Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1.

The lipopolysaccharide (LPS) molecule is an important virulence determinant in Klebsiella pneumoniae. Studies on the serotype O1 LPS were initiated to determine the basis for antigenic heterogeneity previously observed in the O1 side chain polysaccharides and to resolve apparent ambiguities in the reported polysaccharide structure. Detailed chemical analysis, involving methylation and 1H- and 13C-nuclear magnetic resonance studies, demonstrated that the O-side chain polysaccharides of serotype O1 LPS contained a mixture of two structurally distinct D-galactan polymers. The repeating unit structures of these two polymers were identified as [----3)-beta-D-Galf-(1----3)-alpha-D-Galp-(1----] (D-galactan I) and [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] (D-Galactan II). D-Galactan I polysaccharides were heterogeneous in size and were detected throughout the sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) profile of O1 LPS. In contrast, D-galactan II was confined to the higher-molecular-weight region. The structures of the two D-galactans were not influenced by simultaneous synthesis of a capsular K antigen. Apparently, neither of the D-galactans constitutes a common antigen widespread in Klebsiella spp. as determined by immunochemical analysis. Examination of the LPSs in mutants indicated that expression of D-galactan I can occur independently of D-galactan II. Transconjugants of Escherichia coli K-12 strains carrying the his region of K. pneumoniae were constructed by chromosome mobilization with RP4::mini-Mu. In these transconjugants, the O antigen encoded by the his-linked rfb locus was determined to be D-galactan I, suggesting that genes involved in the expression of D-galactan II are not closely linked to the rfb cluster.     

Role of Rfe and RfbF in the initiation of biosynthesis of D-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1.

The 6.6-kb rfb gene cluster from Klebsiella pneumoniae serotype O1 (rfbKpO1) contains six genes whose products are required for the biosynthesis of a lipopolysaccharide O antigen with the following repeating unit structure: -->3-beta-D-Galf-1-->3-alpha-D-Galp-1-->(D-galactan I). rfbFKpO1 is the last gene in the cluster, and its gene product is required for the initiation of D-galactan I synthesis. Escherichia coli K-12 strains expressing the RfbFKpO1 polypeptide contain dual galactopyranosyl and galactofuranosyl transferase activity. This activity modifies the host lipopolysaccharide core by adding the disaccharide beta-D-Galf-1-->3-alpha-D-Galp, representing a single repeating unit of D-galactan I. The formation of the lipopolysaccharide substituted either with the disaccharide or with authentic polymeric D-galactan I is dependent on the activity of the Rfe enzyme. Rfe (UDP-GlcpNAc::undecaprenylphosphate GlcpNAc-1-phosphate transferase) catalyzes the formation of the lipid-linked biosynthetic intermediate to which galactosyl residues are transferred during the initial steps of D-galactan I synthesis. The rfbFKpO1 gene comprises 1,131 nucleotides, and the predicted polypeptide consists of 373 amino acid residues with a predicted M(r) of 42,600. A polypeptide with an M(r) of 42,000 was evident in sodium dodecyl sulfate-polyacrylamide gels when rfbKpO1 was expressed behind the T7 promoter. The carboxy-terminal region of RfbFKpO1 shares similarity with the carboxy terminus of RfpB, a galactopyranosyl transferase which is involved in the synthesis of the type 1 O antigen of Shigella dysenteriae.     

Relationships between rfb gene clusters required for biosynthesis of identical D-galactose-containing O antigens in Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16.

The lipopolysaccharide O antigens of Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16 both contain a repeating unit disaccharide of [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->]; the resulting polymer is known as D-galactan I. In K. pneumoniae serotype O1, the genes responsible for the synthesis of D-galactan I are found in the rfb gene cluster (rfbKpO1). We report here the cloning and analysis of the rfb cluster from S. marcescens serotype O16 (rfbSmO16). This is the first rfb gene cluster examined for the genus Serratia. Synthesis of D-galactan I is an rfe-dependent process for both K. pneumoniae serotype O1 and S. marcescens serotype O16. Hybridization experiments with probes derived from each of the six rfbKpO1 genes indicate that the cloned rfbSmO16 cluster contains homologous genes arranged in the same order. However, the degree of homology at the nucleotide sequence level was sufficiently low that hybridization was detected only under low-stringency conditions. rfbABSmO16 genes were subcloned and shown to encode an ABC-2 (ATP-binding cassette) transporter which is functionally identical to the one encoded by the corresponding rfb genes from K. pneumoniae serotype O1. The amino acid sequences of the predicted RfbA and RfbB homologs showed identities of 75.7% (87.9% total similarity) and 78.0% (86.5% total similarity), respectively. The last gene of the rfbKpO1 cluster, rfbFKpO1, encodes a bifunctional galactosyltransferase which initiates the formation of D-galactan I. RfbFKpO1 and RfbFSmO16 are 57.6% identical (with 71.1% total similarity), and both show similarity with RfpB, the galactosyltransferase involved in the synthesis of Shigella dysenteriae type I O-polysaccharide. The G+C contents of the rfbAB genes from each organism are quite similar, and values are lower than those typical for the species. However, the G+C content of rfbFSmO16 (47.6%) was much higher than that of rfbFKpO1 (37.3%), despite the fact that the average for each species (52 to 60%) falls within the same range.     

Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae

D-Galactan I is an O-antigenic polymer with the repeat unit structure [-->3)-beta-D-Galf-(1-->3)-alpha-D-Galp-(1-->], that is found in the lipopolysaccharide of Klebsiella pneumoniae O1 and other gram-negative bacteria. A genetic locus containing six genes is responsible for the synthesis and assembly of D-galactan I via an ATP-binding cassette (ABC) transporter-dependent pathway. The galactosyltransferase activities that are required for the processive polymerization of D-galactan I were identified by using in vitro reactions. The activities were determined with endogenous lipid acceptors in membrane preparations from Escherichia coli K-12 expressing individual enzymes (or combinations of enzymes) or in membranes reconstituted with specific lipid acceptors. The D-galactan I polymer is built on a lipid acceptor, undecaprenyl pyrophosphoryl-GlcpNAc, a product of the WecA enzyme that participates in the biosynthesis of enterobacterial common antigen and O-antigenic polysaccharide (O-PS) biosynthesis pathways. This intermediate is directed into D-galactan I biosynthesis by the bifunctional wbbO gene product, which sequentially adds one Galp and one Galf residue from the corresponding UDP-sugars to form a lipid-linked trisaccharide. The two galactosyltransferase activities of WbbO are separable by limiting the UDP-Galf precursor. Galactosyltransferase activity in membranes reconstituted with exogenous lipid-linked trisaccharide acceptor and the known structure of D-galactan I indicate that WbbM catalyzes the subsequent transfer of a single Galp residue to form a lipid-linked tetrasaccharide. Chain extension of the D-galactan I polymer requires WbbM for Galp transferase, together with Galf transferase activity provided by WbbO. Comparison of the biosynthetic pathways for D-galactan I and the polymannose E. coli O9a antigen reveals some interesting features that may reflect a common theme in ABC transporter-dependent O-PS assembly systems.     

Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species.

Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries. In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I. The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable. Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization. However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype. The nucleotide sequences for the six rfb genes showed variations in moles percent G+C values and in the values for nucleotide sequence identity, which ranged from 66.9 to 79.7%. The predicted polypeptides ranged from 64.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity). The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb genes through recent lateral transfer events.     

Antigenic polysaccharides of bacteria. 35. Establishment of the structure of polysaccharide chains of lipopolysaccharides of Pseudomonas cepacia IMV 4176 and IMV 4202 (Serotype 3)

On mild acid degradation of the Pseudomonas cepacia strain IMV 4176 lipopolysaccharide, two polysaccharides were obtained, one of which is a homopolymer of N-acetyl-D-galactosamine and the other is composed of equal amounts of N-acetyl-D-galactosamine and D-ribose. Partial hydrolysis with aqueous oxalic acid caused depolymerization of the heteropolysaccharide, and the homopolysaccharide was isolated in the individual state. On the basis of methylation and 13C NMR analysis, it was concluded that both polysaccharides are built up of disaccharide repeating units having the following structures: ----4)-alpha-D-GalpNAc-(1----4)-beta-D-GalpNAc-(1---- and ----4)-alpha-D-GalpNAc-(1----2)-beta-D-Ribf-(1----. The heteropolysaccharide from P. cepacia strain 4176 is identical by the structure of the repeating unit to the O-specific polysaccharide of P. cepacia strain IMV 4202 (serotype 3), Pseudomonas aeruginosa O12 and Serratia marcescens O14.     

Molecular cloning of the rfb region of Klebsiella pneumoniae serotype O1:K20: the rfb gene cluster is responsible for synthesis of the D-galactan I O polysaccharide.

Previous chemical analyses identified two structurally distinct O polysaccharides in the lipopolysaccharide of Klebsiella pneumoniae serotype O1:K20 (C. Whitfield, J. C. Richards, M. B. Perry, B. R. Clarke, and L. L. MacLean, J. Bacteriol. 173:1420-1431, 1991). The polysaccharides were designated D-galactan I and D-galactan II; both are homopolymers of galactose. To begin investigation of the synthesis and expression of these O polysaccharides, we have cloned a 7.3-kb region of the chromosome of K. pneumoniae O1:K20, containing the his-linked rfbkpO1 (O-antigen biosynthesis) gene cluster. In Escherichia coli K-12 and Salmonella typhimurium, rfbkpO1 directed the synthesis of D-galactan I but not D-galactan II. The cloned rfbkpO1 genes did not complement a mutation affecting D-galactan II synthesis in K. pneumoniae CWK37, suggesting that another (unlinked) locus is also required for D-galactan II expression. However, plasmids carrying rfbkpO1 did complement a mutation in K. pneumoniae CWK43 which eliminated expression of both D-galactan I and D-galactan II, indicating that at least one function is common to synthesis of both polymers. Synthesis of D-galactan I was dependent on chromosomal galE and rfe genes. Hybridization experiments indicated that the rfbkpO1 sequences from different serotype O1 Klebsiella isolates showed some restriction fragment length polymorphism.     

The structure of the polysaccharide part of the LPS from Serratia marcescens serotype O19, including linkage region to the core and the residue at the non-reducing end

The structure of the LPS from Serratia marcescens serotype O19 was investigated. Deamination of the LPS released the O-chain polysaccharide together with a fragment of the core oligosaccharide. The following structure of the product was determined by NMR spectroscopy, mass spectrometry, and chemical methods: [carbohydrate structure: see text] The main polymer consists of a repeating disaccharide V-U and is present on average of 18 units per chain as estimated by integration of signals in the NMR spectra. The residue O corresponds to the primer, which initiates biosynthesis of the O-chain, and an oligomer of a disaccharide R-S is an insert between the primer and the main polymer. The polysaccharide has a beta-Kdo residue at the non-reducing end, a feature similar to that observed previously in the LPS from Klebsiella O12.     

Elucidation of the structure of the Pasteurella haemolytica serotype T10 lipopolysaccharide O-antigen by n.m.r. spectroscopy

The aqueous-phase lipopolysaccharide isolated from Pasteurella haemolytica serotype T10 cells by the phenol-water extraction method was found to be S-type lipopolysaccharide which possessed O-antigenic polysaccharide chains composed only of D-galactose residues. Structural analysis of the O-polysaccharide, using a combination of 1D and 2D 1H- and 13C-n.m.r. methods, led to the identification of the disaccharide repeating-unit as [----3)-alpha-D-Galp-(1----3)-beta-D-Galf-(1----]n. The serological cross-reactivity between P. haemolytica serotypes T4 and T10 can now be related to the structural similarity of the antigenic LPS O-polysaccharides.     

Structural studies on the capsular polysaccharide of Klebsiella serotype K40

The capsular polysaccharide of Klebsiella serotype K40 contained D-mannose, D-glucuronic acid, D-galactose, and L-rhamnose in the approximate molar ratios 1:1:1:2. The primary structure of the capsular polysaccharide has been investigated mainly by methylation analysis, periodate oxidation, characterization of oligosaccharides, base degradation reaction, and 1H and 13CNMR spectroscopy. The polysaccharide does not contain any pyruvic acetal or O-acetyl substitution. It has a pentasaccharide repeating unit of the following primary structure: alpha-D-Manp 1----4 ----4)-beta-D-GlcpA-(1----2)-alpha-L-Rhap-(1----3)-beta-D-Ga lp-(1----2)-alpha- L-Rhap-(1----.     

Characterization of the lipopolysaccharide O antigens of Actinobacillus pleuropneumoniae serotypes 9 and 11: antigenic relationships among serotypes 9, 11, and 1.

The antigenic lipopolysaccharide O polysaccharides of capsular serotypes 9 and 11 were examined by chemical, immunological, and nuclear magnetic resonance methods. Immunodiffusion tests carried out on these O antigens indicated that both contained common epitopes which were also shared by Actinobacillus pleuropneumoniae serotype 1. Chemical analysis and high-field nuclear magnetic resonance spectroscopy showed that the O antigens of serotypes 9 and 11 were high-molecular-weight polymers consisting of a backbone of repeating trisaccharide units composed of alpha-L-rhamnopyranosyl and alpha-D-glucopyranosyl residues (2:1). One of the alpha-L-rhamnose units forms a branch point and is stoichiometrically substituted with terminal 2-acetamido-2-deoxy-beta-D-glucose residues in the serotype 11 O polysaccharide, but only to the extent of 25% in the serotype 9 O polysaccharide. Thus, the serotype 9 O polysaccharide contains two different repeating units: a tetrasaccharide unit with the same structure as that of the serotype 11 O polysaccharide and a trisaccharide unit: [formula: see text] where R = beta-D-GlcpNAc for serotype 1 and 11 O polysaccharides, and R = H (75%) and R = beta-D-GlcpNAc (25%) for serotype 9. The structure of the previously determined serotype 1 O polysaccharide (E. Altman, J.-R. Brisson, and M. B. Perry, Biochem. Cell. Biol. 64:17-25, 1986) is identical to that of the serotype 11 O polysaccharide. We propose a more complete serotyping scheme for A. pleuropneumoniae which includes designation of both the capsular (K) and O antigens.     

Structure of the serotype f polysaccharide antigen of Streptococcus mutans

The structure of the serotype f polysaccharide antigen of Streptococcus mutans was determined by methylation analysis, periodate oxidation, and partial methanolysis, and the configuration of the anomeric linkages by 13C-n.m.r. spectroscopy, indicating the trisaccharide repeating unit----3)-alpha-L-Rhap-(1----2)-[alpha-D-Glcp-(1----3)]-alpha-L-+ ++Rhap- (1----. The structure of the backbone of the polysaccharide was confirmed by demonstrating immunological identity between the product of Smith degradation of the S. mutans serotype f antigen and the group A-variant streptococcal polysaccharide.     

Antigenic polysaccharides of bacteria. 34. Structure of O-specific polysaccharide chains of lipopolysaccharides from Pseudomonas cepacia strains IMV 4207 (Serotype A) and IMV 598/2

On the basis of non-destructive analysis by means of 1H and 13C NMR spectroscopy and calculation of specific optical rotation, it was concluded that O-specific polysaccharide of Pseudomonas cepacia strain IMV 4207 (serotype A) has the structure (I): (formula; see text) Two structurally different polysaccharides were found in the ratio of approximately 2.5:1 in P. cepacia strain IMV 598/2 which is serologically related to serotype A in Nakamura classification and serotype 2 in Heidt classification. The minor polysaccharide has the structure (I) whereas the major one possesses the structure (II) which is characteristic of the formerly studied O-specific polysaccharide of P. cepacia strain IMV 4137 belonging to serotype 2: ----4)-beta-D-Galp-(1----2)-alpha-L-Rhap-(1----.     

Partial deletion of the cloned rfb gene of Escherichia coli O9 results in synthesis of a new O-antigenic lipopolysaccharide.

The rfb gene, involved in the synthesis of the O-specific polysaccharide (a mannose homopolymer) of Escherichia coli O9 lipopolysaccharide (LPS), was cloned in E. coli K-12 strains. The O9-specific polysaccharide covalently linked to the R core of K-12 was extracted from the K-12 strains harboring the O9 rfb gene. All the other genes required for the synthesis of rfe-dependent LPS are therefore considered to be present in the K-12 strains. It was found that bacteria harboring some clones with deletions of the ca. 20-kilobase-pair (kbp) BglII-StuI fragment no longer synthesized the O9-specific polysaccharide. However, bacteria harboring clones del 21, del 22, and del 25, which carry deletions of the 10-kbp PstI-StuI fragment, synthesized an O-specific polysaccharide antigenically distinct from E. coli O9 LPS. Although this new O-specific polysaccharide consisted solely of mannose and the mannose residues were combined only through alpha-1,2 linkage, it was still composed of a repeating oligosaccharide unit, possibly a trisaccharide unit,----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----. It is therefore likely that this new O-specific polysaccharide was derived from a part of the O9-specific polysaccharide----3)alpha Man-(1----3)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----2)alpha Man-(1----and that the deleted part of the clones was responsible for the synthesis of alpha-1,3 linkages of the O9-specific polysaccharide.     

Structure of the extracellular gelling polysaccharide produced by Enterobacter (NCIB 11870) species

The gelling polysaccharide produced by a species of Enterobacter (NCIB 11870) contains L-fucose, D-glucose, and D-glucuronic acid in the ratios 1:2:1. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide revealed terminal non-reducing glucose, (1----3)-linked fucose, (1----3,1----4)-linked glucose, and (1----4)-linked glucuronic acid in the ratios 1:1:1.2:0.8. From the results of Smith degradation of the polysaccharide and spectroscopic studies of the acidic tetra- and octa-saccharides produced by bacteriophage-induced enzymic depolymerization of the polysaccharide, the following tetrasaccharide repeating-unit is proposed. (Formula: see text). This repeating-unit is identical to that of the capsular polysaccharide produced by Klebsiella aerogenes serotype K54 except for the absence of O-acetyl groups. The effects of the O-acetyl groups on the secondary structure and rheological properties of these polysaccharides are discussed.     

Structures of the O-specific polymers from the lipopolysaccharides of the reference strains for Pseudomonas cepacia serogroups O3 and O5

The putative O-specific polymers of lipopolysaccharides from two reference strains of Pseudomonas cepacia have been isolated and characterized. Both polymers have disaccharide repeating-units. Structure 1 was established for the O3 polymer, and structure 2 for the O5 polymer. Polymers with the same repeating units have been found previously as the O antigens of other bacteria. ----2)-beta-D-Ribf-(1----4)-alpha-D-GalpNAc-(1---- ----4)-alpha-L-Rhap-(1----3)-beta-D-ManpNAc-(1----     

Structure of the O-chain of the lipopolysaccharide of Pasteurella haemolytica serotype T3

Cell-wall lipopolysaccharide isolated from Pasteurella haemolytica serotype T3 using the phenol-water extraction procedure was shown to be an S type lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Hydrolysis with mild acid afforded a lipid-free, antigenic O-chain polysaccharide. On the basis of one- and two-dimensional 1H and 13C nuclear magnetic resonance studies, in conjunction with microanalytical chemical methods, the O-polysaccharide was determined to be a linear polymer of a disaccharide repeating unit having the structure. [----3)-beta-D-G1cpNAc-(1----4)-alpha-L-Rhap-(1----]n     

Somatic antigens of Pseudomonas aeruginosa. The structure of the O-specific polysaccharide chain of the lipopolysaccharide from P. aeruginosa O13 (Lányi)

The O-specific polysaccharide, obtained on mild acid degradation of lipopolysaccharide of Pseudomonas aeruginosa O13 (Lányi classification), is built up of trisaccharide repeating units involving 2-acetamidino-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), 2-acetamidino-2,6-dideoxy-L-galactose (L-fucosacetamidine, L-FucAm), and a new sialic-acid-like sugar, 5,7-diacetamido-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonuloso n ic acid (Sug), and thus contains simultaneously both acidic and basic functions. Cleavage of the polysaccharide with hydrogen fluoride in methanol revealed the high stability of the glycosidic linkage of the ulosonic acid and afforded methyl glycosides of a disaccharide and a trisaccharide. The structures of the new ulosonic acid and acetamidino group were established by analysing the oligosaccharide fragments by 1H, 13C nuclear magnetic resonance spectrometry, as well as on the basis of their chemical conversions: alkaline hydrolysis of the acetamidino group into acetamido group, reductive deamination with lithium borohydride into the ethylamino group and acetylation with acetic anhydride in pyridine accompanied by intramolecular acylation of the acetamidino function by the ulosonic acid to form a six-membered lactam ring. Identification of the oligosaccharide fragments and comparative analysis of the 13C nuclear magnetic resonance spectra of the oligosaccharides and polysaccharide revealed the following structure of the repeating unit: ----3)D-QuiNAcp(alpha 1----3)Sugp(alpha 2----3)L-FucAmp(alpha 1----.     

Structure of the K95 antigen from Escherichia coli O75:K95:H5, a capsular polysaccharide containing furanosidic KDO-residues

The structure of the K95 antigenic capsular polysaccharide (K95 antigen) of Escherichia coli O75:K95:H5 was elucidated by determination of the composition, 1H- and 13C-n.m.r. spectroscopy, periodate oxidation, and methylation analysis. The K95 polysaccharide, which contains furanosidic 3-deoxy-D-manno-2-octulosonic acid (KDOf) residues, consists of----3)-beta-D-Rib-(1----8)-KDOf-(2----repeating units, has a molecular weight of approximately 25,000 (approximately 65 repeating units), and is randomly O-acetylated (1 acetyl group per repeating unit at unknown positions).