1 by gel-permeation chromatography on Sephacryl S-200 HR Part of refraction index elution pro®le of LPS (A) Silver-stained SDS/PAGE (B) and Western blot with mAb 2625 (C) and mAb LPS-1 (D) of fractionated LPS The dotted lines in the top panel indicate fractions and the dashed lines in the lower panels indicate the borders of di€erent, appropriately aligned SDS/polyacrylamide gels or Western blots Fractionation of LPS by GPC To determine the location of the mAb 2625 epitope, LPS from wild-type RC1 and mutant 5215 was fractionated by GPC on Sephacryl S-200 HR The refraction index elution pro®le of the GPC of wild-type RC1 LPS (Fig 4A) indicated the same characteristic bimodal distribution of long and short O-chain LPS species as in SDS/PAGE (Fig 3A) A similar pro®le showing a low amount of long O-chain LPS species and a relatively high amount of short O-chain LPS species was observed for mutant 5215 LPS (Fig 5A) Silver-stained SDS/PAGE after GPC of the wildtype RC1 LPS revealed 33 fractions (nos 96±128) containing LPS species with different OPS chain-lengths (Fig 4B) A ladder-like pattern was observed with differences of up to 10 sugar residues within each fraction and with overlapping about 6±8 residues in each pair of neighbouring fractions In Western blot analysis, mAb 2625 bound only to the wild-type LPS species from fractions 96±116, and did not bind to LPS molecules below a certain size, i.e a certain OPS chain-length (Fig 4C) In contrast, mAb LPS-1 exclusively bound to the LPS species from fractions 113±126, and did not bind to LPS molecules above a certain size (Fig 4D) This rather sharp margin clearly Fig Fractionation of LPS from L pneumophila mutant 5215 by gelpermeation chromatography on Sephacryl S-200 HR Part of refraction index elution pro®le of LPS (A) Western blot with mAb 2625 (B) of fractionated LPS The dotted lines in the top panel indicate fractions and the dashed lines in the lower panel indicate the borders of di€erent, appropriately aligned Western blots showed that the mAb 2625 epitope is only present or accessible in LPS species having a speci®c OPS chain-length of  15 and more residues of legionaminic acid Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur J Biochem 269) 565 As followed from Fig 3, all LPS-containing fractions (nos 96±128) from mutant 5215 bound mAb LPS-1 (Fig 5B) Preparation and characterization of OPS An OPS attached to the core oligosaccharide (PS) was prepared by mild acid hydrolysis of LPS from each strain (wild-type RC1, mutant 5215, and phase variant 811) to cleave the lipid A moiety followed by GPC on Sephadex G-50 (S) Based on the ®nding that the OPS is linked to the terminal L-rhamnose residue of the LPS core oligosaccharide (RhaII) [9] and the observation that 48% aqueous HF selectively cleaves the glycosidic linkage of 6-deoxy sugars in polysaccharides [38], a protocol was elaborated to remove core oligosaccharide constituents from PS When the intact LPS was treated with 48% aqueous HF and separated by SDS/PAGE, Western blot analysis revealed that although the OPS of the LPS was partially cleaved, the mAb 2625 epitope in the remaining LPS species was not affected After de-O-acetylation of the LPS, the HF treatment completely cleaved the OPS of the LPS In this case, silver-stained SDS/PAGE showed only low-molecular-mass molecules resembling rough-type LPS, which did not react with mAb 2625 in Western blot In contrast to the glycosidic linkage of 6-deoxy sugars, e.g L-rhamnose [38], that of legionaminic acid and its N-linked substituents are stable under the same conditions [14] Therefore, PS was de-O-acetylated, treated with 48% aqueous HF, and the resultant modi®ed polysaccharide (PSNH4OH/HF) was fractionated by tandem GPC on Toyopearl TSK HW-50 (S) and Fractogel TSK HW-40 (S) to separate long-, middle-, and short-chain OPS species The middle-chain species were present in a low amount (e.g see pool III in Fig 6) Only a negligible difference in the elution pro®les of PSNH4OH/HF from wild-type RC1, mutant 5215, and phase variant 811 was observed Fig Fractionation of OPS (PSNH4OH/HF) from L pneumophila wildtype RC1 by tandem gel-permeation chromatography on Toyopearl TSK HW-50 (S) and Fractogel TSK HW-40 (S) Pools I, III, and V correspond to long-, middle-, and short-chain PSNH4OH/HF, respectively; pools II, IV, and VI correspond to intermediate fractions OPS from each strain separated into pools I to VI (Fig 6) were investigated by 1H NMR spectroscopy The 1H NMR spectra (only pools I, III, and V are shown in Fig 7) indicated that legionaminic acid with its major substituents, 5-N-acetimidoyl and 7-N-acetyl groups (Fig 8, structure 1) remained intact (see also Tables and 2) The only anomeric proton signals present in the spectra were those of two L-rhamnose residues (RhaI H1 dH 5.06 and RhaII H1 dH 4.99; Fig 9A±C), which was in accordance with identi®cation of only L-rhamnose by GLC analysis of the alditol acetates derived from PSNH4OH/HF Therefore, the isolated PSNH4OH/HF species from all three strains were composed of legionaminic acid and L-rhamnose, whereas the major portion of the core oligosaccharide was cleaved by HF treatment A 1H,13C HMQC experiment demonstrated that PSNH4OH/HF contained at the reducing end either an Rha disaccharide ® 3)-a-L-RhaII-(1 ® 3)-L-RhaI ( 70%) or a single Rha residue ® 3)-L-RhaII ( 30%), in both cases the reducing Rha residue being predominantly, but not exclusively, a-con®gured The disaccharide was identi®ed by the H1 chemical shift of RhaII (compare published data [9]) and a characteristic down®eld displacement of the C1 signal from dC 95.17 in nonlinked Rha to dC 103.21 in a-linked RhaII (compare to published data in [45]) Identi®cation of N-methylated derivatives of legionaminic acid N-Methyl groups in bacterial polysaccharides occur rarely [46] and published data are only scarce Therefore, careful NMR spectroscopic analysis was used to elucidate the structure of N-methylated derivatives of legionaminic acid Comparison of the 1H NMR spectra of the OPS of pool III from all three investigated strains revealed four signals between 2.9 and 3.3 p.p.m (all singlets; Fig 9, panels A and D), whose presence correlated with the reactivity of mAb 2625 with LPS in Western blot The signals were observed in long- and middle-chain OPS from wild-type RC1, but in no OPS from mutant 5215 (Fig 9, panels B and E) In phase variant 811, these signals were recognized in the OPS of the same pools as in wild-type RC1 but were 10- to 20-fold less intense (Fig 9, panels C and F) Except for the region of the four signals, the 1H NMR spectra of the polysaccharides from all three strains were almost identical (compare Fig 9, panels A±C) The middle-chain OPS of pool III from wildtype RC1 having 15±20 residues of legionaminic acid was the smallest one that displayed these signals and, moreover, the relative intensity of these signals was the highest compared to other pools from the same strain Therefore, further structural investigation was performed with this preparation The 1H NMR chemical shifts and the grouping of the signals in pairs (one pair at dH 3.30 and 3.19, and the other at dH 3.03 and 2.95; see below) resembled those of N-methyl groups in stereoisomers of N-acetimidoyl-N-methylglycine (N-acetimidoylsarcosine) at dH 3.70 (E) and dH 3.60 (Z) [47] The corresponding 13C NMR chemical shifts (one pair at dC 42.95/42.89 and 40.74/40.60, and the other at dC 33.73 and 32.56, respectively) and a DEPT-135 NMR experiment con®rmed N-linked methyl groups The 1H,13C HMQC experiment revealed also a third, minor pair of signals at dH 2.97 and 2.94 and dC 30.93 and 29.86, which were partially 566 O Kooistra et al (Eur J Biochem 269) Ó FEBS 2002 Fig 1D 1H NMR spectra of the long-, middle-, and short-chain PSNH4OH/HF (pools I, III, and V; panels A±C) from wild-type RC1 The integral traces for selected signals with the corresponding integration values referenced to the anomeric proton signal of RhaII (H1 dH 4.99, integration value set at 0.7) in the ® 3)a-L-RhaII-(1 ® 3)-L-RhaI disaccharide are superimposed on the spectra The insets show the region between 2.8 and 3.4 p.p.m extended 5-fold Bold numbers refer to structures shown in Fig For abbreviations see legend to Fig superimposed on the signals of the major higher-®eld pair in the 1H NMR spectrum (Fig 10, right panel) In the 1H,13C HMBC spectrum (Fig 10, left panel), the proton signals of the major lower-®eld pair at dH 3.30 and 3.19 cross-correlated to the lower-®eld carbon signals at dC 40.74/40.60 and 42.95/42.89 Furthermore, both proton signals correlated to the signals of the same nonprotonated carbon (dC 167.03) and the same C-methyl group (dC 16.63) of an N-acetimidoyl group In a NOESY experiment, the two N-methyl signals correlated to each other These data suggested that both N-methyl groups are linked to the same nitrogen of an N-acetimidoyl group, i.e that an (N,N-dimethylacetimidoyl)amino group is present (Fig 8, structure 2) This group is linked to C5 of a legionaminic acid residue, which is substituted with a nonmethylated N-acetimidoyl group in the other legionaminic acid residues of the OPS The proton signals of the major higher-®eld pair at dH 3.03 and 2.95 correlated in the 1H,13C HMBC spectrum to different nonprotonated carbon signals at dC 169.02 and dC 168.23 and different C-methyl carbon signals at dC 20.49 and dC 20.21 of another N-acetimidoyl group, respectively (Fig 10, left panel) In addition, each proton signal correlated to a signal of a nitrogen-bearing carbon (C5) of legionaminic acid (dC 55.93 and 57.32) Taken these data together, it was concluded that there is present a 5-acetimidoyl(N-methyl)amino group that occurs as two stereoisomers (Fig 8, structure 3) An N-methylacetamido group could be excluded based on identi®cation, using 2D NMR experiments, of a nonmethylated 7-acetamido group of this particular legionaminic acid residue (Table 2) A methylamino group was excluded based on published data of the methylamino derivative of L-fucose in the LPS of Bordetella pertussis strain 1414 [48,49], in which the N-methyl group gave a single singlet in the 1H NMR spectra Similarly, the minor pair of signals at dH 2.97 and 2.94 was assigned to a 5-(N-methylacetimidoyl)amino group A NOESY experiment (Fig 11) was applied to stereochemical analysis of the N-methylated acetimidoylamino (acetamidine) groups in and 3, which may occur as stereoisomers due to a partial double-bond character of the linkages at both nitrogens A strong NOE correlation was observed between the lower-®eld signal from each major pair of the N-methyl signals (dH 3.30 in and dH 3.03 in 3) and the corresponding C-methyl signals of the N-acetimidoyl group, but only a weak or no cross-peak could be detected for the higher-®eld signal (dH 3.19 in and dH 2.95 in 3) Hence, the lower- and higher-®eld 1H NMR signals of Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur J Biochem 269) 567 Fig Structures of 5-acetimidoylamino-7-acetamido-3,5,7,9-tetradeoxy-D-glycero-D-galacto-non-2-ulosonic acid (5-N-acetimidoyl-7-Nacetyllegionaminic acid, 1) and its N-methylated derivatives, 5-N-(N,N-dimethylacetimidoyl)-7-N-acetyllegionaminic acid (2-E) and two stereoisomers of 5-N-acetimidoyl-7-N-acetyl-5-N-methyllegionaminic acid (3-E and 3-Z) belonged to the N-methyl groups at N1 in trans and cis orientation to N2 of the acetamidine group, respectively (Fig 8; the descriptors cis and trans for the two N-methyl groups in are used only to designate their positions relative to N2 and not refer to stereoisomerism) The lower-®eld H NMR signal of belonged to the N-methyl group at N2 in the E isomer and the higher-®eld signal to that in the Z isomer, in which N1 is in trans and cis orientation to C5 of legionaminic acid, respectively (Fig 8) Like 3, may theoretically occur as two stereoisomers, E and Z, with regard to the partial double bond at N2, while the NMR spectra showed the presence of only one isomer Molecular modelling data (not shown) suggested that this isomer is 2-E with trans orientation of N1 to C5 as 2-Z would be sterically hindered by interaction of one of the N-methyl groups at N1 (that in cis orientation to N2) with the pyranose ring of legionaminic acid The N-methyl signal in gave strong NOE correlations to three more proton signals, which were assigned to H4, H7, and either H6 or, less likely, H8 of legionaminic acid The assignment was performed by correlations between signals for H4 (dH 3.33 E, dH 3.37 Z) and H3ax,3eq (axial: dH 1.86 E, dH 1.89 Z, equatorial: dH 2.48 E, dH 2.51 Z) in COSY, and for H7 (dH 3.68 E, dH 3.75 Z) and CˆO of the 7-acetamido group (dC 175.91 E, dC 175.74 Z) in the 1H,13C HMBC experiment In addition, signals for H9 (dH 1.17 E, dH 1.16 Z) were found by a weak NOE correlation with the N-methyl group, whereas signals for H6 and H8 appeared to coincide (dH 4.19 E, dH 4.13 Z) A signal for H5 of could not be reliably identi®ed, most likely, owing to its coincidence with the H5 signal of the major, non-N-methylated derivative The NOESY data suggested that in the predominant conformation of both 3-E and 3-Z the N-methyl group at N2 has an axial (or close to axial) orientation related to the pyranose ring of legionaminic acid and lies on or close to a plane formed by H4, H6, and H7 In contrast, in no NOE correlation was observed between the N-methyl groups and any proton of legionaminic acid, evidently owing to the remoteness of the N-methyl groups at N1 from the pyranose ring Long-, middle-, and short-chain PSNH4OH/HF from wildtype RC1 were investigated by 1D 1H NMR spectroscopy and signal integration was performed to calculate the average chain-length of the PSNH4OH/HF and the distribution of N-methylated legionaminic acid derivatives The signal of a-L-RhaII H1 (dH 4.99) in the ® 3)-a-L-RhaII-(1 ® 3)-LRhaI disaccharide was used as a reference, because it was the Table 1H and 13C NMR chemical shifts for N-methylated acetimidoylamino groups in pool III of the OPS from wild-type RC1 NMe, N-methyl group; NAmCH3 and NAmCˆN, C-methyl group and nonprotonated carbon of the 5-acetimidoylamino group, respectively ND, not determined Derivative of legionaminic acid dH (p.p.m.) 5-N-Acetimidoyl (1) 5-N-(N,N-Dimethylacetimidoyl) (2-E) 5-N-(N-Methylacetimidoyl) (minor) 5-N-Acetimidoyl-5-N-methyl (3) dC (p.p.m.) 5-N-Acetimidoyl (1) 5-N-(N,N-Dimethylacetimidoyl) (2-E) 5-N-(N-Methylacetimidoyl) (minor) 5-N-Acetimidoyl-5-N-methyl (3) NAmCH3 NMe 3.19 (cis) 2.97 (E) 2.95 (E) 3.30 (trans) 2.94 (Z) 3.03 (Z) 40.74 (cis) 40.60 (cis) 30.93 (E) 32.56 (E) 42.95 42.89 29.86 33.73 (trans) (trans) (Z) (Z) NAmCˆN 2.24 2.19 2.25 (E) 2.30 (E) ND 2.19 (Z) 20.08 16.63 ND 20.21 (E) 167.61 167.03 ND 167.22 (E) 20.49 (Z) 168.23 (E) 167.05 (Z) 169.02 (Z) 568 O Kooistra et al (Eur J Biochem 269) Ó FEBS 2002 Table 1H and 13C NMR chemical shifts for and in the OPS pool III from wild-type RC1 NAcCH3 and NAcCˆO, C-methyl group and nonprotonated carbon of the 7-acetamido group, respectively ND, not determined Derivative of legionaminic acid 5-N-Acetimidoyl -7-N-acetyl (1) 5-N-Acetimidoyl (E) -7-N-acetyl-5-N- (Z) methyl- (3) 5-N-Acetimidoyl -7-N-acetyl (1) 5-N-Acetimidoyl (E) -7-N-acetyl-5-N- (Z) methyl- (3) d (p.p.m.) H3ax 1.61 H3eq 2.62 H4 4.02 H5 3.53 H6 3.95 H7 3.89 H8 3.89 H9 1.20 NAcCH3 2.08 1.86 1.89 2.48 2.51 3.33 3.37 ND ND 4.19 4.13 3.68 3.75 4.19 4.13 1.17 1.16 2.17 ND C1 174.47 C2 101.86 C3 39.40 C4 71.86 C5 54.30 C6 72.76 C7 55.53 C8 67.92 C9 19.65 NAcCH3 NAcCˆO 23.23 175.32 ND ND ND ND 32.04x 31.86 72.66 71.23 57.32 55.93 68.41 68.37 55.01 54.94 68.41 68.37 19.44 19.41 23.71 23.67 only anomeric proton present in a single anomeric con®guration Integration of the signals of 1D 1H NMR spectra (Fig 7) indicated that the average chain-length of long-, middle-, and short-chain PSNH4OH/HF (Fig 6, pools I, III, and V) is about 40, 18, and 10 legionaminic acid residues The ratio of the 5-N-(N,N-dimethylacetimidoyl)-7-N-acetyl and 5-N-acetimidoyl-5-N-methyl-7-N-acetyl derivatives of legionaminic acid was : in long-chain and : in middle-chain PSNH4OH/HF, respectively Based on the relative intensities of the proton signals it was concluded that 175.91 175.74 only one legionaminic acid residue is N-methylated in each polysaccharide chain above a speci®c length DISCUSSION Phase variation in L pneumophila has dramatic effects on the virulence of the bacteria in various in vitro and in vivo models Phase variation can be monitored with the aid of mAb 2625 that is speci®c for an epitope in the LPS of L pneumophila Sg subgroups OLDA and Oxford The Fig 600-MHz 1H NMR spectra of pool III of the OPS (PSNH4OH/HF) from three L pneumophila strains Left panel: the full spectra of pool III from wild-type RC1 (A), mutant 5215 (B), and phase variant 811 (C) Assignment was made using 2D NMR experiments and published data [7] Right panels: part of spectra of pool III from wildtype RC1 (D), mutant 5215 (E), and phase variant 811 (F) NMecis and NMetrans, N-methyl groups in 2-E (the descriptors cis and trans designate the positions of the N-methyl groups relative to N2); NMe, N-methyl group in the stereoisomers 3-E and 3-Z; NAcCH3 and NAmCH3 , C-methyl groups of 7-acetamido and 5-acetimidoylamino substituents, respectively Bold numbers refer to structures shown in Fig Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur J Biochem 269) 569 Fig 10 Sections of 2D 1H,13C HMBC (left panel) and HMQC (right panel) spectra of pool III of the OPS (PSNH4OH/HF) from L pneumophila wildtype RC1 Spectra are aligned in F1 dimension The corresponding 13C and 1H NMR spectra are displayed along F1 and F2 axes Spectra were recorded at 600 MHz and 27 °C For abbreviations see legend to Fig Cross-peaks marked by   belong to an unknown minor isomer of legionaminic acid Fig 11 Part of a NOESY spectrum of pool III of the OPS (PSNH4OH/HF) from L pneumophila wild-type RC1 The spectrum was recorded at 600 MHz and 27 °C using a mixing time 300 ms For abbreviations see legend to Fig Cross-peaks marked by   belong to an unknown minor isomer of legionaminic acid 570 O Kooistra et al (Eur J Biochem 269) mAb 2625 epitope is present in wild-type cells and is lost in the avirulent switch mutant upon phase variation [24] The loss of the reactivity with mAb 2625 affects the epitope of another LPS-speci®c monoclonal antibody, mAb LPS-1, which is speci®c for the outer core region of the LPS from L pneumophila Sg strains [12,24,43] In Western blot, the mAb LPS-1 binding pattern of LPS from wild-type RC1 was different from that of phase variant 811 A similar alteration has been observed for LPS from the spontaneous mutant 137 derived from wild-type strain 5097 [17] LPS of the mutants did not bind to mAb 2625, but showed a stronger binding to mAb LPS-1 compared to the corresponding wild-type LPS It was suggested that an alteration in the LPS structure enhances the accessibility of the mAb LPS-1 epitope and that the altered structure may be located in the core oligosaccharide [24] However, the latter could not be con®rmed by chemical studies Comprehensive analysis, using NMR spectroscopy and MALDI-TOF MS, of the core oligosaccharides from all three strains (wild-type RC1, mutant 5215, and phase variant 811) revealed no structural deviation from each other and from the strain Philadelphia core oligosaccharide studied earlier [9,10,12] Fractionation of LPS by GPC and subsequent Western blot analysis enabled us to determine the location of the mAb 2625 epitope in the long and middle O-chain LPS species of wild-type strain RC1 Remarkably, mAb LPS-1 bound only to LPS species lacking the mAb 2625 epitope, thus suggesting that the mAb 2625 epitope interferes with the binding of mAb LPS-1 The OPS of L pneumophila Sg is a polylegionaminic acid (Fig 8, structure 1), which could not be depolymerized to monomers [7] Therefore, detailed NMR spectroscopic studies were performed with selectively degraded polysaccharides fractionated by tandem GPC Comparative analysis of the fractionated OPS from wild-type RC1 and mutant 5215 revealed minor components, which occurred only in the wild-type polysaccharide species above a speci®c chain-length and whose presence correlated with the reactivity of mAb 2625 with LPS in Western blot The minor components were identi®ed as 5-N-(N,Ndimethylacetimidoyl)-7-N-acetyllegionaminic acid (2) and 5-N-acetimidoyl-7-N-acetyl-5-N-methyllegionaminic acid (3) In addition, 5-N-(N-methylacetimidoyl)-7-N-acetyllegionaminic acid was detected in a negligible amount, which could either be a biosynthetic precursor or a degradation product of No N-methylated acetimidoylamino group has been hitherto found in bacterial polysaccharides Only one N-methylated legionaminic acid residue is present in each polysaccharide chain above a certain length This fact may be the reason for the failure to identify the N-methylated derivatives in previous studies of the nonfractionated OPS of L pneumophila using 13C NMR spectroscopy [8] Investigation of binding af®nities by surface plasmon resonance biomolecular interaction analysis revealed that only long- and middle-chain PSNH4OH/HF from wild-type RC1 bind to mAb 2625 but not short-chain PSNH4OH/HF The same holds true for any PSNH4OH/HF from phase variant 811 or mutant 5215 [26] As binding to mAb LPS-1 is dependent on O-acetylation [12], de-O-acetylated PSNH4OH/HF does not bind to mAb LPS-1 at all Ó FEBS 2002 However, interference of the mAb 2625 epitope with binding of the core-speci®c mAb LPS-1 in Western blot suggested that the N-methylated legionaminic acid residue is located in the OPS close to or, most likely, next to the core, i.e is linked to the terminal L-rhamnose residue (RhaII) of the core oligosaccharide of long- and middle-chain LPS species Unfortunately, this link could not be proved directly, as no interresidue NOE correlation could be observed between any proton of either or and RhaII A lack of the mAb LPS-1 epitope due to a core modi®cation can be excluded as no such modi®cation was found by detailed structural studies of the puri®ed core oligosaccharides from wild-type RC1, mutant 5215, and phase variant 811 [12] Therefore, there is a strong indication for the location of the N-methylated legionaminic acid residue proximal to the LPS core oligosaccharide, though its location at any other position of the OPS could not be strictly excluded The expression of the N-methyl-associated epitope is suppressed in the phase variant 811 The presence of 10- to 20-fold lower amounts of and in strain 811 could be due to the presence of a 10% portion of wild-type revertant cells in the phase-variant population [24] As mutant 5215 is as virulent as wild-type RC1 but completely lacks the N-methylated derivatives of legionaminic acid, it can be excluded that the N-methyl groups are determinants of virulence More likely, different phenotypic alterations may occur upon phase variation and the loss of the mAb 2625 epitope is one of them Phase variation in L pneumophila was found to in¯uence other LPS biosynthesis pathways involved in assembly of lipid A Phase variant 811 expressed a different fatty acid pro®le as compared to wild-type RC1 and mutant 5215 [44] Although the characteristic secondary long-chain fatty acids, such as 28:0(27-oxo) and 27:0(1,27dioic), are present in all three strains, positions and 2¢ of the lipid A backbone are predominantly occupied by 16:0(3-OH) and 18:0(3-OH) in avirulent phase variant 811 but by 20:0(3-OH) in the two virulent strains [44] Similar studies of avirulent wild-type 5097 and the derived spontaneous mutant 137 revealed N-methylation in the OPS of the wild-type but not of the mutant strain In mutant 137, a point mutation inactivated the ORF gene encoding methyl transferase [17], which is probably, at least in part, responsible for N-methylation N-Methyl groups were also found in the OPS from strain Philadelphia 1, in which their 1H NMR chemical shifts are in¯uenced by 8-O-acetylation of the N-methylated legionaminic acid residue [12] Interestingly, the 8-O-acetyl group masks the mAb 2625 epitope, which has become accessible to the antibody only after chemical de-O-acylation of the LPS [12] Previously, at least four epitopes have been described on the de-O-acetylated LPS from strain Philadelphia 1, which were recognized by monoclonal antibodies produced against non-Philadelphia strains of L pneumophila Sg [19] The N-methyl groups are presumably present in all and unique to Sg strains of L pneumophila, as the ORF 8±12 operon occurs in Sg strains, but neither in strains from other L pneumophila serogroups, nor in non-pneumophila strains [17] Although present as minor components, the N-methylated residues of legionaminic acid contribute to the immunospeci®city of L pneumophila The investigation of binding of mAb 2625 to the N-methylated legionaminic acid Ó FEBS 2002 N-Methylation in Legionella pneumophila LPS (Eur J Biochem 269) 571 derivatives is described in the accompanying paper [26] Being hydrophobic and having a signi®cant electronic effect, the N-methyl groups may adjust the appropriate hydrophobicity and the appropriate charge to the legionaminic acid residue, which intervenes between the highly hydrophobic outer core region of the LPS and the OPS having a zwitterionic character Another putative function of the N-methylated derivatives may be related to the fact that phase variation is mediated by a genetic element of possibly phage origin The N-methyl groups located close to the core oligosaccharide may play a role of a signal or a receptor for phage particles, though no bacteriophage has been yet described for L pneumophila However, the most interesting question is in which way the different phenotypic alterations that occur upon phase variation in L pneumophila might be connected Our current hypothesis is that phase variation affects a regulatory factor, which in¯uences LPS biosynthesis, virulence, and serum resistance Future studies will focus on the elucidation of such factor and the mechanism of its ef®cacy ACKNOWLEDGEMENTS We thank Dr B Lindner and Mrs H Luthje for performing MALDIÈ TOF MS, H.-P Cordes for running NMR spectra, and H Moll for expert help with GLC-MS The skilful technical assistance of Mrs K Jakob is gratefully acknowledged This work was supported by grants from the Deutsche Forschungsgemeinschaft, LU 514/2±2 (E L and M F.) and ZA 149/3±2 (U Z.), grant 436 RUS 113/314/0 from the Deutsche Forschungsgemeinschaft (Y A K and U Z.), and grant 00-04-04009 from the Russian Foundation for Basic Research (Y A K.) 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