INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Like other members of the family Enterobacteriaceae, the lipopolysaccharides (LPSs)¹ of Klebsiella pneumoniae consist of three structural domains, (i) the hydrophobic lipid A, which is a major component of the outer leaflet of the Gram-negative outer membrane, and (ii) the core oligosaccharide, which is linked to lipid A and provides the attachment site for (iii) the long chain polysaccharide (O antigen; O chain). Typically, structural diversity is greater in the regions of LPS extending from the cell surface (i.e. the O chains). Varying chemical structures in the O chains gives rise to a number of serologically distinct O antigens. In the Klebsiellae there are 11 known O-chain structures, but structural similarities lead to some serological cross-reactivities, so the actual number of unique O serotypes is less (1, 2). Several of the O-antigens are based on a structure designated D-galactan I with a repeat unit comprising [-3)--Galf-(1-3)--Galp-(1-].² When present alone, this structure provides the 2a antigen (3), but it can be capped by additional structural domains or modified by side chain acetyl or galactosyl residues to generate additional unique antigens (3-6). For example, in the most clinically prevalent serotype, the O1 antigen, D-galactan I chains are capped by a domain with a different repeat unit structure, [-3)--Galp-(1-3)--Galp-(1-] (D-galactan II). Genetic (7) and chemical (8, 9) analyses indicate that D-galactan I chains are linked directly to the lipid A core structure, whereas D-galactan II is confined to the distal end of some of the available D-galactan I chains (10). D-Galactan II provides the epitope(s) that defines the O1 antigen (9), and its presence is required for the resistance of the bacteria to complement-mediated killing in the host; K. pneumoniae mutants that only produce D-galactan I are therefore serum-sensitive (11, 12). However, not all Klebsiella O serotypes are based on D-galactan I. The prevalent O3 and O5 serotypes comprise mannan O chains with structures identical to the Escherichia coli O9 and O8 antigens, respectively (13, 14). The remaining Klebsiella O-chain structures are heteropolymers.

The biosynthesis of the polysaccharide O chain has been investigated in some serotypes of K. pneumoniae by biochemical and genetic experimental approaches. Much of the data for the O3 and O5 serotypes is derived from the related equivalent systems in E. coli. However, the genetic loci required for synthesis of the corresponding O chain structures in E. coli (O8_ec and O9_ec) and K. pneumoniae (O5_kp and O3_kp, respectively) are essentially identical and reflect lateral gene transfer events (15, 16). Biosynthesis data is also available for D-galactan I (17, 18). These polymannans and polygalactans are polymerized by a processive glycosyltransfer mechanism, i.e. sequential addition of monosaccharides to the non-reducing end of the nascent chain. Polymer growth occurs on a "primer" (or acceptor) consisting of undecaprenyl pyrophosphoryl (und-PP)-linked GlcNAc. This compound is formed by WecA, a UDP-GlcNAc:undecaprenyl phosphate GlcNAc-1-phosphate transferase that forms und-PP-GlcNAc (19-21). Specific mannosyltransferases or galactosyltransferases then extend the und-PP-GlcNAc acceptor (17, 18, 21). The glycosyltransferase reactions occur at the inner face of the cytoplasmic membrane, and once chain extension is complete, the nascent O chain is transferred across the cytoplasmic membrane by an ABC (ATP binding cassette) transporter (21, 22). At the periplasmic face of the membrane, the O chain is transferred from the lipid intermediate to preformed lipid A core by the activity of the ligase enzyme waaL to complete the LPS molecule (for review, see Refs. 23 and 24).

A crucial open question in the biosynthesis of these O chains is the mechanism(s) by which the chain length is modulated. Examination of LPS molecules isolated from these bacteria shows a typical "modal" (i.e. restricted) distribution of O-chain lengths. The mechanism of LPS O-chain length determination is crucial for the biology of these organisms because it contributes to resistance to complement-mediated serum killing. The O5_kp and O8_ec antigens have been reported to contain 3-O-methyl-D-mannose at their non-reducing termini (13), leading to speculation that terminal structural elements may act as terminating signals for chain extension (24). Other serotypes have not been investigated.

Recently we have found that polysaccharide O chain can be detached from lipid A of K. pneumoniae LPS by treatment with nitrous acid, which destroys the glycosidic linkage of the -GlcN residue, present in the core oligosaccharide (25) (Fig. 1). The polysaccharides thus obtained were short enough to allow direct structural determination of the end groups by NMR spectroscopy. Here we present data of the analysis of the end groups in the nitrous acid-released O chains from K. pneumoniae serotypes O1, O2a, O2a,c, O3, O4, O5, and O12. Most of these O chains have non-reducing terminal modifications, suggesting this is a common feature in Klebsiella O antigens. The data also identifies the ligation site for O antigens in the LPS core and shows it to be conserved in different serotypes.

View larger version (9K): [in this window] [in a new window]   Fig. 1.   Structure of core oligosaccharide from Klebsiella LPS.

where L-glycero-D-manno-heptose is non-stoichiometric. Analysis of the LPSs from a waaL (O-polysaccharide ligase-deficient) mutant showed that the -GlcNAc in the linkage region is derived from the O-chain biosynthesis pathway, and the -3-deoxy-D-manno-octulosonic acid represents the core residue to which the O chain is ligated. Consistent with the common ligation site, the WaaL proteins from these serotypes are essentially identical. Polysaccharides from serotypes O1, O2a, and O2a,c contain no special groups at the non-reducing end, whereas polysaccharides from serotypes O3, O4, O5, and O12 have residues at the non-reducing end that are not found in the polymer repeating units.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Lipopolysaccharide Isolation-- K. pneumoniae CWK2 (serotype O1:K20) has been described elsewhere (9). The O-serotype reference strains, O3:K11 (strain 390), O4:K42 (1702), O5:K57 (4425/51), and O12:K80 (708) were provided by Dr. M. Trautmann (Department of Medical Microbiology and Hygiene, University of Ulm, Germany). Cells of additional serotype O12 strains K. pneumoniae type strain 708 (O12:K80, NRCC 6136), clinical isolate 134/94 (NRCC 6137), clinical isolate KD 420 (NRCC 6138), and clinical isolate 74/94 (NRCC 6139) were obtained from Statens Seruminstitut (Copenhagen, Denmark). LPSs from smooth strains were isolated as described (26). To isolate LPSs from the waaL mutant, K. pneumoniae strain CWG399 was grown in a fermenter (10L) in LB for 21h. The cells were then harvested and lyophilized, and the LPS was extracted from dry cells by the phenol/chloroform/light petroleum ether method of Galanos et al. (27), with a yield of 5.8% of the bacterial dry mass.

Amplification of the waaL Genes from K. pneumoniae and Generation of a Chromosomal waaL Mutation-- To analyze the sequences of the waaL genes from serotypes O1, O3, O4, O5, and O12, the genes were PCR-amplified from the appropriate strains with primers KPwaa1 (5'-GCGGCCTGGATCCGACCAACT-3') and KPwaa2 (5'-AGGCGAAGCAGGTACCCTGTGAAGA-3'). The primers were based on the sequence of K. pneumoniae C3 (O1:K66) waaL, available in GenBank^TM (accession number AF146532). The sequences of the waaL genes determined in this work are available in GenBank^TM as accession numbers AF482003 (serotype O1, strain CWK2), AF482004 (O3), AF482005 (O4), AF482006 (O5), and AF482007 (O12). The chromosomal waaL gene in K. pneumoniae CWK2 (O1) was inactivated using a non-polar gentamicin (aacC1) resistance cassette. To construct the waaL::aacC1 insertion, portions of the waaL gene and flanking DNA were PCR-amplified from K. pneumoniae CWK2 chromosomal DNA using PwoI polymerase. The products from primers KPwaa1 and KPwaa12 (5'-AGCATGATATCCACCGGCAGA-3') and KPwaa14 (5'-TCGCAGGGGATATCTATCATCAG-3') and KPwaa15 (5'-GGGATATCAGGTACCGGTGTTAAC-3') were digested with BamHI, EcoRV, and KpnI at sites introduced by the primers (underlined) and ligated to BamHI and KpnI sites in pBCSK+. This produced a construct containing a waaL gene with a 503-bp deletion and an internal EcoRV site. The waaL deletion-derivative gene and flanking DNA was removed on a BamHI and KpnI fragment and ligated to a similarly digested positive-selection suicide delivery vector that is based on pKO3 (28). The SmaI-digested aacC1 cassette was then ligated into the EcoRV site within waaL. This construct was transferred into CWK2 by electroporation, and allelic exchange was generated a strain that was resistant to gentamicin (aacC1 insertion) and sensitive to chloramphenicol (i.e. loss of vector). The resulting mutant was designated CWG399, and the correct insertion was verified by PCR. To confirm that the mutant was only defective in waaL activity, the mutation was complemented using a plasmid carrying waaL. The waaL open reading frame was PCR-amplified using PwoI and primers Kpwaa1 and KPwaa2 (see above). The PCR fragment was ligated to vector pRK404 (29) that was prepared by digestion with HindIII and end-filled using Klenow. The resulting plasmid was designated pWQ161. Nucleotide sequences were determined at the Guelph Molecular Supercenter (University of Guelph, Guelph, ON, Canada).

Examination of LPSs by SDS-PAGE and Western Immunoblotting-- For SDS-PAGE analysis, LPS was isolated from proteinase K-digested whole cell lysates (30). The LPS was separated on a 15% SDS-PAGE gel and visualized by silver staining and by Western immunoblot with rabbit anti-D-galactan I-specific antibodies (17).

Preparation of the Polysaccharides Released by Deamination-- The methods for deamination have been described elsewhere (25). Briefly, LPS (200 mg) was dissolved in water (20 ml). NaNO₂ (100 mg) and acetic acid (1 ml) were then added, and after a 6-h incubation at 25 °C, the lipid-containing part of the LPS was removed by ultracentrifugation (120,000 × g, 2h). The supernatant was fractionated by gel filtration chromatography on Sephadex G50 (superfine). Polysaccharides were purified on a Hamilton PRP ×100 anion exchange high performance liquid chromatography column in a gradient of 0-1 M NaCl, and the collected peaks were desalted by gel filtration.

NMR Spectroscopy and General Methods-- Chemical shifts obtained by NMR spectroscopy were assigned using two-dimensional homo- and heteronuclear experiments at 799.96 MHz for proton and 201.12 MHz for carbon using acetone as reference for proton (2.225 ppm) and 1,4-dioxane for carbon (67.4 ppm). Spectra were recorded at 40 °C in D₂O on a Varian UNITY INOVA 800 in 5-mm tubes. The double quantum-filtered phase-sensitive COSY experiment was performed using the Varian standard program tndqcosy, with 0.37-s acquisition time and 4096 data points in the F2 dimension. The data matrix was zero-filled in the F1 dimension to give a matrix of 4096 × 2048 points and was resolution-enhanced in both dimensions by a shifted sine-bell function before Fourier transformation. Similarly, the nuclear Overhauser experiment was performed using the Varian standard tnnoesy, with a mixing time of 100 ms. The total correlation spectroscopy experiment was performed using standard Varian program tntocsy with a spinlock time of 80 ms. The heteronuclear experiments were performed using standard pulse-field gradient programs gHSQC, gHSQCTOCSY, and gHMBC. The spectra were assigned using the computer program Pronto (31). Mass spectra were recorded by electrospray ionization Fourier transform-ion cyclotron mass spectrometry (ESI FT-ICR MS) utilizing an Apex II with a 7-Tesla super-conducting magnet (Bruker Daltonics, Bremen, Germany). GLC, GLC-mass spectroscopy, methylation, and monosaccharide analyses were performed as previously described (32).

O-Deacylated LPSs were prepared by treatment of the LPS with anhydrous hydrazine (1 ml for 50 mg of LPS, 40 °C, 1 h). After treatment, the samples were diluted with water and dialyzed.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Compositional Analysis of O-chain-containing LPS Fragments and Determination of Repeating Unit Structures-- LPSs from K. pneumoniae serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were treated with NaNO₂/acetic acid, and the lipid A-containing part was removed by ultracentifugation. The soluble products were fractionated by gel and anion exchange chromatography to give polysaccharide fractions in addition to the two previously described (25) oligosaccharides 1 and 2, originating from O-chain-deficient LPS molecules.

View larger version (9K): [in this window] [in a new window]   Structure II.

The polysaccharide fractions were eluted from gel filtration chromatography as one or two peaks of different molecular mass, and the lower mass fractions were used for structural analysis. The polysaccharides were passed through an anion exchange column in water, and the void volume fractions were used in further studies; this treatment significantly improved the quality of NMR spectra, removing background signals derived from contaminants.

The monosaccharide analysis (GLC of alditol acetates) of the polysaccharides (data not shown) revealed the presence of the expected repeating unit monosaccharides (Fig. 2) and of glucosamine and L-glycero-D-manno-heptose in quantities about 20 times less than those of the repeating unit components. Monosaccharide analysis of the polysaccharide from serotype O5_kp also revealed small amount of 3-O-methylmannose, which was previously reported to be present at the non-reducing end of the polymeric chain (13, 33). GLC analysis of the acetylated products of acid methanolysis of all polysaccharides showed the presence of Kdo.

View larger version (31K): [in this window] [in a new window]   Fig. 2.   Structures of the O-chain glycans isolated from the deaminated LPSs of different K. pneumoniae serotypes. The serotypes are indicated on the right. The repeating unit domains are boxed, and with the exception of serotype O12, the structures are identical to those already published in the literature. The O-repeating units are defined from chemical and biosynthesis data. The letter designation for the residues corresponds to the NMR data in Tables I-VIII.

The polysaccharides were reduced with NaBH₄ and methylated, after which the methylated products were hydrolyzed and converted to partially methylated 1d-alditol acetates. Gas chromatography-mass spectrometry analysis led to the identification of the major products derived from repeating units (Fig. 2) and a number of small peaks of the intensity about 20-fold less than major products. Among these minor products, 1,3,4-tri-O-methyl-6-O-acetyl-2,5-anhydromannitol, 2,3,4,6,7-penta-O-methyl-1,5-di-O-acetyl-1d-heptitol, and 2-(N-methylacetamido)-2-deoxy-4,6-di-O-methyl-1,3,5-tri-O-acetyl-1d-glucitol were identified in all strains. These derivatives originated from the monosaccharide residues M, P, and F at the reducing terminus. In serotype O12, the derivative of 3-substituted GlcNAc was present in a large proportion because it is also a component of the repeating unit (34). Methylation of the polysaccharides from serotypes O1, O2a, and O2a,c gave minor amounts of the derivatives of non-substituted galactofuranose. Methylation of the polysaccharide from serotype O12 showed the presence of minor amounts of non-substituted and 3-substituted rhamnopyranose. The methylated products obtained from strain O5 contained 2,3,4,6-tetra-O-methyl-1,5-di-O-acetyl-1d-mannitol.

The NMR spectra of all polymers contained, in addition to the signals of the components of repeating units, clearly visible signals of the residues of Kdo and anomeric signals of Hep P and of the hydrated aldehyde group of 2,5-anhydromannose (2,5-anhMan) (Figs. 3 and 4). The spectra were interpreted using two-dimensional techniques, and most of the proton and carbon signals of repeating unit monosaccharides were assigned. The signals of terminal regions were well visible on the correlation spectra (Fig. 5) but could be only partially assigned because of low signal intensities and overlap problems. Nevertheless, ¹H and ¹³C NMR signals for atoms 1-5 were found in most cases (Tables I-VIII), and interglycoside NOE and HMBC correlations were identified. These data were sufficient for the identification of the constituent monosaccharides and for sequence determination of the repeating unit and terminal regions of the polymers. Monosaccharides were identified on the basis of vicinal coupling constants and chemical shifts of the signals, and their connectivity was identified from NOE and HMBC data. Interglycoside NOE correlations between H-1 and transglycosidic proton as well as adjacent protons were observed. HMBC spectra contained correlations from H-1 to the transglycosidic carbon atom, indicating linkage position, and contained useful information for the signal assignment of intraresidual correlations in -pyranoses to C-2 (weak) and C-3 and C-5 (strong) and for -pyranose correlations to C-2 and C-3. For Kdo residues, HMBC correlations from H-3 to its own C-2 and from C-2 to transglycosidic carbon atom were identified.

View larger version (30K): [in this window] [in a new window]   Fig. 3.   1H NMR spectra of the polysaccharides, obtained from K. pneumoniae serotypes O1, O4, and O12. The lower trace shows the isolated core oligosaccharide fragment found at the reducing end of the polysaccharides.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   1H NMR spectra of the polysaccharides, obtained from K. pneumoniae serotypes O3 and O5. Methyl group signals are denoted with asterisks (*).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Parts of the HSQC 1H,13C correlation spectrum of the polysaccharide from K. pneumoniae O4. Left, low level cut showing the signals of terminal residues; right, high level cut, containing only signals of the repeating unit.

View this table: [in this window] [in a new window]   Table I NMR data for the common part of the polysaccharides Average chemical shifts for all analyzed polysaccharides ±0.02 ppm for 1H NMR and ±0.2 ppm for 13C NMR data are shown. Residues marked a or b are derived from oligosaccharides with or without -Hep (P; Fig. 2), respectively. anhMan, 2.5-anhydrimannose.

View this table: [in this window] [in a new window]   Table II NMR data for K. pneumoniae O1 polysaccharide Residues marked with a "prime" belong to the non-reducing end-repeating unit, whereas those residues marked a "double prime" belong to the reducing end-repeating unit. Residues marked a or b are derived from oligosaccharides with or without -Hep (P; Fig. 2), respectively.

View this table: [in this window] [in a new window]   Table III NMR data for K. pneumoniae O2a polysaccharide Residues marked with a "prime" belong to the non-reducing end-repeating unit, whereas those residues marked with a "double prime" belong to the reducing end-repeating unit. Residues marked a or b are derived from oligosaccharides with or without -Hep (P; Fig. 2), respectively.

View this table: [in this window] [in a new window]   Table IV NMR data for K. pneumoniae O2a,c polysaccharide Residues marked with a "prime" belong to the non-reducing end-repeating unit, whereas those marked with a "double prime" and asterisk refer to residues in the reducing end-repeating unit and the residue linking the two types of repeating units, respectively. Residues marked a or b are derived from oligosaccharides with or without -Hep (P; Fig. 2), respectively.

View this table: [in this window] [in a new window]   Table V NMR data for K. pneumoniae O3 polysaccharide Residues marked with a "prime" belong to the non-reducing end-repeating unit. Residues marked a or b are derived from oligosaccharides with or without -Hep (P; Fig. 2), respectively.

View this table: [in this window] [in a new window]   Table VI NMR data for K. pneumoniae O4 polysaccharide Residues marked with a "prime" belong to the non-reducing end-repeating unit, whereas those marked with a "double prime" belong to the reducing end-repeating unit. Residues marked a or b are derived from oligosaccharides with or without -Hep (P; Fig. 2), respectively.

View this table: [in this window] [in a new window]   Table VII NMR data for K. pneumoniae O5 polysaccharide Residues marked with a "prime" belong to the non-reducing end-repeating unit, whereas those marked with a "double prime" belong to the reducing end-repeating unit. Residues marked a or b are derived from oligosaccharides with or without -Hep (P; Fig. 2), respectively.

Structure of the Linkage Region between O Chain and Core Oligosaccharide-- Detailed analysis of the spectra of all polysaccharides revealed the presence of the same fragment at the reducing end of each of the polymeric chains,

View larger version (7K): [in this window] [in a new window]   Structure III.

where P is H or -Hep (Table I). The residue F was substituted either by a repeating unit (serotypes O1, O2a, O2a,c, O3, O4, O12) or by another fragment (serotype O5).

Linkage Region Structure in the Glycan from Serotype O1-- NMR analysis of the polysaccharide derived from K. pneumoniae O1 LPSs revealed that the structure of the O chain was as described previously (9, 11). It consisted of two types of repeating units, [-3)--Galf-(1-3)--Galp-(1-] (D-galactan I) and [-3)--Galp-(1-3)--Galp-(1-] (D-galactan II), with the D-galactan I attached to -GlcNAc F residue at position 3. D-Galactan II was found attached to the non-reducing end of some D-galactan I chains. No structural elements that are not components of the repeating units were found either between galactan I and galactan II or at the non-reducing end of the polysaccharide chain. Although structures with variable lengths of D-galactan II are present, the shortest fractions, containing 0-3 repeating units of D-galactan II, were used for NMR structural analysis. Molecules with higher molecular weights did not yield spectra with well resolved terminal residues. Both terminal galactofuranose and galactopyranose were found in methylation analysis, showing that populations of the polymer can have either D-galactan I or D-galactan II units at the non-reducing end, in support of previous results (9, 10). NMR analysis identified Galf as the non-reducing terminal residue in those O chains lacking D-galactan II, but the expected terminal Galp residues from molecules containing D-galactan II could not be resolved in the spectra. The NMR data were consistent with a direct linkage between the Galp residue of the first repeating unit and -GlcNAc (residue F) of the core linkage region.

The structure of the O1 polymer was confirmed by electrospray ionization mass spectrometry data (Fig. 6). The mass spectrum contained a number of signals with maximum at 4151.4 Da, corresponding to 11 repeating units with the expected fragment with non-hydrated 2,3-anhydromannose residue at the reducing end; signals of the polymers with 9-16 repeating units were observed. Two series of signals were present, reflecting partial substitution with Hep residue P.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Mass spectrum of the polysaccharide, obtained by deamination of the K. pneumoniae O1 LPS. The numbers indicate the numbers of repeating units in a given molecular species. Peaks marked with a (+) reflect molecular species where residue P is a heptose, whereas species with a hydrogen at residue P are unmarked.

Linkage Region Structure in the Glycan from Serotype O2a-- As reported previously, this polymer had only the galactan I type of repeating units (3, 6). The non-reducing end of the polymer was comprised of a residue of Galf, in agreement with the results from D-galactan I chains in the O1 antigen.

Linkage Region Structure in the Glycan from Serotype O2a,c-- The analysis of the O2a,c polymer revealed the same two repeating units reported previously reported (3), [-5)--Galf-(1-3)--GlcNAc-(1-] (2c antigen) and [-3)--Galf-(1-3)--Galp-(1-] (D-galactan I). D-Galactan I chains were linked to -GlcNAc F, and the 2c antigen was found at the non-reducing terminus of some D-galactan I chains. As in the case of the serotypes O1 and O2a, the structure of the polymer contained no unique residues at the non-reducing terminus. The only detectable terminal residue was Galf, and this could arise from either repeating unit. No novel residues were detected between two types of the repeating units.

Linkage Region Structure in the Glycan from Serotype O3-- Analysis of the polymer from serotype O3 revealed the presence of the previously described repeating unit (35). NMR spectra of this polysaccharide as well as of the polymer obtained from O3 LPS by conventional mild acid hydrolysis contained the signals of two methyl groups (_H/_C, 3.55/54.1, 3.56/54.0 ppm) (Fig. 4). The intensities of these signals were consistent with their presence at the end of the polymeric chain. However, the origin of these signals remained unclear. No methylated monosaccharides were detected in GLC-mass spectrometry analysis of the alditol acetates, alditol acetates obtained from the dephosphorylated polysaccharide, or acetylated products of the methanolysis of the polysaccharide. Furthermore, no components other than mannose, glucosamine, D,L-heptose, and Kdo were detected by conventional analytical methods. The methyl signals gave no NOE to any proton and no HMBC correlations, in contrast to what is normally observed for the methyl groups present as ether substituents (e.g. in the polymer from serotype O5). Interestingly, the same signals are visible on the published ¹H NMR spectrum of the polymer from Hafnia alvei, which was found to be identical to the Klebsiella O3 and E. coli O9 polysaccharides (36). Methylation analysis of the O3 polymer showed the presence of a very small amount of terminal mannose, which was not detected in the NMR spectra. One modified mannose residue, T, can be identified, but the nature of modification could not be resolved by currently available methods nor could the frame for the repeating unit be identified from the NMR data. The residues N and Q between the repeating unit and GlcNAc F probably do not belong to the repeating unit and are similar to analogous residues in the serotype O5 polysaccharide. This conclusion agrees with genetic and biosynthesis data.

Linkage Region Structure in the Glycan from Serotype O4-- The polymer obtained from Klebsiella O4 contained the previously described repeating unit (37). NMR data showed that it is linked directly to the GlcNAc F through an -Gal residue. The ¹H NMR spectrum contained additional signals from a Kdo residue (Fig. 3). Comparison of the NMR data for this Kdo residue with published data for methyl - and -pyranosides identified it as having -pyranose configuration (38). HMBC correlation between Kdo C-2 (identified from HMBC correlation to H-3 protons) and Ribf H-2 identified the non-reducing terminus as -Kdo-(2-2)--D-Ribf.

Linkage Region Structure in the Glycan from Serotype O5-- NMR data for the serotype O5 polysaccharide (Fig. 4) revealed the presence of the previously described repeating unit as well as a 3-O-methylated mannose residue (13). However, the repeating unit domain was not linked directly to the -GlcNAc F residue. Instead, the structure contains a bridging disaccharide located between the repeating unit and GlcNAc F.

View larger version (6K): [in this window] [in a new window]   Structure IV.

The non-reducing end of the polymer was represented by the residue of 3-O-methyl--mannopyranose residue T, which does not belong to the repeating unit. The position of the methyl group was identified using NOE correlation.

Linkage Region Structure in the Glycan from Serotype O12-- The structure of the repeating unit of the polysaccharide from serotype O12 differed from that previously published, in which the rhamnose residue was reported to be substituted at position 3 (34): [-3)--GlcNAc-(1-3)--Rha-(1-].

Methylation analysis showed the presence of 3-substituted N-acetylglucosamine and 4-substituted rhamnose residues as major components, and this was confirmed by NMR experiments. NOE and HBMC data revealed a repeating unit containing rhamnose residues substituted at position 4. A small amount of 3-substituted rhamnose detected in methylation analysis is derived from the terminal region of the polymer. To eliminate the possibility that the differences in the two O12 structures reflected different sources of LPS isolation, LPSs from four additional isolates of K. pneumoniae serotype O12 were examined. These included the type strain 708 (serotype O12:K80) from Statens Seruminstitut (Denmark) collection used for the structure in the published literature (34) and three other clinical isolates (see "Experimental Procedures"). All isolated polysaccharides had the same repeating unit structure containing 4-substituted Rha residues. The revised O12 repeating unit structure is therefore [-3)--D-GlcNAc-(1-4)--L- Rha-(1-].

No additional monosaccharides were found between the repeating unit and GlcNAc F. The polymer contained a residue of Kdo at the non-reducing end (Fig. 2) and NMR data for this Kdo residue showed a -pyranose configuration (38). As in the case of the serotype O4, the attachment position of Kdo was found on the basis of the observed HMBC correlation between C-2 of Kdo residue and H-3 of rhamnose residue A'. The terminal residue of -Kdo is thus linked to O-3 of the Rha residue, whereas Rha present within the repeating unit itself is substituted at position 4. A small amount of non-substituted Rha was evident, derived from the few chains lacking the terminal -Kdo.

The electrospray ionization mass spectrum of the polymer contained, as in the case of O1 polysaccharide, two series of peaks due to the partial presence of Hep P at 5493 (no Hep P), 5684 (with Hep P), 5842, 6034, 6191, and 6384 Da, corresponding to the molecules containing 13-15 repeating units (data not shown). Masses corresponded to equal number of Rha and GlcNAc residues, which indicates that Rha A', substituted with Kdo, does not belong to the repeating unit.

Structure of the Core Oligosaccharide in CWG399, an O-antigen Ligase-deficient Mutant-- To unequivocally determine which residues in the linkage region are derived from the core and which are provided by the O-chain biosynthesis pathway, an O-chain ligase-deficient waaL mutant was constructed by allelic exchange. Within the waa locus reported from K. pneumoniae serotype O1:K66 is one open reading frame predicting a protein that resembles known WaaL proteins. These proteins all have multiple transmembrane segments and a characteristic periplasmic domain toward the C terminus and are, therefore, readily identified by examination of hydrophobicity-hydrophilicity plots (Fig. 7A). To confirm its identity, the waaL gene from K. pneumoniae serotype O1 was mutated with a non-polar cassette in strain CWG399. As expected, the waaL mutant lacked smooth LPSs containing the O1 antigen due to the ligation defect (Fig. 7B). The LPS from CWG399 was isolated and de-O-acylated, and the products were subjected to mass spectrometry. The resulting spectrum (Fig. 7C) resembled that seen from the subset of rough (O-deficient) LPS isolated from strains with predominantly smooth (O-substituted) LPS. The largest major peak with mass ion 3057.9 corresponds to an LPS molecule containing the entire core structure. The remaining species reflect the variable substitution with -GalA and -D,L-Hep residues, and these structures have already been documented (26). Ligation could be restored in CWG399 by the addition of pWQ161 carrying the wild-type waaL gene. As this data was being completed, a similar mutation/complementation approach was used to confirm the identity of waaL in the O1:K66 isolate, although the structure of the LPS in the mutant was not determined (39). The core structure for CWG399 lacks the O chain as expected as well as the -GlcNAc residues found in the linkage site structure.

View larger version (38K): [in this window] [in a new window]   Fig. 7.   Characterization of the LPS in a waaL (O-antigen ligase-deficient) mutant of K. pneumoniae. Panel A, the waaL gene encoding the O-antigen ligase was identified from the GenBankTM accession (AF146532) for the waa locus from K. pneumoniae strains C3 (O1:K66), based on the characteristic hydropathy profile shared with other known ligases such as the one from E. coli core type R1 (53). The gene was mutated by allelic exchange to generate strain CWG399. Panel B shows the PAGE profile of LPS from the waaL mutant (CWG399) and its parent (CWK2), demonstrating the loss of high molecular weight O-chain-substituted LPS in the mutant and the restoration of the wild-type LPS profile by plasmid pWQ161-encoded waaL. The immunoblot was developed using rabbit polyclonal serum raised against the D-galactan I component of the O polysaccharide. Panel C shows the mass spectrum obtained from the de-O-acylated LPS of K. pneumoniae CWG399 and the deduced structure of the core oligosaccharide. Residues J, K, and P are all non-stoichiometric additions as reported previously (26) and lead to the heterogeneity in core structures derived from CWG399.

Identification of a Conserved waaL gene in K. pneumoniae-- The waaL genes were amplified by PCR from K. pneumoniae serotypes O1, O3, O4, O5, and O12, and the sequences were determined. The sequences for the two predicted waaL proteins from the O1 isolates shared 97.5% identity and 98.9% overall similarity. When the O1 sequence (from CWK2) was compared with the predicted proteins from the other serotypes, similarly high levels of conservation were found: O3_kp (99.7% identity; 100% overall similarity), O4_kp (99.7%; 100%), O5_kp (98.1%; 99.4%), and O12_kp (96.1%; 98.9%). A partial waaL sequence is also available from the in progress K. pneumoniae genome project (genome.wustl.edu/gsc/Projects/K.pneumoniae), although the O serotype of the source strain (MGH78578) is apparently unknown. This sequence also shows a very high level of conservation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previously we reported that the rough (O-chain-deficient) LPS fractions isolated from K. pneumoniae serotypes O1, O₂, O3, O4, O5, O8, and O12 contain a conserved fragment that is released by deamination with nitrous acid, converting GlcN (residue M) into anhydro-mannose residue (26) (Fig. 1). Here we show that this structural motif forms part of a linkage region between the core oligosaccharide and the repeating unit domain of the O chain with the structure below.

View larger version (7K): [in this window] [in a new window]   Structure V.

The -GlcNAc was not detected in the products derived from the core oligosaccharide in previous studies, suggesting that it forms part of the O-chain polysaccharide. Conclusive evidence for this proposal was obtained by the absence of -GlcNAc in the core oligosaccharide isolated from the O-antigen ligase (waaL)-deficient mutant CWG399, which by definition, must have a complete core. To our knowledge this is the first definitive structure from a K. pneumoniae strain with a genetically defined LPS defect. Previous analyses have involved core fractions isolated from strains with smooth LPS or cores from undefined mutants lacking O chains and could therefore represent truncated structures. The observation of a conserved linkage region in the LPS of different Klebsiella serotypes is consistent with the finding that the LPS core oligosaccharides of isolates representing different O serotypes share a common epitope recognized by a monoclonal antibody, although the precise epitope has not been identified (40).

Biosynthesis of the D-galactan I O chain from serotype O1_kp (17) and the polymannan O8_ec and O9_ec antigens (which are identical to O5_kp and O3_kp, respectively) (41) all occur on an und-PP-GlcNAc primer. The original in vitro studies involving the polymannans identified a requirement for the Rfe enzyme (now renamed as WecA) in O-chain assembly and pointed to und-PP-Glc as a possible primer (41-44). It was proposed that the Glc residue at the reducing terminus of the mannan was transferred to the lipid A core with the mannan in the ligation reaction (45). However, subsequent analysis of the role of WecA in enterobacterial common antigen synthesis established the enzyme as being a UDP-GlcNAc:undecaprenyl phosphate GlcNAc-1-phosphate transferase (19). Consistent with this assignment, in vivo studies with the O8_ec confirmed the involvement of und-PP-GlcNAc as the primer (20). Later biosynthesis experiments with the O9 antigen resolved the contradictory literature by showing that both und-PP-Glc and Und-PP-GlcNAc could serve as primers for in vitro mannan assembly with und-PP-GlcNAc being more efficient (21). The synthesis of und-PP-Glc in the original work was presumably due to the reaction conditions employed and relaxed specificity of WecA in vitro. The definitive structure of the linkage domain between the O chain and core oligosaccharide and the structure of CWG399 LPS confirms und-PP-GlcNAc as the donor of the -GlcNAc residue located at the reducing terminus of the glycan chain in Klebsiella. The definitive structure of the linkage domain between the O chain and core oligosaccharide and the structure of CWG399 LPS confirms und-PP-GlcNAc as the donor of the -GlcNAc residue located at the reducing terminus of the glycan chain. From the structure presented here, the und-PP-linked O chain in each serotype is extended from a reducing terminal -GlcNAc and is then transferred to an identical attachment site in the core. Consequently, the waaL (O-antigen ligase) enzyme in each serotype would perform an identical reaction, therefore explaining the high degree of identity found in the predicted waaL proteins from K. pneumoniae serotypes O1, O3, O4, O5, and O12 (96.1-99.7%).

Analysis of the structure of the core-attachment region and repeat-unit structure of these O serotypes provides additional insight into the biosynthetic process. In the O1, O2a, O2a,c, O4, and O12 polymers, there is a direct transition from the primer GlcNAc into the first chemically defined repeat unit of the O chain. However, with the exception of O12, dedicated glycosyltransferase activities must be necessary for the addition of the first residue of the repeating unit onto the GlcNAc primer, because the resulting linkage is not found elsewhere in the O chain. For example, in the biosynthesis of D-galactan I, the bifunctional WbbO enzyme transfers one residue each of Galp and Galf to und-PP-GlcNAc to form the structure -3)--Galf-(1-3)--Galp-(1-3)-GlcNAc (18). In subsequent chain extension, only the galactofuranosyl activity of WbbO participates in repeating unit synthesis and an additional enzyme (WbbM) provides galactopyranosyltransferase activity. From a biosynthetic perspective, the initial Galp residue forms an "adaptor" that is extended by multiple rounds of processive glycosyltransferase activity to form the repeat unit domain. Genetic and biosynthesis data are not available for the O4_kp and O12_kp O chains, but the structures of the deamidation-released glycans provide some insight. In serotype O4_kp, it is evident that there is a requirement for one galactosyltransferase activity that acts once per O chain to form -Gal-(1-3)--GlcNAc in the adaptor region and another Gal transferase that generates -Gal-(1-2)--Ribf linkages in the repeat unit. In contrast, the presence of GlcNAc within the repeating unit of serotype 12 affords the possibility that a single rhamnosyltransferase adds Rha residues to either the primer GlcNAc or a GlcNAc residue within the repeating unit.

The biosynthetic pathways for the O3 and O5 polymannans both involve the same primer-adaptor sequence with the structure below.

View larger version (6K): [in this window] [in a new window]   Structure VI.

Biosynthetic data obtained from the E. coli polymannose antigens can be directly related to O5_kp and O3_kp, because the relevant biosynthetic enzymes are conserved. In the prototype O9a_ec system, the WbdC (formerly MtfC) mannosyltransferase transfers the initial mannosyl residue to und-PP-GlcNAc (21). This reaction occurs only once per polymannose chain, so the resulting residue (Q, Fig. 2) does not form part of the repeat unit. The next transfer is mediated by WbdB (formerly MtfB), which is proposed to add 2 -(1-3)-linked mannosyl residues (21). Examination of data base sequences shows that the WbdA and WbdC proteins from E. coli serotypes O8, O9a, and O9 and Klebsiella O3 and O5 are virtually identical (>95%). Furthermore, the WbdB-WbdC pairs from E. coli O9a and O8 are functionally interchangeable.³ From this collective data, WbdC and WbdB appear to play identical roles in the initial biosynthetic steps of each member of this polymannose O-chain family. After formation of this adaptor, the pathways for biosynthesis of the repeating unit domains of the polymannan O chains diverge. In O9_ec (O3_kp), the repeat unit is apparently formed by the activities of WbdB and WbdA. Defined sequence changes in the WbdA homologues (46) result in structural differences between O9_ec (O3_kp) and a variant (O9a_ec) containing a tetrasaccharide repeat unit differing by one -(1-2)-linked mannosyl residue from the pentasaccharide in O9_ec/O3_kp. WbdA is capable of adding blocks of -(1-2)-linked mannosyl residues, presumably 2 in O9a_ec and 3 in O9_ec (O3_kp). The proposed biosynthetic pathway suggests that polymer extension occurs by alternating activities of WbdB and WbdA, but definitive proof of such an alternating mechanism is not yet available. The gene cluster for O8_ec/O5_kp biosynthesis lacks wbdA, and the sequences of these parts of the O-antigen biosynthesis loci differ. Differences in the sequences of glycosyltransferases would be expected because the O8_ec/O5_kp antigen contains a -linked mannose in its repeating unit. It seems unlikely that WbdB is involved in later chain extension stages of O8_ec/O5_k synthesis because, unlike the O9_ec/O3_kp polymers, there is no -Man-(1-3)--Man linkage within the repeat unit.

Despite differences in enzyme specificities, the requirement for a specific glycosyltransferase in forming an adaptor region between the -GlcNAc and the repeat unit domain of the O chain is a common feature in the K. pneumoniae polysaccharides studied here and is likely to be involved in all polymers whose assembly involves processive glycosyl transfer.

The mechanism that regulates O chain length in processive synthesis mechanisms is unknown (23). The observation that the O8_ec/O5_kp polymannans have 3-O-methylmannose at their reducing termini led to speculation that such residues act as molecular markers for chain termination (13). We are aware of two other examples where a modified residue is found at the non-reducing terminus of an O chain. One is in Vibrio cholerae, O1 where 2-O-methyl groups are found (47). Although V. cholerae mutants lacking this residue are not impaired in the ability to synthesize O antigen, they do lose the epitope associated with seroconversion from type Ogawa to Inaba (48). In the O polysaccharide of Bordetella bronchiseptica, a unique 2,3,4-triamino-2,3,4-trideoxy--galacturonamide derivative is found at the chain terminus (49). The observation that the O4_kp and O12_kp O chains terminate in Kdo residues extends the range of known terminal modifications to include glycosyl residues. The K. pneumoniae O1 and O2a,c provide somewhat different situations in which a D-galactan I chain is capped by an additional structural domain rather than a single residue. It is not yet clear whether this is an extreme form of chain termination, but in the absence of such capping in mutants lacking D-galactan I or in other isolates with the O2a antigen, the size distribution of D-galactan I differs (9).

Gene content and, in some cases, functional analyses of their respective O-chain biosynthesis loci, provide insight into a common feature in the biosynthesis of O antigens in V. cholerae O1 (36), B. bronchiseptica (52), and Klebsiella O3 and O5 (and O8_ec and O9_ec) (21). These O chains are assembled by an ABC transporter-dependent mechanism in which the transporter exports nascent polymer across the cytoplasmic membrane to the periplasm where ligation occurs (for review, see Ref. 24). The presence of non-reducing terminal modifications may therefore provide a unifying theme in the biosynthesis of O chains that are extended by processive glycosyltransfer to the non-reducing terminus. This process clearly differs from the other major pathway for O-antigen biosynthesis. In the Wzy-dependent pathway, und-PP-linked repeating units provide substrates for a blockwise polymerization mechanism that minimally involves the Wzy protein. Growth of the und-PP-linked glycan occurs by the addition of repeating units to the reducing terminus and the polymerization reaction occurs at the periplasmic face of the inner membrane (for review, see Ref. 24). The Wzz protein is required for chain-length determination in this pathway, and it is thought to coordinate the activity of the polymerase, O-antigen ligase, or both by a mechanism that has yet to be elucidated (for review, Ref. 23). There is no requirement for novel non-reducing terminal residues to delineate chain termination in the Wzy-dependent pathway, and none have been identified in structural analyses of many representative O antigens.

The termination of O chains at a specific residue explains the observation that purified smooth LPS from these bacteria gives rise to a typical ladder of molecules on SDS-PAGE, with the molecules differing by increments of one repeat unit. In contrast, chain completion at any residue would yield a distribution in size increments of a single glycosyl residue, resulting in an unresolved smear on SDS-PAGE. Although the terminal modifications may provide a mechanism of chain termination in polymers that grow by processive transfer of glycosyl residues to the non-reducing terminus, a crucial open question is how the process is regulated. The presence and chain length of the LPS O chain plays a critical role in the resistance of Klebsiella to complement-mediated serum killing (11, 12, 50, 51). Thus the regulation of O-chain termination represents an important element in the biology of significant pathogens like K. pneumoniae.

	ACKNOWLEDGEMENTS

We thank Donald Krajcarsky (National Research Council, Canada) and Buko Lindner (Forschungszentrum Borstel, Federal Republic of Germany) for contributions to mass spectrometry analysis. The spectra at 800 MHz were obtained at the Varian Unity Inova spectrometer of the Danish Instrument Center for NMR Spectroscopy of Biological Macromolecules.

	FOOTNOTES

^* This work was supported by funding from the Canadian Bacterial Diseases Network (to M. B. P. and C. W.) and the Natural Sciences and Engineering Research Council (to C. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed: Inst. for Biological Sciences, National Research Council, 100 Sussex Dr., Ottawa, ON K1A 0R6, Canada. Tel.: 613-990-03-97; Fax: 613-952-90-92; E-mail: evguenii.vinogradov@nrc.ca.

Supported by a Postgraduate Scholarship from the Natural Sciences and Engineering Research Council.

This author acknowledges receipt of a Canada Research Chair award.

Published, JBC Papers in Press, April 30, 2002, DOI 10.1074/jbc.M202683200

² All monosaccharides have the D configuration except L-Rha.

³ P. Amor and C. Whitfield, unpublished data.

	ABBREVIATIONS

The abbreviations used are: LPS, lipopolysaccharide; Hep, L-glycero-D-manno-heptose; GalA, galacturonic acid; Kdo, 3-deoxy-D-manno-octulosonic acid; P, phosphate; und-PP, undecaprenyl pyrophosphoryl; NOE, nuclear Overhauser effect; HMBC, heteronuclear multiple bond coherence.

	REFERENCES

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