Show legend Show as text

Structure type: polymer chemical repeating unit
Compound class: EPS, cell wall polysaccharide
Contained glycoepitopes: IEDB_136095,IEDB_136906,IEDB_137472,IEDB_141794,IEDB_142488,IEDB_146664,IEDB_151528,IEDB_190606,IEDB_983931,SB_192,SB_7

• Article ID: 106
  Tone-Shimokawa Y, Toida T, Kawashima T "Isolation and structural analysis of polysaccharide containing galactofuranose from the cell walls of Bifidobacterium infantis" - Journal of Bacteriology 178(1) (1996) 317-320
  

  We isolated cell wall polysaccharides (PS-1 and PS-2) from Bifidobacterium infantis Reuter ATCC 15697 and found that the backbone of PS-2 is →3)-β-D-Galf-(1→3)-α-D-Galp-(1→ in which β-D-Galf and α-D-Galp are partially substituted at O-6 with β-D-Glcp. This is the first report of the presence of this disaccharide backbone in a gram-positive bacterium; it resembles the O antigen of some bacteria.

  structural, polysaccharide, analysis, cell, cell wall, structural analysis, isolation, Bifidobacterium, galactofuranose

  NCBI PubMed ID: 8550438
  Publication DOI: 10.1128/jb.178.1.317-320.1996
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Institutions: Biochemical Research Laboratory, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 228, Japan.
  Methods: methylation, NMR-2D, NMR, Smith degradation
  
   
  
  Bifidobacterium infantis Reuter ATCC 15697
  CSDB ID 5215 (all data & tools)
• Article ID: 5832
  Pyclik M, Srutkova D, Schwarzer M, Gorska S "Bifidobacteria cell wall-derived exo-polysaccharides, lipoteichoic acids, peptidoglycans, polar lipids and proteins - their chemical structure and biological attributes" - International Journal of Biological Macromolecules 147 (2020) 333-349
  

  A variety of health benefits has been documented to be associated with the consumption of probiotic bacteria, namely bifidobacteria and lactobacilli. Thanks to the scientific advances in recent years we are beginning to understand the molecular mechanisms by which bacteria in general and probiotic bacteria in particular act as host physiology and immune system modulators. More recently, the focus has shifted from live bacteria towards bacteria-derived defined molecules, so called postbiotics. These molecules may represent safer alternative compared to the live bacteria while retaining the desired effects on the host. The excellent source of effector macromolecules is the bacterial envelope. It contains compounds that are pivotal in the adhesion phenomenon, provide direct bacteria-to-host signaling capacity and the associated physiological impact and immunomodulatory properties of bacteria. Here we comprehensively review the structure and biological role of Bifidobacterium surface and cell wall molecules: exopolysaccharides, cell wall polysaccharides, lipoteichoic acids, polar lipids, peptidoglycans and proteins. We discuss their involvement in direct signaling to the host cells and their described immunomodulatory effects.

  exopolysaccharide, Bacterial antigens, Bifidobacterium, peptidoglycan, lipoteichoic acid, probiotics

  NCBI PubMed ID: 31899242
  Publication DOI: 10.1016/j.ijbiomac.2019.12.227
  Journal NLM ID: 7909578
  Publisher: Butterworth-Heinemann
  Correspondence: schwarzer@biomed.cas.cz; sabina.gorska@hirszfeld.pl
  Institutions: Laboratory of Microbiome Immunobiology, Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland, Laboratory of Gnotobiology, Institute of Microbiology of the Czech Academy of Sciences, Novy Hradek, Czech Republic
  
   
  
  Bifidobacterium longum ssp. infantis ATCC 15697
  CSDB ID 6477 (all data & tools)

Show legend Show as text

Structure type: polymer chemical repeating unit
Trivial name: D-galactan I, galactan I, galactan, D-galactan, microbial carbohydrate determinants, D-galactan-I
Compound class: CPS, EPS, O-polysaccharide, O-antigen, LPS, cell wall polysaccharide
Contained glycoepitopes: IEDB_136095,IEDB_136906,IEDB_137472,IEDB_141794,IEDB_151528,IEDB_190606,SB_7

• Article ID: 106
  Tone-Shimokawa Y, Toida T, Kawashima T "Isolation and structural analysis of polysaccharide containing galactofuranose from the cell walls of Bifidobacterium infantis" - Journal of Bacteriology 178(1) (1996) 317-320
  

  We isolated cell wall polysaccharides (PS-1 and PS-2) from Bifidobacterium infantis Reuter ATCC 15697 and found that the backbone of PS-2 is →3)-β-D-Galf-(1→3)-α-D-Galp-(1→ in which β-D-Galf and α-D-Galp are partially substituted at O-6 with β-D-Glcp. This is the first report of the presence of this disaccharide backbone in a gram-positive bacterium; it resembles the O antigen of some bacteria.

  structural, polysaccharide, analysis, cell, cell wall, structural analysis, isolation, Bifidobacterium, galactofuranose

  NCBI PubMed ID: 8550438
  Publication DOI: 10.1128/jb.178.1.317-320.1996
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Institutions: Biochemical Research Laboratory, Morinaga Milk Industry Co., Ltd., Zama, Kanagawa 228, Japan.
  Methods: methylation, NMR-2D, NMR, Smith degradation
  
   
  
  Bifidobacterium infantis Reuter ATCC 15697, Pasteurella haemolytica
  CSDB ID 334 (all data & tools)
• Article ID: 183
  Vinogradov E, Frirdich E, MacLean LL, Perry MB, Petersen BO, Duus JO, Whitfield C "Structures of lipopolysaccharides from Klebsiella pneumoniae. Eluicidation of the structure of the linkage region between core and polysaccharide O chain and identification of the residues at the non- reducing termini of the O chains" - Journal of Biological Chemistry 277(28) (2002) 25070-25081
  

  Deamination of LPSs from Klebsiella pneumoniae released O-chain polysaccharides together with a fragment of the core oligosaccharide. The structures of the products from serotypes O1, O2a, O2a,c, O3, O4, O5, and O12 were determined by NMR spectroscopy and chemical methods, identifying the linkage region between the O antigens and the core as well as novel residues at the non-reducing ends of the polysaccharides. All serotypes had an identical linkage between the O chain and core.

  lipopolysaccharides, oligosaccharide, structure, core oligosaccharide, NMR spectroscopy, O-antigens, linkage, region, O-chain, Klebsiella pneumoniae, reducing

  NCBI PubMed ID: 11986326
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: evguenii.vinogradov@nrc.ca
  Institutions: Institute for Biological Sciences, National Research Council, Ottawa, Ontario K1A 0R6, Canada, Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada, Carlsberg Laboratory, Department of Chemistry, Gamle Carlsberg Vej 10, DK-2500 Valby, Copenhagen, Denmark
  Methods: NMR-2D, NMR, serological methods, genetic methods
  
   
  
  Klebsiella pneumoniae O1, Klebsiella pneumoniae O2a, Klebsiella pneumoniae O2a,c
  CSDB ID 5760 (all data & tools)
• Article ID: 215
  Warnecke JM, Nitschke M, Moolenaar CEC, Rietschel ET, Hartmann RK, Mamat U "The 5'-proximal hairpin loop of lbi RNA is a key structural element in repression of D-galactan II biosynthesis in Klebsiella pneumoniae serotype O1" - Molecular Microbiology 36(3) (2000) 697-709
  

  The lbi (lipopolysaccharide biosynthesis interfering) RNA of phage Acm1, an untranslated RNA transcript of 97 nucleotides, previously shown to affect Opolysaccharide biosynthesis in various Escherichia coli strains, was found to downregulate the synthesis of the D-galactan II component of the O-specific polysaccharide in Klebsiella pneumoniae serotype O1. Enzymatic and Pb21 probing experiments revealed that lbi RNA consists of two consecutive stemloop structures, the 50-proximal hairpin loop of 15 nucleotides being particularly accessible to single strand-specific probes. Based on the assumption that the 50-proximal hairpin loop may be involved in an antisense interaction with cellular target RNAs, we randomly mutagenized one or two of its central nucleotides. Expression of mutated lbi RNA variants in K. pneumoniae serotype O1 relieved at least partly the repression of D-galactan II formation. In addition, a truncated version of lbi RNA lacking the 30-proximal hairpin loop was almost as efficient as the wild-type RNA in downregulating D-galactan II synthesis. The results obtained indicate that the 50-proximal hairpin loop of lbi RNA functions as a key structural element in the mechanism leading to the inhibition of D-galactan II biosynthesis in K. pneumoniae serotype O1.

  biosynthesis, structural, serotype, Klebsiella, Klebsiella pneumoniae, D-galactan, repression, RNA

  NCBI PubMed ID: 10844658
  Journal NLM ID: 8712028
  Publisher: Blackwell Publishing
  Correspondence: umamat@fz-borstel.de
  Institutions: Institute of Biochemistry, Department of Nephrology, Medical University of Lubeck, Ratzeburger Allee160, D-23538 Lubeck, Germany, Department of Immunochemistry and Biochemical Microbiology, Research Center Borstel, Center for Medicine and Biosciences, Borstel, Germany
  Methods: genetic methods
  
   
  
  Klebsiella pneumoniae
  CSDB ID 5562 (all data & tools)
• Article ID: 247
  Guan S, Clarke AJ, Whitfield C "Functional analysis of the galactosyltransferases required for biosynthesis of D-galactan I, a component of the lipopolysaccharide O1 antigen of Klebsiella pneumoniae" - Journal of Bacteriology 183(11) (2001) 3318-3327
  

  D-Galactan I is an O-antigenic polymer with the repeat unit structure [→3)-β-D-Galf-(1→3)-α-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

  Lipopolysaccharide, biosynthesis, antigen, Klebsiella pneumoniae, D-galactan, galactosyltransferase

  NCBI PubMed ID: 11344139
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: cwhitfie@uoguelph.ca
  Institutions: Department of Microbiology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
  
   
  
  Klebsiella pneumoniae O1
  CSDB ID 6876 (all data & tools)
• Article ID: 288
  Kelly RF, Whitfield C "Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species" - Journal of Bacteriology 178(17) (1996) 5205-5214
  

  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 α-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.

  expression, O-antigen, antigens, gene cluster, Klebsiella, D-galactan, rfb gene cluster, species

  NCBI PubMed ID: 8752339
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: cwhitfle@uoguelph.ca
  Institutions: Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, NIG 2W1
  
   
  
  Klebsiella pneumoniae O1
  CSDB ID 7192 (all data & tools)
• Article ID: 393
  Szabo M, Bronner D, Whitfield C "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" - Journal of Bacteriology 177(6) (1995) 1544-1553
  

  The lipopolysaccharide O antigens of Klebsiella pneumoniae serotype O1 and Serratia marcescens serotype O16 both contain a repeating unit disaccharide of [→3)-β-D-Galf-(1→3)-α-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.

  Lipopolysaccharide, biosynthesis, LPS, O-antigen, antigens, gene cluster, Klebsiella, Serratia marcescens, Klebsiella pneumoniae, Serratia, rfb gene cluster

  NCBI PubMed ID: 7533758
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: cwhitfie@micro.uoguelph.ca
  Institutions: Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, N1G 2W1
  Methods: genetic methods
  
   
  
  Klebsiella pneumoniae O1, Serratia marcescens O16
  CSDB ID 8358 (all data & tools)
• Article ID: 472
  Shankar-Sinha S, Valencia GA, Janes BK, Rosenberg JK, Whitfield C, Bender RA, Standiford TJ, Younger JG "The Klebsiella pneumoniae O antigen contributes to bacteremia and lethality during murine pneumonia" - Infection and Immunity 72(3) (2004) 1423-1430
  

  Bacterial surface carbohydrates are important pathogenic factors in gram-negative pneumonia infections. Among these factors, O antigen has been reported to protect pathogens against complement-mediated killing. To examine further the role of O antigen, we insertionally inactivated the gene encoding a galactosyltransferase necessary for serotype O1 O-antigen synthesis (wbbO) from Klebsiella pneumoniae 43816. Analysis of the mutant lipopolysaccharide by sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed the absence of O antigen. In vitro, there were no detectable differences between wild-type K. pneumoniae and the O-antigen-deficient mutant in regard to avid binding by murine complement C3 or resistance to serum- or whole-blood-mediated killing. Nevertheless, the 72-h 50% lethal dose of the wild-type strain was 30-fold greater than that of the mutant (2 x 10(3) versus 6 x 10(4) CFU) after intratracheal injection in ICR strain mice. Despite being less lethal, the mutant organism exhibited comparable intrapulmonary proliferation at 24 h compared to the level of the wild type. Whole-lung chemokine expression (CCL3 and CXCL2) and bronchoalveolar inflammatory cell content were also similar between the two infections. However, whereas the wild-type organism produced bacteremia within 24 h of infection in every instance, bacteremia was not seen in mutant-infected mice. These results suggest that during murine pneumonia caused by K. pneumoniae, O antigen contributes to lethality by increasing the propensity for bacteremia and not by significantly changing the early course of intrapulmonary infection.

  O-antigen, Klebsiella, Klebsiella pneumoniae, pneumonia

  NCBI PubMed ID: 14977947
  Journal NLM ID: 0246127
  Publisher: American Society for Microbiology
  Correspondence: jyounger@umich.edu
  Institutions: Department of Emergency Medicine, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA
  
   
  
  Klebsiella pneumoniae 43816
  CSDB ID 8954 (all data & tools)
• Article ID: 620
  Aucken HM, Wilkinson SG, Pitt TL "Re-evaluation of the serotypes of Serratia marcescens and separation into two schemes based on lipopolysaccharide (O) and capsular polysaccharide (K) antigens" - Microbiology 144 (1998) 639-653
  

  Chemical and serological analysis has revealed that many of the 29 O serotype reference strains of Serratia marcescens contain both neutral and acidic polysaccharides which correspond to LPS O antigens and capsular K antigens, respectively. New O and K antigen typing schemes have therefore been devised, based on the known chemical structures of the surface polysaccharides of the organism. These schemes were designed to allow the specific detection of these antigens on unknown strains using ELISAs. O antigens were detected using whole cells cultured in broth then autoclaved to remove capsular material, while K antigens were detected using formolized whole cells which had been cultured on glycerol agar to enhance capsule production. After testing with the 29 reference strains as well as 423 distinct clinical strains, it was apparent that different aspects of chemical structure were associated with different degrees of serological reactivity and the typing schemes were modified further to accommodate this. In general, the O antigen repeating unit structures were chemically simple with di- or trisaccharide backbones. Serological specificity was often provided solely by the presence or absence of an O-acetyl substituent, or a change in the linkage between two sugar residues. Five of the O serotypes in the new scheme were represented by 12 of the 29 reference strains, while three reference strains lacked O antigens altogether, resulting in the elimination of 10 of the original O types. In contrast, the K antigen repeating unit structures were more complex and chemically diverse, having at least four sugar residues. Three K types were each seen in two reference strains while 12 of the 29 reference strains were acapsular. Thus, the resulting schemes contain 19 O types and 14 K types and allow the definitive serotype identification of S. marcescens.

  Lipopolysaccharide, antigen, LPS, structure, K-antigen, O-antigen, capsular polysaccharide, Serratia marcescens, Serratia, serotyping, O-serotype

  NCBI PubMed ID: 9534235
  Journal NLM ID: 0376646
  Publisher: Washington, DC: Kluwer Academic/Plenum Publishers
  Correspondence: haucken@phls.co.uk
  Institutions: Laboratory of Hospital Infection, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK, School of Chemistry, The University, Hull HU6 7RX, UK
  Methods: NMR, SDS-PAGE, ELISA, chromatography, Quellung reaction, serum adsorption assays
  
   
  
  Serratia marcescens O20
  CSDB ID 263 (all data & tools)
• Article ID: 1307
  Zamze S, Martinez-Pomares L, Jones H, Taylor PR, Stillion RJ, Gordon S, Wong SY "Recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor" - Journal of Biological Chemistry 277(44) (2002) 41613-41623
  

  The in vitro binding of the macrophage mannose receptor to a range of different bacterial polysaccharides was investigated. The receptor was shown to bind to purified capsular polysaccharides from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. Binding was Ca(2+)- dependent and inhibitable with d-mannose. A fusion protein of the mannose receptor containing carbohydrate recognition domains 4-7 and a full-length soluble form of the mannose receptor containing all domains external to the transmembrane region both displayed very similar binding specificities toward bacterial polysaccharides, suggesting that domains 4-7 are sufficient for recognition of these structures. Surprisingly, no direct correlation could be made between polysaccharide structure and binding to the mannose receptor, suggesting that polysaccharide conformation may play an important role in recognition. The full-length soluble form of the mannose receptor was able to bind simultaneously both polysaccharide via the carbohydrate recognition domains and sulfated oligosaccharide via the cysteine-rich domain. The possible involvement of the mannose receptor, either cell surface or soluble, in the innate and adaptive immune responses to bacterial polysaccharides is discussed

  lipopolysaccharides, structure, Streptococcus pneumoniae, capsular polysaccharides, recognition, Klebsiella pneumoniae, Mannose, macrophage, receptor

  NCBI PubMed ID: 12196537
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: susanne.zamze@jenner.ac.uk
  Institutions: Edward Jenner Institute for Vaccine Research, Compton, Berkshire RG20 7NN, United Kingdom and the Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom
  
   
  
  Klebsiella pneumoniae O1
  CSDB ID 6088 (all data & tools)
• Article ID: 1403
  Clarke BR, Bronner D, Keenleyside WJ, Severn WB, Richards JC, Whitfield C "Role of Rfe and RfbF in the initiation of biosynthesis of D-galactan I, the lipopolysaccharide O antigen from Klebsiella pneumoniae serotype O1" - Journal of Bacteriology 177(19) (1995) 5411-5418
  

  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: 33-b-D-Galf-133-a-D-Galp-13(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 b-D-Galf-133-a-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 Mr of 42,600. A polypeptide with an Mr of 42,000 was evident in sodium dodecyl sulfate-polyacrylamide gels when rfbKpO1 was expressed behind the T7 promoter. The carboxyterminal 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.

  Lipopolysaccharide, biosynthesis, antigen, LPS, role, serotype, O-antigen, O antigen, Klebsiella, Klebsiella pneumoniae, D-galactan, initiation

  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  Correspondence: cwhitfie@micro.uoguelph.ca
  Institutions: Department of Microbiology, University of Guelph, Guelph, Canada
  Methods: genetic methods, biochemical methods
  
   
  
  Klebsiella pneumoniae O1
  CSDB ID 6533 (all data & tools)
• Article ID: 1674
  Whitfield C, Valvano MA "Biosynthesis and expression of cell-surface polysaccharides in gram-negative bacteria" - Advances in Microbial Physiology 35 (1993) 135-246
  

  This chapter provides an overview of the molecular mechanisms involved in synthesis and expression of cell-surface polysaccharides in Gram-negative bacteria. Biosynthesis of many cell-surface components, including polysaccharides, involves enzymes and enzyme complexes found in the cytoplasmic membrane. The peptidoglycan layer is located immediately external to the cytoplasmic membrane and this layer is required for cell shape and rigidity. Gram-negative bacteria possess a periplasm that contains a variety of proteins and enzymes, including some involved in import and export of macromolecules. Biosynthesis of bacterial cell-surface polysaccharides involves a series of sequential processes: (1) biosynthesis of activated precursors in the cytoplasm, (2) formation of repeating units, (3) polymerization of repeating units, and (d) export of polysaccharides to the cell surface. The assembly of polysaccharide repeating units and subsequent polymerization reactions occur at the cytoplasmic membrane, using precursors synthesized in the cytoplasm. Genes for biosynthesis of cell-surface polysaccharides are chromosomal and are arranged in clusters of one or more transcriptional units. The synthesis of lipopolysaccharide (LPS) may be subject to complex regulation, but on-off switching is not possible due to the essential structural requirement for the lipid A-core LPS molecule. Most bacteria use extracellular polysaccharides (EPSs) for protection, and many regulatory strategies are directed to modulating EPS synthesis in response to appropriate environmental cues. Application of genetic and biochemical approaches has facilitated detailed analysis of complex, multicomponent systems, such as those involved in synthesis of cell-surface polysaccharides.

  NCBI PubMed ID: 8310880
  Publication DOI: 10.1016/S0065-2911(08)60099-5
  Journal NLM ID: 0117147
  Institutions: Department of Microbiology, University of Guelph, Ontario, Canada, Department of Microbiology, University of Guelph, Guelph, Ontario, Canada, Department of Microbiology and Immunology, University of Western Ontario, London, Ontario, Canada, N6A 5C1
  
   
  
  Klebsiella pneumoniae 1, Klebsiella pneumoniae 2a, Pasteurella haemolytica 4, Pasteurella haemolytica 10, Serratia marcescens 16, Serratia marcescens 20
  CSDB ID 101044 (all data & tools)
• Article ID: 1778
  Knirel YA, Kochetkov NK "The structure of lipopolysaccharides of gram-negative bacteria. III. The structure of O-antigens: A review" - Biochemistry (Moscow) 59(12) (1994) 1325-1383
  

  This review summarizes data on the composition and structure of the O-antigens, the polysaccharide chains of the outer-membrane lipopolysaccharides (LPS) of Gram-negative bacteria defining the immunospecificity of these microbial cells. Special reference is given to some structural features of the O-antigens, such as the presence of unique monosaccharides and noncarbohydrate components, masked regularity, and the occurrence in one microorganism of LPS with structurally different polysaccharide chains. Antigenic relationships between microorganisms belonging to different taxonomic groups are discussed.

  structure, O-antigen, chemical composition, bacterial lipopolysaccharides, Salmonella livingstone C1

  NCBI PubMed ID: 7533007
  Journal NLM ID: 0376536
  Publisher: Nauka/Interperiodica
  Institutions: Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
  
   
  
  Klebsiella pneumoniae O1 I, Klebsiella pneumoniae O2a,2b,2c, Klebsiella pneumoniae O8, Serratia marcescens O16, Serratia marcescens O20, Serratia plymuthica 1 I, Pasteurella haemolytica T4, Pasteurella haemolytica T10
  CSDB ID 108573 (all data & tools)
• Article ID: 1910
  Perry MB, Babiuk LA "Structure of the polysaccharide chain of Pasteurella haemolytica (serotype 4) lipopolysaccharide" - Canadian Journal of Biochemistry and Cell Biology = Revue canadienne de biochimie et biologie cellulaire 62 (1984) 108-114
  

  The antigenic polysaccharide side chain of the cell wall lipopolysaccharide of Pasteurella haemolytica (serotype 4) was investigated by methylation, periodate oxidation, partial hydrolysis, and 13C and 1H nuclear magnetic resonance analysis methods and was found to be a simple unbranched linear polymer composed of a disaccharide repeating unit having the structure —3)-α-D-Galp-(1—3)-β-D-Galf-(1—.

  Publication DOI: 10.1139/o84-016
  Journal NLM ID: 8302763
  Publisher: Ottawa: National Research Council Of Canada
  Institutions: Division of Biological Sciences, National Research Council of Canada, Ottawa, Ontario K1A OR6 Canada
  Methods: 13C NMR, 1H NMR, methylation, periodate oxidation, GLC-MS, gel filtration, SDS-PAGE, sugar analysis, GLC, paper chromatography, optical rotation measurement
  
   
  
  Pasteurella haemolytica 4
  CSDB ID 112121 (all data & tools)
• Article ID: 2133
  Richards JC, Leitch RA "Elucidation of the structure of the Pasteurella haemolytica serotype T10 lipopolysaccharide O-antigen by NMR spectroscopy" - Carbohydrate Research 186(2) (1989) 275-286
  

  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)-α-D-Galp-(1→3)-β-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.

  NCBI PubMed ID: 2472201
  Publication DOI: 10.1016/0008-6215(89)84041-8
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Institutions: Division of Biological Sciences, National Research Council of Canada, Ottawa, Ont.
  Methods: 13C NMR, 1H NMR
  
   
  
  Pasteurella haemolytica T10
  CSDB ID 115616 (all data & tools)
• Article ID: 2149
  Oxley D, Wilkinson SG "Structures of neutral glycans isolated from the lipopolysaccharides of reference strains for Serratia marcescens serogroups O16 and O20" - Carbohydrate Research 193 (1989) 241-248
  

  Neutral polymers have been isolated from the lipopolysaccharides of the reference strains for Serratia marcescens O16 and O20, serogroups which exhibit significant cross-reactivity. Both organisms produce a galactan with the disaccharide repeating-unit shown, and which apparently accounts for the serological observations. The same galactan has also been reported as the O4-specific polysaccharide of Pasteurella haemolytica. In S. marcescens O16, the galactan is apparently accompanied by a polymer of 2-substituted β-D-ribofuranosyl residues.

  NCBI PubMed ID: 2692814
  Publication DOI: 10.1016/0008-6215(89)85122-5
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Institutions: School of Chemistry, The University, Hull, Great Britain
  Methods: 13C NMR, 1H NMR, methylation, GLC-MS, GLC
  
   
  
  Serratia marcescens 16, Serratia marcescens 20
  CSDB ID 115766 (all data & tools)
• Article ID: 2153
  Oxley D, Wilkinson SG "Structure of the O-specific galactan from the lipopolysaccharide of the reference strain for Serratia marcescens serogroup O24" - Carbohydrate Research 195 (1989) 117-122
  

  The putative O-specific polysaccharide for Serratia marcescens serogroup O24 is a galactan with a branched, trisaccharide repeating-unit of the structure shown. The structure of the backbone is identical to that of the linear galactans isolated from the reference strains for S. marcescens serogroups O16 and O20, presumably accounting for the serological cross-reactions observed. (Formula: see text)

  NCBI PubMed ID: 2699831
  Publication DOI: 10.1016/0008-6215(89)85094-3
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Institutions: School of Chemistry, The University, Hull (Great Britain)
  Methods: 13C NMR, 1H NMR, methylation, GLC-MS, GLC
  
   
  
  Serratia marcescens 24
  CSDB ID 115800 (all data & tools)
• Article ID: 2234
  Kol O, Wieruszeski JM, Strecker G, Fournet B, Zalisz R, Smets P "Structure of the O-specific polysaccharide chain of Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide. A complementary elucidation" - Carbohydrate Research 236 (1992) 339-344
  

  No abstract available

  NCBI PubMed ID: 1291059
  Publication DOI: 10.1016/0008-6215(92)85028-X
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Institutions: Laboratoire de Chimie Biologique de l'Université des Sciences et Techniques de Lille-Flandres Artois, Villeneuve d'Ascq, France.
  Methods: 13C NMR, 1H NMR, periodate oxidation, GLC-MS, sugar analysis, GLC, HPLC
  
   
  
  Klebsiella pneumoniae O1:K2
  CSDB ID 116981 (all data & tools)
• Article ID: 2489
  Aucken HM, Oxley D, Wilkinson SG "Structural and serological characterisation of an O-specific polysaccharide from Serratia plymuthica" - FEMS Microbiology Letters 111 (1993) 295-300
  

  The surface polysaccharides of a strain of Serratia plymuthica were characterised and shown to consist of a linear, acidic galactoglucomannan as well as a major and a minor neutral galactan. Immunoblotting results demonstrated cross-reactions between this strain and others with similar galactans (S. marcescens O16 and O20, Klebsiella O1, and Pasteurella haemolytica T4 and T10).

  NCBI PubMed ID: 7691682
  Journal NLM ID: 7705721
  Publisher: Blackwell Publishing
  Institutions: Laboratory of Hospital Infection, Central Public Health Laboratory, London, UK
  
   
  
  Serratia plymuthica
  CSDB ID 126040 (all data & tools)
• Article ID: 2592
  Whitfield C, Richards JC, Perry MB, Clarke BR, MacLean LL "Expression of two structurally distinct D-galactan O antigens in the lipopolysaccharide of Klebsiella pneumoniae serotype O1" - Journal of Bacteriology 173 (1991) 1420-1431
  
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  
   
  
  Klebsiella pneumoniae 1
  CSDB ID 131766 (all data & tools)
• Article ID: 2598
  Whitfield C, Perry MB, MacLean LL, Yu SH "Structural analysis of the O-antigen side chain polysaccharides in the lipopolysaccharides of Klebsiella serotypes O2(2a), O2(2a,2b), and O2(2a,2c)" - Journal of Bacteriology 174 (1992) 4913-4919
  
  Journal NLM ID: 2985120R
  Publisher: American Society for Microbiology
  
   
  
  Klebsiella sp. 2
  CSDB ID 131783 (all data & tools)
• Article ID: 2777
  Kelly RF, Severn WB, Richards JC, Perry MB, MacLean LL, Tomas JM, Merino S, Whitfield C "Structural variation in the O-specific polysaccharides of Klebsiella pneumoniae serotype O1 and O8 lipopolysaccharide: evidence for clonal diversity in rfb genes" - Molecular Microbiology 10 (1993) 615-625
  

  The O-polysaccharide fraction of the lipopolysaccharide from Klebsiella pneumoniae serotype O8 was found to comprise two galactose-containing homopolymers. Structural analysis, using chemical and high-field nuclear magnetic resonance (NMR) techniques, established that the K. pneumoniae O8 polysaccharides are composed of the linear, disaccharide repeating units [formula: see text] K. pneumoniae O8 mutant RFK-1 was isolated by resistance to phage KO1-2; strain RFK-1 expressed only D-galactan I-OAc. The 1H- and 13C-NMR resonances from this O-polysaccharide indicate that all of the O-acetyl groups within the K. pneumoniae O8 polysaccharide are carried on D-galactan I and O-acetylation occurs only on the β-D-galactofuranose residues; 60% of the available β-D-galactofuranose residues are non-acetylated. The O-acetylation of the remaining residues is equally distributed between the O-2 and O-6 positions. The carbohydrate backbone structures in the O8 polysaccharide are identical to D-galactan I and II expressed by K. pneumoniae O1, accounting for the antigenic cross-reaction between strains belonging to serotypes O1 and O8. However, the O1 polysaccharides are not acetylated and the O-acetyl groups present in the K. pneumoniae serotype O8 polysaccharides provide a structural basis for their recognition as distinct serotypes. The rfb (O-polysaccharide biosynthesis) gene cluster of K. pneumoniae serotype O1 determines the synthesis of D-galactan I. rfbKpO1-specific gene probes were used to examine conservation in the rfb gene clusters of other K. pneumoniae serotypes which produce D-galactan I. Six O1 strains were examined and all showed hybridization with rfbKpO1 probes under conditions of high stringency. Three serotype O2 strains produce D-galactan I and these strains also contained DNA sequences recognized by rfbKpO1 probes under high stringency. The physical maps of these homologous rfb chromosomal regions showed some polymorphism. Surprisingly, the rfbKpO8 region from K. pneumoniae serotype O8 was only recognized by rfbKpO1 probes under low-stringency hybridization conditions, providing evidence for two substantially different clonal groups of rfb genes from K. pneumoniae strains with structurally related O-antigens.

  NCBI PubMed ID: 7526122
  Journal NLM ID: 8712028
  Publisher: Blackwell Publishing
  Institutions: Department of Microbiology, University of Guelph, Ontario, Canada
  
   
  
  Klebsiella pneumoniae 8
  CSDB ID 144074 (all data & tools)
• Article ID: 3487
  Katzenellenbogen E, Toukach FV, Kocharova NA, Korzeniowska-Kowal A, Gamian A, Shashkov AS, Knirel YA "Structure of a phosphoethanolamine-containing O-polysaccharide of Citrobacter freundii strain PCM1443 from serogroup O39 and its relatedness to the Klebsiella pneumoniae O1 polysaccharide" - FEMS Immunology and Medical Microbiology 53(1) (2008) 60-64
  

  Lipopolysaccharide was extracted from cells of Citrobacter freundii PCM 1443 from serogroup O39 and degraded by mild acid hydrolysis to give an O-polysaccharide. Based on enzymatic and methylation analyses, along with 1H and 13C nuclear magnetic resonance spectroscopy, it was found that the lipopolysaccharide studied has two different linear polysaccharide chains of d-galactan type containing 3-substituted galactose residues. One of the galactans has the disaccharide repeating units of α-D-galactopyranose and β-D-galactofuranose and the other is comprised of α-D-galactopyranose and β-D-galactopyranose, the latter being substituted in 25% repeats with PEtN at O-6. An immunoblotting assay demonstrated that the lipopolysaccharide of C. freundii PCM 1443 is serologically related to that of Klebsiella pneumoniae O1, which contains the same galactan chains but is devoid of phosphoethanolamine.

  Lipopolysaccharide, serological specificity, Citrobacter freundii, Galactan, O-Polysaccharide structure

  NCBI PubMed ID: 18371072
  Journal NLM ID: 9315554
  Publisher: Elsevier
  Correspondence: katzenel@iitd.pan.wroc.pl
  Institutions: L Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla, Wroclaw, Poland
  Methods: 13C NMR, 1H NMR, methylation, GLC-MS, NMR-2D, SDS-PAGE, sugar analysis, acid hydrolysis, serological methods
  
   
  
  Citrobacter freundii O39 PCM 1443
  CSDB ID 23000 (all data & tools)
• Article ID: 3640
  Kelly RF, Perry MB, MacLean LL, Whitfield C "Structures of the O-antigens of Klebsiella serotypes 02 (2a,2e), 02 (2a,2e,2h), and 02 (2a,2f,2g), members of a family of related D-galactan O-antigens in Klebsiella spp." - Journal of Endotoxin Research 2(2) (1995) 131-140
  

  The structures of the lipopolysaccharide O-antigens from Klebsiella serotypes 02(2a,2f,2g), O2(2a,2e) and O2(2a,2e,2h) have been determined. These O-polysaccharides are part of a family of related structures, which share a D-galactan I backbone. D-galactan I has the repeating unit structure: [→3)-β-D-Galf-(1→ 3)-α-D-Galp-(1→]. The O-polysaccharide of serotype O2(2a,2f,2g) differs from other known O-polysaccharides in Klebsiella spp. Each of the main-chain Galp residues in the 02(2a,2f,2g) O-polysaccharide is substituted with an α-(1→4)-linked D-Galp residue, to form a trisaccharide repeating unit. The LPS O-polysaccharides of serotypes O2(2a,2e) and O2(2a,2e,2h) both contain α-(1→2)-linked D-Galp substituents on the main-chain Galp residues, and resemble serotype O9. The only difference between the 09, O2(2a,2e), and O2(2a,2e,2h) carbohydrate structures involves the stoichiometry of addition of side chain-D-Galp residues. However, the polymers from serotypes O2(2a,2e), O2(2a,2e,2h) and 09 are all modified by O-acetylation and these modifications may contribute to altered antigenic factors. The structures reported here resolve ambiguities between previous chemical and serological analyses of 02 antigens. Genetic analyses showed that enzymes involved in the addition of α-D-Galp residues are encoded by genes outside the rfbO9 (O-antigen biosynthesis) region and this provides an explanation for occasional non-stoichiometric addition of side chain α-D-Galp residues.

  structure, serotype, O-antigen, endotoxin, Klebsiella, D-galactan

  Publication DOI: 10.1177/096805199500200208
  Journal NLM ID: 9433350
  Publisher: Maney Publishing
  Institutions: Canadian Bacterial Diseases Network, Department of Microbiology, University of Guelph, Guelph, Ontario, Institute for Biological Sciences, National Research Council of Canada, Ottawa, Ontario, Canada
  Methods: genetic methods
  
   
  
  Klebsiella sp. O2
  CSDB ID 23607 (all data & tools)
• Article ID: 3769
  Richards MR, Lowary TL "Chemistry and biology of galactofuranose-containing polysaccharides" - Chembiochem: a European Journal of Chemical Biology 10(12) (2009) 1920-1938
  

  The thermodynamically less stable form of galactose-galactofuranose (Galf)-is essential for the viability of several pathogenic species of bacteria and protozoa but absent in this form in mammals, so the biochemical pathways by which Galf-containing glycans are assembled and catabolysed are attractive sites for drug action. This potential has led to increasing interest in the synthesis of molecules containing Galf residues, their subsequent use in studies directed towards understanding the enzymes that process these residues and the identification of potential inhibitors of these pathways. Major achievements of the past several years have included an in-depth understanding of the mechanism of UDP-galactopyranose mutase (UGM), the enzyme that produces UDP-Galf, which is the donor species for galactofuranosyltransferases. A number of methods for the synthesis of galactofuranosides have also been developed, and practitioners in the field now have many options for the initiation of a synthesis of glycoconjugates containing either α- or β-Galf residues. UDP-Galf has also been prepared by a number of approaches, and it appears that a chemoenzymatic approach is currently the most viable method for producing multi-milligram amounts of this important intermediate. Recent advances both in the understanding of the mechanism of UGM and in the synthesis of galactofuranose and its derivatives are highlighted in this review.

  biosynthesis, carbohydrates, glycoconjugates, glycosylation, organic synthesis

  NCBI PubMed ID: 19591187
  Publication DOI: 10.1002/cbic.200900208
  Journal NLM ID: 100937360
  Publisher: Weinheim, Germany: Wiley Interscience
  Correspondence: tlowary@ualberta.ca
  Institutions: Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, University of Alberta, Gunning-Lemieux Chemistry Centre, Edmonton, AB, Canada
  
   
  
  Klebsiella pneumoniae, Citrobacter freundii
  CSDB ID 23824 (all data & tools)
• Article ID: 3880
  Kos V, Whitfield C "A membrane-located glycosyltransferase complex required for biosynthesis of the D-galactan I lipopolysaccharide O antigen in Klebsiella pneumoniae" - Journal of Biological Chemistry 285(25) (2010) 19668-19687
  

  D-galactan I is polysaccharide with the disaccharide repeat unit structure [→3-β-D-Galf-(1→3)-α-D-Galp-(1→]. This glycan represents the lipopolysaccharide O antigen found in many Gram-negative bacteria, including several Klebsiella pneumoniae O serotypes. The polysaccharide is synthesized in the cytoplasm prior to its export via an ATP-binding cassette transporter. Sequence analysis predicts three galactosyltransferases in the D-galactan I genetic locus. They are WbbO (belonging to glycosyltransferase (GT) family 4), WbbM (GT-family 8), and WbbN (GT-family 2). The WbbO and WbbM proteins are each predicted to contain two domains, with the GT modules located towards their C-termini. The N-terminal domains of WbbO and WbbM exhibit no similarity to proteins with known function. In vivo complementation assays suggest that all three glycosyltransferases are required for D-galactan I biosynthesis. Using a bacterial two-hybrid system and confirmatory co-purification strategies, evidence is provided for protein:protein interactions among the glycosyltransferases, creating a membrane-located enzyme complex dedicated to D-galactan I biosynthesis.

  Lipopolysaccharide, structure, serotype, O-antigen, gram negative bacteria, Klebsiella pneumoniae, D-galactan, galactosyltransferase

  NCBI PubMed ID: 20410291
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: cwhitfie@uoguelph.ca
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada
  Methods: SDS-PAGE, Western blotting, serological methods, genetic methods, biochemical methods, bioinformatic analysis
  
   
  
  Klebsiella pneumoniae
  CSDB ID 25313 (all data & tools)
• Article ID: 4307
  Greenfield LK, Whitfield C "Synthesis of lipopolysaccharide O-antigens by ABC transporter-dependent pathways" - Carbohydrate Research 356 (2012) 12-24
  

  The O-polysaccharide (O-PS; O-antigen) of bacterial lipopolysaccharides is made up of repeating units of one or more sugar residues and displays remarkable structural diversity. Despite the structural variations, there are only three strategies for O-PS assembly. The ATP-binding cassette (ABC)-transporter-dependent mechanism of O-PS biosynthesis is widespread. The Escherichia coli O9a and Klebsiella pneumoniae O2a antigens provide prototypes, which are distinguished by the fine details that link glycan polymerization and chain termination at the cytoplasmic face of the inner membrane to its export via the ABC transporter. Here, we describe the current understanding of these processes. Since glycoconjugate assembly complexes that utilize an ABC transporter-dependent pathway are widespread among the bacterial kingdom, the models described here are expected to extend beyond O-PS biosynthesis systems

  Lipopolysaccharide, O-polysaccharide, ATP-binding cassette transporter, Escherichia coli O9a, Klebsiella pneumoniae O2a

  NCBI PubMed ID: 22475157
  Publication DOI: 10.1016/j.carres.2012.02.027
  Journal NLM ID: 0043535
  Publisher: Elsevier
  Correspondence: C. Whitfield
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
  
   
  
  Klebsiella pneumoniae O1, Klebsiella pneumoniae O2a, Serratia marcescens O16
  CSDB ID 28377 (all data & tools)
• Article ID: 4318
  Huang JX, Azad MA, Yuriev E, Baker MA, Nation RL, Li J, Cooper MA, Velkov T "Molecular Characterization of Lipopolysaccharide Binding to Human a-1-Acid Glycoprotein" - Journal of Lipids 2012 (2012) 475153
  

  The ability of AGP to bind circulating lipopolysaccharide (LPS) in plasma is believed to help reduce the proinflammatory effect of bacterial lipid A molecules. Here, for the first time we have characterized human AGP binding characteristics of the LPS from a number of pathogenic Gram-negative bacteria: Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia, Pseudomonas aeruginosa, and Serratia marcescens. The binding affinity and structure activity relationships (SAR) of the AGP-LPS interactions were characterized by surface plasma resonance (SPR). In order to dissect the contribution of the lipid A, core oligosaccharide and O-antigen polysaccharide components of LPS, the AGP binding affinity of LPS from smooth strains, were compared to lipid A, Kdo2-lipid A, R(a), R(d), and R(e) rough LPS mutants. The SAR analysis enabled by the binding data suggested that, in addition to the important role played by the lipid A and core components of LPS, it is predominately the unique species- and strain-specific carbohydrate structure of the O-antigen polysaccharide that largely determines the binding affinity for AGP. Together, these data are consistent with the role of AGP in the binding and transport of LPS in plasma during acute-phase inflammatory responses to invading Gram-negative bacteria.

  O-antigen, lipid A, gram negative bacteria, lipopolysaccharide-binding, AGP binding, SPR

  NCBI PubMed ID: 23316371
  Publication DOI: 10.1155/2012/475153
  Journal NLM ID: 101553819
  Publisher: Cairo: Hindawi Pub Corp
  Correspondence: Tony Velkov
  Institutions: Institute for Molecular Bioscience, The University of Queensland, 306 Carmody Road, St. Lucia, QLD 4072, Australia, Drug Development and Innovation, Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia, Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, VIC 3052, Australia, Priority Research Centre in Reproductive Science, School of Environmental and Life Sciences, University of Newcastle, Callaghan, NSW 2308, Australia
  Methods: molecular modeling, fluorescence spectroscopy, surface plasmon resonance (SPR)
  
   
  
  Klebsiella pneumoniae O2a,c
  CSDB ID 28399 (all data & tools)
• Article ID: 4329
  Knirel YA "Structure of O-antigens" - Book: Bacterial lipopolysaccharides: Structure, chemical synthesis, biogenesis and interaction with host cells (2011) Chapter 3, 41-115
  

  The lipopolysaccharide (LPS) is the major constituent of the outer leaflet of the outer membrane of Gram-negative bacteria. Its lipid A moiety is embedded in the membrane and serves as an anchor for the rest of the LPS molecule. The outermost repetitive glycan region of the LPS is linked to the lipid A through a core oligosaccharide (OS), and is designated as the O-specific polysaccharide (O-polysaccharide, OPS) or O-antigen. The O-antigen is the most variable portion of the LPS and provides serological specificity, which is used for bacterial serotyping. The OPS also provides protection to the microorganisms from host defenses such as complement mediated killing and phagocytosis, and is involved in interactions of bacteria with plants and bacteriophages. Studies of the OPSs ranging from the elucidation of their chemical structures and conformations to their biological and physico-chemical properties help improving classification schemes of Gram-negative bacteria. Furthermore, these studies contributed to a better understanding of the mechanisms of pathogenesis of infectious diseases, as well as provided information to develop novel vaccines and diagnostic reagents.

  Lipopolysaccharide, synthesis, lipopolysaccharides, structure, Bacterial, host, O-antigen, O antigen, cell, O antigens, O-antigens, chemical, interaction, cells, PDF, chemical synthesis, biogenesis

  Publication DOI: 10.1007/978-3-7091-0733-1_3
  Publisher: Springer
  Correspondence: knirel@ioc.ac.ru
  Editors: Knirel YA, Valvano MA
  Institutions: Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia
  
   
  
  Citrobacter freundii O39, Klebsiella pneumoniae O1, Klebsiella pneumoniae O6, Klebsiella pneumoniae O2a, Klebsiella pneumoniae O2a,b, Klebsiella pneumoniae O2a,c, Hafnia alvei Y166/91, Mannheimia haemolytica T4, Mannheimia haemolytica T10
  CSDB ID 27524 (all data & tools)
• Article ID: 4728
  Stowell SR, Arthur CM, McBride R, Berger O, Razi N, Heimburg-Molinaro J, Rodrigues LC, Gourdine J, Noll AJ, von Gunten S, Smith DF, Knirel YA, Paulson JC, Cummings RD "Microbial glycan microarrays define key features of host-microbial interactions" - Nature Chemical Biology 10(6) (2014) 470-476
  

  Genomic approaches continue to provide unprecedented insight into the microbiome, yet host immune interactions with diverse microbiota can be difficult to study. We therefore generated a microbial microarray containing defined antigens isolated from a broad range of microbial flora to examine adaptive and innate immunity. Serological studies with this microarray show that immunoglobulins from multiple mammalian species have unique patterns of reactivity, whereas exposure of animals to distinct microbes induces specific serological recognition. Although adaptive immunity exhibited plasticity toward microbial antigens, immunological tolerance limits reactivity toward self. We discovered that several innate immune galectins show specific recognition of microbes that express self-like antigens, leading to direct killing of a broad range of Gram-negative and Gram-positive microbes. Thus, host protection against microbes seems to represent a balance between adaptive and innate immunity to defend against evolving antigenic determinants while protecting against molecular mimicry.

  molecular mimicry, glycan, interaction, Gram-negative, galectins, Gram-positive, innate immunity, Glycomics

  Publication DOI: 10.1038/nchembio.1525
  Journal NLM ID: 101231976
  Publisher: New York, NY: Nature Publishing Group
  Correspondence: jpaulson@scripps.edu; rdcummi@emory.edu
  Institutions: Department of Biochemistry and the Glycomics Center, Emory University School of Medicine, Atlanta, Georgia, USA, Department of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California, USA, Department of and Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA
  Methods: serological methods
  
   
  
  Serratia marcescens O20
  CSDB ID 30479 (all data & tools)
• Article ID: 4894
  Yeh KM, Chiu SK, Lin CL, Huang LY, Tsai YK, Chang JC, Lin JC, Chang FY, Siu LK "Surface antigens contribute differently to the pathophysiological features in serotype K1 and K2 Klebsiella pneumoniae strains isolated from liver abscesses" - Gut Pathogens 8 (2016) 4
  

  BACKGROUND: The virulence role of surface antigens in a single serotype of Klebsiella pneumoniae strain have been studied, but little is known about whether their contribution will vary with serotype. METHOD: To investigate the role of K and O antigen in hyper-virulent strains, we constructed O and K antigen deficient mutants from serotype K1 STL43 and K2 TSGH strains from patients with liver abscess, and characterized their virulence in according to the abscess formation and resistance to neutrophil phagocytosis, serum, and bacterial clearance in liver. RESULTS: Both of K1 and K2-antigen mutants lost their wildtype resistance to neutrophil phagocytosis and hepatic clearance, and failed to cause abscess formation. K2-antigen mutant became serum susceptible while K1-antigen mutant maintained its resistance to serum killing. The amount of glucuronic acid, indicating the amount of capsular polysaccharide (CPS, K antigen), was inversed proportional to the rate of phagocytosis. O-antigen mutant of serotype K1 strains had significantly more amount of CPS, and more resistant to neutrophil phagocytosis than its wildtype counterpart. O-antigen mutants of serotype K1 and K2 strains lost their wildtype serum resistance, and kept resistant to neutrophil phagocytosis. While both mutants lacked the same O1 antigen, O-antigen mutant of serotype K1 became susceptible to liver clearance and cause mild abscess formation, but its serotype K2 counterpart maintained these wildtype virulence. CONCLUSION: We conclude that the contribution of surface antigens to virulence of K. pneumoniae strains varies with serotypes.

  virulence, K-antigen, serotype, O-antigen, capsular polysaccharide, Klebsiella pneumoniae, vaccine, phagocytosis, surface antigen

  NCBI PubMed ID: 26893615
  Publication DOI: 10.1186/s13099-016-0085-5
  Journal NLM ID: 101474263
  Publisher: London: BioMed Central
  Correspondence: fychang@ndmctsgh.edu.tw; lksiu@nhri.org.tw
  Institutions: Infection Control Office, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Kung Road, Neihu, 114 Taipei City Taiwan, School of Respiratory Therapy, College of Medicine, Taipei Medical University, Taipei, Taiwan, Department of Internal Medicine, Division of Pulmonary Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan, Department of Internal Medicine, Division of Infectious Diseases and Tropical Medicine, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Kung Road, Neihu, 114 Taipei City Taiwan, Institute of Infectious Diseases and Vaccine Research, National Health Research Institutes, 35 Keyan Road, Zhunan, 35053 Miaoli, Taiwan, Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan
  Methods: PCR, Western blotting, serological methods, genetic methods, serum bactericidal assays, statistical analysis, human neutrophil phagocytosis assay
  
   
  
  Klebsiella pneumoniae O1
  CSDB ID 11622 (all data & tools)
• Article ID: 4901
  Mann E, Whitfield C "A widespread three-component mechanism for the periplasmic modification of bacterial glycoconjugates" - Canadian Journal of Chemistry 94(11) (2016) 883-893
  

  The diverse structures of bacterial glycoconjugates are generally established during the early stages of synthesis by the activities of nucleotide sugar-dependent glycosyltransferases active in the cytoplasm. However, in some cases, further modifications of varying complexity occur after the glycoconjugate is exported to the periplasm. These processes are distinguished by the involvement of polyprenyl monosphosphoryl donors and require glycosyltransferases possessing GT-C folds. Established prototypes are found in modifications of some bacterial lipopolysaccharides, where 4-amino-4-deoxy-l-arabinose is added to lipid A and glucose side branches are used to modify O-antigens. Here we review the current understanding of these systems and describe similarities to other periplasmic glycan modifications in bacteria and the N-glycosylation pathway for assembly of eukaryotic glycoproteins.

  LPS, O-antigen, lipid A, glycosyltransferase, glycoconjugate biosynthesis, GT-C enzymes, polyprenol lipid

  Publication DOI: 10.1139/cjc-2015-0594
  Journal NLM ID: 0372705
  Publisher: National Research Council of Canada Canada
  Correspondence: Chris Whitfield
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada
  
   
  
  Raoultella terrigena R1Gly, Klebsiella pneumoniae NTUH-K2044
  CSDB ID 11639 (all data & tools)
• Article ID: 5043
  Stojkovic K, Szijarto V, Kaszowska M, Niedziela T, Hartl K, Nagy G, Lukasiewicz J "Identification of D-Galactan-III As Part of the Lipopolysaccharide of Klebsiella pneumoniae Serotype O1" - Frontiers in Microbiology 8 (2017) 684
  

  Klebsiella pneumoniae is a Gram-negative, ubiquitous bacterium capable of causing severe nosocomial infections in individuals with impaired immune system. Emerging multi-drug resistant strains of this species and particularly carbapenem-resistant strains pose an urgent threat to public health. The lipopolysaccharide (LPS) O-antigen is the main surface antigen. It contributes to the virulence of this species and determines the O-serotype of K. pneumoniae isolates. Among the nine main O-serotypes of K. pneumoniae, O1-and O2-type pathogens are causative agents of over 50% of all infections. Serotype O1, the most common O-serotype, expresses complex LPS consisting of d-galactan-I (a polymer built of →3)-β-d-Galf-(1→3)-α-d-Galp-(1→ repeating units) capped by d-galactan-II (built of [→3)-α-d-Galp-(1→3)-β-d-Galp-(1→] repeating units). Galactan-I is present as the sole polymer in O2 serotype. Recently, in case of serotype O2, conversion of galactan-I to galactan-III (→3)-β-d-Galf-(1→3)-[α-d-Galp-(1→4)]-α-d-Galp-(1→) was reported. Substitution of →3)-α-d-Galp by a branching terminal α-d-Galp was dependent on the presence of the gmlABC operon and had a major impact on the antigenicity of the galactan polymer. Genetic analysis indicated that 40% of the O1 clinical isolates also carry the gmlABC locus; therefore we aimed to characterize the corresponding phenotype of LPS O-antigens. The presence of galactan-III among O1 strains was proven using galactan-III-specific monoclonal antibodies and confirmed by structural analyses performed using sugar and methylation analysis as well as classical and high-resolution magic angle spinning NMR spectroscopy. By using an isogenic mutant pair, we demonstrated that galactan-III expression was dependent on the presence of glycosyltransferases encoded by gmlABC, as was shown previously for the O2 serotype. Furthermore, the galactan-II structures in O1gml+ strains remained unaffected corroborating no functional interactions between the biosynthesis of galactan-III and galactan-II polymers.

  Lipopolysaccharide, O-antigen, Klebsiella pneumoniae, Galactan, HR-MAS NMR, d-galactan-I, d-galactan-III, serotype O1

  NCBI PubMed ID: 28487676
  Publication DOI: 10.3389/fmicb.2017.00684
  Journal NLM ID: 101548977
  Publisher: Lausanne: Frontiers Research Foundation
  Correspondence: Jolanta Lukasiewicz
  Institutions: Arsanis Biosciences GmbH, Vienna, Austria, Laboratory of Microbial Immunochemistry and Vaccines, Department of Immunochemistry, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of SciencesWroclaw, Poland
  Methods: 13C NMR, 1H NMR, methylation, gel filtration, NMR-2D, sugar analysis, mild acid hydrolysis, immunoblotting, HR-MAS NMR, cloning, bioinformatic analysis, dialysis
  
   
  
  Klebsiella pneumoniae O1 Kp4, Klebsiella pneumoniae O1 Kp4/pSU2718, Klebsiella pneumoniae O2 Kp30
  CSDB ID 11966 (all data & tools)
• Article ID: 5044
  Szijarto V, Guachalla LM, Hartl K, Varga C, Banerjee P, Stojkovic K, Kaszowska M, Nagy E, Lukasiewicz J, Nagy G "Both clades of the epidemic KPC-producing Klebsiella pneumoniae clone ST258 share a modified galactan O-antigen type" - International Journal of Medical Microbiology 306(2) (2016) 89-98
  

  Klebsiella pneumoniae ST258 is a globally disseminated, extremely drug resistant, nosocomial clone with limited treatment options. We show that the vast majority of ST258 isolates express modified d-galactan-I lipopolysaccharide O-antigen, termed hereinafter as D-galactan-III. The genetic determinant required for galactan-III synthesis was identified as a distinct operon adjacent to the rfb (wb) locus encoding D-galactan-I synthesis. The three genes within the operon encode predicted glycosyltransferases. Testing an isogenic transformant pair revealed that expression of D-galactan-III, in comparison to D-galactan-I, conferred improved survival in the presence of human serum. Eighty-three percent of the more than 200 ST258 draft genome sequences currently available carries the corresponding operon and hence these isolates are predicted to express galactan-III antigens. A D-galactan-III specific monoclonal antibody (mAb) was shown to bind to extracted LPS from a panel of ST258 isolates. The same mAb confirmed accessibility of galactan-III in surface staining of ST258 irrespective of the distinct capsular antigens expressed by both clades described previously. Based on these data, the galactan-III antigen may represent an attractive target for active and passive immunization approaches against K. pneumoniae ST258.

  monoclonal antibody, LPS O-antigen, d-galactan-I, d-galactan-III, Klebsiella pneumoniae ST258, Serotype O2

  NCBI PubMed ID: 26723873
  Publication DOI: 10.1016/j.ijmm.2015.12.002
  Journal NLM ID: 100898849
  Publisher: Jena, Germany: Urban & Fischer Verlag
  Correspondence: Gabor Nagy
  Institutions: Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Wroclaw, Poland, Arsanis Biosciences GmbH, Vienna, Austria
  Methods: 13C NMR, 1H NMR, methylation, gel filtration, NMR-2D, sugar analysis, ELISA, mild acid hydrolysis, genetic methods, immunoblotting, serum bactericidal assays, HR-MAS NMR, statistical analysis, cloning, bioinformatic analysis, immunization, flow cytometry analysis
  
   
  
  Klebsiella pneumoniae O2 Kp26, Klebsiella pneumoniae O2 PCM 27, Klebsiella pneumoniae O2 Kp26/pKP100, Klebsiella pneumoniae O2 Kp26/pSU2718
  CSDB ID 12240 (all data & tools)
• Article ID: 5063
  Ucieklak K, Koj S, Pawelczyk D, Niedziela T "Structural Masquerade of Plesiomonas shigelloides Strain CNCTC 78/89 O-Antigen-High-Resolution Magic Angle Spinning NMR Reveals the Modified d-galactan I of Klebsiella pneumoniae" - International Journal of Molecular Sciences 18(12) (2017) 2572
  

  The high-resolution magic angle spinning nuclear magnetic resonance spectroscopy (HR-MAS NMR) analysis of Plesiomonas shigelloides 78/89 lipopolysaccharide directly on bacteria revealed the characteristic structural features of the O-acetylated polysaccharide in the NMR spectra. The O-antigen profiles were unique, yet the pattern of signals in the, spectra along with their (1)H,13C chemical shift values, resembled these of d-galactan I of Klebsiella pneumoniae. The isolated O-specific polysaccharide (O-PS) of P. shigelloides strain CNCTC 78/89 was investigated by 1H and 13C NMR spectroscopy, mass spectrometry and chemical methods. The analyses demonstrated that the P. shigelloides 78/89 O-PS is composed of →3)-α-d-Galp-(1→3)-β-d-Galf2OAc-(1→ disaccharide repeating units. The O-acetylation was incomplete and resulted in a microheterogeneity of the O-antigen. This O-acetylation generates additional antigenic determinants within the O-antigen, forms a new chemotype, and contributes to the epitopes recognized by the O-serotype specific antibodies. The serological cross-reactivities further confirmed the inter-specific structural similarity of these O-antigens.

  Lipopolysaccharide, O-antigen, NMR spectroscopy, O-acetylation, HR-MAS, Plesiomonas shigelloides, d-galactan I

  NCBI PubMed ID: 29186063
  Publication DOI: 10.3390/ijms18122572
  Journal NLM ID: 101092791
  Publisher: Basel, Switzerland: MDPI
  Correspondence: tomasz.niedziela@iitd.pan.wroc.pl
  Institutions: Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, 53-114 Wroclaw, Poland
  Methods: 13C NMR, 1H NMR, gel filtration, SDS-PAGE, sugar analysis, ELISA, mild acid hydrolysis, MALDI-TOF MS, immunoblotting, HR-MAS NMR, acetylation
  
   
  
  Plesiomonas shigelloides CNCTC 78/89, Klebsiella pneumoniae Kp20
  CSDB ID 12335 (all data & tools)
• Article ID: 5127
  Bellich B, Lagatolla C, Tossi A, Benincasa M, Cescutti P, Rizzo R "Influence of Bacterial Biofilm Polysaccharide Structure on Interactions with Antimicrobial Peptides: A Study on Klebsiella pneumoniae" - International Journal of Molecular Sciences 19(6) (2018)
  

  Biofilms are complex systems produced by bacteria and constituted by macromolecular matrix embedding cells. They provide advantages to bacteria including protection against antimicrobials. The protection given by biofilms produced by Klebsiella pneumoniae strains towards antimicrobial peptides of the innate immune system was investigated. In particular, the role of matrix bacterial exopolysaccharides was explored. Three clinical strains producing exopolysaccharides with different chemistry were selected and the interaction of purified biofilm polysaccharides with two bovine cathelicidins was studied by circular dichroism spectroscopy and microbiological assays to establish their influence on the peptide’s antimicrobial activity. The spectroscopic data indicated a different extent of interaction with the two peptides, in a manner dependent on their sugar composition, and in particular the presence of rhamnose residues correlated with a lower interaction. The extent of interaction was then related to the protection towards antimicrobial peptides, conferred by the addition of the different exopolysaccharides, in minimum inhibitory concentration (MIC) assays against a reference Escherichia coli strain. Microbiological results were in very good agreement with spectroscopic data, confirming the active role of matrix polysaccharides in determining a biofilm’s protective capacity and indicating lower protection levels afforded by rhamnose containing exopolysaccharides.

  Escherichia coli, antimicrobial peptides, bacterial biofilm, bacterial protection, exopolysaccharides biological activity, Cathelicidins, matrix exopolysaccharides

  NCBI PubMed ID: 29882774
  Publication DOI: 10.3390/ijms19061685
  Journal NLM ID: 101092791
  Publisher: Basel, Switzerland: MDPI
  Correspondence: R. Rizzo
  Institutions: Department of Life Sciences, University of Trieste, Via Licio Giorgieri 1, 34127 Trieste, Italy
  Methods: CD, antimicrobial peptide sensitivity assays
  
   
  
  Klebsiella pneumoniae KpTs101, Klebsiella pneumoniae O1
  CSDB ID 12869 (all data & tools)
• Article ID: 5142
  Clarke BR, Ovchinnikova OG, Kelly SD, Williamson ML, Butler JE, Liu B, Wang L, Gou X, Follador R, Lowary TL, Whitfield C "Molecular basis for the structural diversity in serogroup O2-antigen polysaccharides in Klebsiella pneumoniae" - Journal of Biological Chemistry 293(13) (2018) 4666-4679
  

  Klebsiella pneumoniae is a major health threat. Vaccination and passive immunization are considered as alternative therapeutic strategies for managing Klebsiella infections. Lipopolysaccharide O antigens are attractive candidates because of the relatively small range of known O-antigen polysaccharide structures, but immunotherapeutic applications require a complete understanding of the structures found in clinical settings. Currently, the precise number of Klebsiella O antigens is unknown because available serological tests have limited resolution, and their association with defined chemical structures is sometimes uncertain. Molecular serotyping methods can evaluate clinical prevalence of O serotypes but require a full understanding of the genetic determinants for each O-antigen structure. This is problematic with Klebsiella pneumoniae because genes outside the main rfb (O-antigen biosynthesis) locus can have profound effects on the final structure. Here, we report two new loci encoding enzymes that modify a conserved polysaccharide backbone comprising disaccharide repeat units [→3)-α-d-Galp-(1→3)-β-d-Galf-(1→] (O2a antigen). We identified in serotype O2aeh a three-component system that modifies completed O2a glycan in the periplasm by adding 1,2-linked α-Galp side-group residues. In serotype O2ac, a polysaccharide comprising disaccharide repeat units [→5)-β-d-Galf-(1→3)-β-d-GlcpNAc-(1→] (O2c antigen) is attached to the non-reducing termini of O2a-antigen chains. O2c-polysaccharide synthesis is dependent on a locus encoding three glycosyltransferase enzymes. The authentic O2aeh and O2c antigens were recapitulated in recombinant Escherichia coli hosts to establish the essential gene set for their synthesis. These findings now provide a complete understanding of the molecular genetic basis for the known variations in Klebsiella O-antigen carbohydrate structures based on the O2a backbone.

  polysaccharide, O antigen, polysaccharide structure, Gram-negative bacteria, glycosyltransferase, serotyping, lipopolysaccharide (LPS), carbohydrate structure, nuclear magnetic resonance (NMR), Klebsiella pneumonia

  NCBI PubMed ID: 29602878
  Publication DOI: 10.1074/jbc.RA117.000646
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: cwhitfie@uoguelph.ca
  Institutions: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, 23 Hongda St. TEDA, Tianjin, China, LimmaTech Biologics AG, 8952 Schlieren, Switzerland, and the Department of Chemistry and Alberta Glycomics Centre, University of Alberta, Edmonton, AB, Canada, the Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1
  Methods: 13C NMR, 1H NMR, gel filtration, NMR-2D, PCR, DNA sequencing, SDS-PAGE, DNA techniques, bioinformatic analysis, mmunoblotting
  
   
  
  Klebsiella pneumoniae O2a, Escherichia coli DH5α
  CSDB ID 12886 (all data & tools)
• Article ID: 5457
  Kelly SD, Clarke BR, Ovchinnikova OG, Sweeney RP, Williamson ML, Lowary TL, Whitfield C "Klebsiella pneumoniae O1 and O2ac antigens provide prototypes for an unusual strategy for polysaccharide antigen diversification" - Journal of Biological Chemistry 294(28) (2019) 10863-10876
  

  A limited range of different structures is observed in O-antigenic polysaccharides (OPSs) from Klebsiella pneumoniae lipopolysaccharides. Among these, several are based on modifications of a conserved core element of serotype O2a OPS, which has a disaccharide repeat structure [→3)-α-Galp-(1→3)-β-Galf-(1→]. Here, we describe the enzymatic pathways for a highly unusual modification strategy involving the attachment of a second glycan repeat unit structure to the non-reducing terminus of O2a. This occurs by the addition of the O1 [→3)-α-Galp-(1→3)-β-Galp-(1→] or O2c [→3)-β-GlcpNAc-(1→5)-β-Galf-(1→] antigens. The organization of the enzyme activities performing these modifications differs, with the enzyme WbbY possessing two glycosyltransferase catalytic sites solely responsible for O1-antigen polymerization and forming a complex with the O2a glycosyltransferase, WbbM. In contrast, O2c polymerization requires glycosyltransferases WbmV and WbmW, which interact with one another, but apparently not with WbbM. Using defined synthetic acceptors and site-directed mutants to assign the activities of the WbbY catalytic sites, we found that the C-terminal WbbY domain is a UDP-Galp-dependent GT-A galactosyltransferase adding β-(1→3)-linked D-Galp, whereas the WbbY N terminus includes a GT-B enzyme adding α-(1→3)-linked D-Galp These activities build the O1 antigen on a terminal Galp in the O2a domain. Using similar approaches, we identified WbmV as the UDP-GlcNAc-dependent transferase and noted that WbmW represents a UDP-Galf-dependent enzyme and that both are GT-A members. WbmVW polymerizes the O2c antigen on a terminal Galf Our results provide mechanistic and conceptual insights into an important strategy for polysacharide antigen diversification in bacteria.

  polysaccharide, O antigen, Gram-negative bacteria, glycosyltransferase, Klebsiella pneumoniae, serotyping, lipopolysaccharide (LPS), antigenic diversity, cell surface, enzyme complex

  NCBI PubMed ID: 31138653
  Publication DOI: 10.1074/jbc.RA119.008969
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: Chris Whitfield
  Institutions: Alberta Glycomics Centre and Department of Chemistry, University of Alberta, Edmonton, AB, Canada, Department of Molecular and Cellular Biology, University of Guelph, Ontario, Canada
  Methods: 13C NMR, 1H NMR, gel filtration, NMR-2D, PCR, DNA sequencing, SDS-PAGE, TLC, chemical synthesis, HPLC, UV, immunoblotting, LC-MS, bioinformatic analysis, CC, LC-ESI-MS, enzymatic synthesis, fluorescent labeling
  
   
  
  Klebsiella pneumoniae O2a, Klebsiella pneumoniae O8
  CSDB ID 798 (all data & tools)
• Article ID: 5726
  Artyszuk D, Izdebski R, Maciejewska A, Kaszowska M, Herud A, Szijarto V, Gniadkowski M, Lukasiewicz J "The Impact of Insertion Sequences on O-Serotype Phenotype and Its O-Locus-Based Prediction in Klebsiella pneumoniae O2 and O1" - International Journal of Molecular Sciences 21(18) (2020) 6572
  

  Klebsiella pneumoniae is a nosocomial pathogen, pointed out by the World Helth Organisation (WHO) as 'critical' regarding the highly limited options of treatment. Lipopolysaccharide (LPS, O-antigen) and capsular polysaccharide (K-antigen) are its virulence factors and surface antigens, determining O- and K-serotypes and encoded by O- or K-loci. They are promising targets for antibody-based therapies (vaccines and passive immunization) as an alternative to antibiotics. To make such immunotherapy effective, knowledge about O/K-antigen structures, drift, and distribution among clinical isolates is needed. At present, the structural analysis of O-antigens is efficiently supported by bioinformatics. O- and K-loci-based genotyping by polymerase chain reaction (PCR) or whole genome sequencing WGS has been proposed as a diagnostic tool, including the Kaptive tool available in the public domain. We analyzed discrepancies for O2 serotyping between Kaptive-based predictions (O2 variant 2 serotype) and the actual phenotype (O2 variant 1) for two K. pneumoniae clinical isolates. Identified length discrepancies from the reference O-locus resulted from insertion sequences (ISs) within rfb regions of the O-loci. In silico analysis of 8130 O1 and O2 genomes available in public databases indicated a broader distribution of ISs in rfbs that may influence the actual O-antigen structure. Our results show that current high-throughput genotyping algorithms need to be further refined to consider the effects of ISs on the LPS O-serotype.

  Lipopolysaccharide, NMR, LPS, O-antigen, Klebsiella, O-serotype, Kaptive, WGS

  NCBI PubMed ID: 32911792
  Publication DOI: 10.3390/ijms21186572
  Journal NLM ID: 101092791
  Publisher: Basel, Switzerland: MDPI
  Correspondence: Jolanta.lukasiewicz@hirszfeld.pl
  Institutions: Arsanis Biosciences GmbH, Vienna, Austria, Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Laboratory of Genomics & Bioinformatics, 53-114 Wroclaw, Poland, Department of Molecular Microbiology, National Medicines Institute, 00-725 Warsaw, Poland
  Methods: 13C NMR, 1H NMR, genetic methods, HR-MAS NMR, serotyping, sequence analysis, Kaptive Web analysis
  
   
  
  Klebsiella pneumoniae BIDMC 7B, Klebsiella pneumoniae ABC152, Klebsiella pneumoniae O2a, Klebsiella pneumoniae O1, Klebsiella pneumoniae Kp26
  CSDB ID 32054 (all data & tools)
• Article ID: 5751
  Clarke BR, Ovchinnikova OG, Sweeney RP, Kamski-Hennekam ER, Gitalis R, Mallette E, Kelly SD, Lowary TL, Kimber MS, Whitfield C "A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family" - Nature Chemical Biology 16(4) (2020) 450-457
  

  Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→3)-α-Galp-(1→3)-β-Galf-(1→). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.

  Lipopolysaccharide, O-antigen, mechanism, galactofuranose, glycosyltransferase, Klebsiella pneumoniae, O-antigen polymerase, vaccine, transporter, glycans, O-serotypes, undecaprenol

  NCBI PubMed ID: 32152541
  Publication DOI: 10.1038/s41589-020-0494-0
  Journal NLM ID: 101231976
  Publisher: New York, NY: Nature Publishing Group
  Correspondence: mkimber@uoguelph.ca; cwhitfie@uoguelph.ca
  Institutions: Department of Chemistry, University of Alberta, Edmonton, AB, Canada, Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
  Methods: 13C NMR, 1H NMR, gel filtration, NMR-2D, PCR, SDS-PAGE, DNA techniques, ESI-MS, biochemical methods, bioinformatic analysis, enzyme structure, topological information
  
   
  
  Klebsiella pneumoniae O2a
  CSDB ID 32083 (all data & tools)
• Article ID: 5869
  Whitfield C, Williams DM, Kelly SD "Lipopolysaccharide O-antigens-bacterial glycans made to measure" - Journal of Biological Chemistry 295(31) (2020) 10593-10609
  

  Lipopolysaccharides are critical components of bacterial outer membranes. The more conserved lipid A part of the lipopolysaccharide molecule is a major element in the permeability barrier imposed by the outer membrane and offers a pathogen-associated molecular pattern recognized by innate immune systems. In contrast, the long-chain O-antigen polysaccharide (O-PS) shows remarkable structural diversity and fulfills a range of functions, depending on bacterial lifestyles. O-PS production is vital for the success of clinically important Gram-negative pathogens. The biological properties and functions of O-PSs are mostly independent of specific structures, but the size distribution of O-PS chains is particularly important in many contexts. Despite the vast O-PS chemical diversity, most are produced in bacterial cells by two assembly strategies, and the different mechanisms employed in these pathways to regulate chain-length distribution are emerging. Here, we review our current understanding of the mechanisms involved in regulating O-PS chain-length distribution and discuss their impact on microbial cell biology.

  polysaccharide, O-antigen, Gram-negative bacteria, glycosyltransferase, outer membrane, lipopolysaccharide (LPS), glycan biosynthesis, bacterial pathogenesis, antigenic diversity, cell surface

  NCBI PubMed ID: 32424042
  Publication DOI: 10.1074/jbc.REV120.009402
  Journal NLM ID: 2985121R
  Publisher: Baltimore, MD: American Society for Biochemistry and Molecular Biology
  Correspondence: cwhitfie@uoguelph.ca
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada
  
   
  
  Klebsiella pneumoniae O2a
  CSDB ID 32233 (all data & tools)
• Article ID: 5871
  Yakovlieva L, Walvoort MTC "Processivity in Bacterial Glycosyltransferases" - ACS Chemical Biology 15(1) (2020) 3-16
  

  Extracellular polysaccharides and glycoproteins of pathogenic bacteria assist in adherence, autoaggregation, biofilm formation, and host immune system evasion. As a result, considerable research in the field of glycobiology is dedicated to study the composition and function of glycans associated with virulence, as well as the enzymes involved in their biosynthesis with the aim to identify novel antibiotic targets. Especially, insights into the enzyme mechanism, substrate binding, and transition-state structures are valuable as a starting point for rational inhibitor design. An intriguing aspect of enzymes that generate or process polysaccharides and glycoproteins is the level of processivity. The existence of enzymatic processivity reflects the need for regulation of the final glycan/glycoprotein length and structure, depending on the role they perform. In this Review, we describe the currently reported examples of various processive enzymes involved in polymerization and transfer of sugar moieties, predominantly in bacterial pathogens, with a focus on the biochemical methods, to showcase the importance of studying processivity for understanding the mechanism.

  processivity, Apparent processivity, Distributivity, Hyperglycosylation, Intrinsic processivity, Semiprocessive

  NCBI PubMed ID: 31750644
  Publication DOI: 10.1021/acschembio.9b00619
  Journal NLM ID: 101282906
  Publisher: Washington, DC: American Chemical Society
  Correspondence: m.t.c.walvoort@rug.nl
  Institutions: Stratingh Institute for Chemistry, University of Groningen, 9747 AG Groningen, The Netherlands
  
   
  
  Klebsiella pneumoniae
  CSDB ID 7679 (all data & tools)
• Article ID: 6289
  Palusiak A "Proteus mirabilis and Klebsiella pneumoniae as pathogens capable of causing co-infections and exhibiting similarities in their virulence factors" - Frontiers in Cellular and Infection Microbiology 12 (2022) 991657
  

  The genera Klebsiella and Proteus were independently described in 1885. These Gram-negative rods colonize the human intestinal tract regarded as the main reservoir of these opportunistic pathogens. In favorable conditions they cause infections, often hospital-acquired ones. The activity of K. pneumoniae and P. mirabilis, the leading pathogens within each genus, results in infections of the urinary (UTIs) and respiratory tracts, wounds, bacteremia, affecting mainly immunocompromised patients. P. mirabilis and K. pneumoniae cause polymicrobial UTIs, which are often persistent due to the catheter biofilm formation or increasing resistance of the bacteria to antibiotics. In this situation a need arises to find the antigens with features common to both species. Among many virulence factors produced by both pathogens urease shows some structural similarities but the biggest similarities have been observed in lipids A and the core regions of lipopolysaccharides (LPSs). Both species produce capsular polysaccharides (CPSs) but only in K. pneumoniae these antigens play a crucial role in the serological classification scheme, which in Proteus spp. is based on the structural and serological diversity of LPS O-polysaccharides (OPSs). Structural and serological similarities observed for Klebsiella spp. and Proteus spp. polysaccharides are important in the search for the cross-reacting vaccine antigens.

  Lipopolysaccharide, core oligosaccharide, Proteus mirabilis, Klebsiella pneumoniae, virulence factors

  NCBI PubMed ID: 36339335
  Publication DOI: 10.3389/fcimb.2022.991657
  Journal NLM ID: 101585359
  Publisher: Lausanne: Frontiers Media SA
  Correspondence: A. Palusiak
  Institutions: Laboratory of General Microbiology, Department of Biology of Bacteria, Institute of Microbiology, Biotechnology and Immunology, University of Łódź, Łódź, Poland
  
   
  
  Klebsiella pneumoniae O2a, Klebsiella pneumoniae O2a,b, Klebsiella pneumoniae O2a,c, Klebsiella pneumoniae O1, Klebsiella pneumoniae O6
  CSDB ID 21088 (all data & tools)
• Article ID: 6412
  Kelly SD, Ovchinnikova OG, Muller F, Steffen M, Braun M, Sweeney RP, Kowarik M, Follador R, Lowary TL, Serventi F, Whitfield C "Identification of a second glycoform of the clinically prevalent O1 antigen from Klebsiella pneumoniae" - Proceedings of the National Academy of Sciences of the USA 120(29) (2023) e2301302120
  

  Carbapenemase and extended beta-lactamase-producing Klebsiella pneumoniae isolates represent a major health threat, stimulating increasing interest in immunotherapeutic approaches for combating Klebsiella infections. Lipopolysaccharide O antigen polysaccharides offer viable targets for immunotherapeutic development, and several studies have described protection with O-specific antibodies in animal models of infection. O1 antigen is produced by almost half of clinical Klebsiella isolates. The O1 polysaccharide backbone structure is known, but monoclonal antibodies raised against the O1 antigen showed varying reactivity against different isolates that could not be explained by the known structure. Reinvestigation of the structure by NMR spectroscopy revealed the presence of the reported polysaccharide backbone (glycoform O1a), as well as a previously unknown O1b glycoform composed of the O1a backbone modified with a terminal pyruvate group. The activity of the responsible pyruvyltransferase (WbbZ) was confirmed by western immunoblotting and in vitro chemoenzymatic synthesis of the O1b terminus. Bioinformatic data indicate that almost all O1 isolates possess genes required to produce both glycoforms. We describe the presence of O1ab-biosynthesis genes in other bacterial species and report a functional O1 locus on a bacteriophage genome. Homologs of wbbZ are widespread in genetic loci for the assembly of unrelated glycostructures in bacteria and yeast. In K. pneumoniae, simultaneous production of both O1 glycoforms is enabled by the lack of specificity of the ABC transporter that exports the nascent glycan, and the data reported here provide mechanistic understanding of the capacity for evolution of antigenic diversity within an important class of biomolecules produced by many bacteria.

  Lipopolysaccharide, O antigen, Klebsiella pneumoniae, antigenic diversity, vaccine candidate

  NCBI PubMed ID: 37428935
  Publication DOI: 10.1073/pnas.2301302120
  Journal NLM ID: 7505876
  Publisher: National Academy of Sciences
  Correspondence: F. Serventi ; C. Whitfield
  Institutions: Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON N1G 2W1, Canada, LimmaTech Biologics AG, Schlieren 8952, Switzerland, Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada, Institute of Biological Chemistry, Academia Sinica, Taipei, Nangang 11529, Taiwan, Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan
  Methods: 13C NMR, 1H NMR, NMR-2D, SDS-PAGE, ESI-MS, mild acid hydrolysis, Western blotting, genetic methods, enzyme assay, LC-MS, bioinformatic analysis, SEC, phylogenetic analysis, monoclonal antibodies
  
   
  
  Klebsiella pneumoniae O2a
  CSDB ID 20757 (all data & tools)
• Article ID: 6482
  Wantuch PL, Knoot CJ, Robinson LS, Vinogradov E, Scott NE, Harding CM, Rosen DA "Capsular polysaccharide inhibits vaccine-induced O-antigen antibody binding and function across both classical and hypervirulent K2:O1 strains of Klebsiella pneumoniae" - PLoS Pathogens 19(5) (2023) e1011367
  

  Klebsiella pneumoniae presents as two circulating pathotypes: classical K. pneumoniae (cKp) and hypervirulent K. pneumoniae (hvKp). Classical isolates are considered urgent threats due to their antibiotic resistance profiles, while hvKp isolates have historically been antibiotic susceptible. Recently, however, increased rates of antibiotic resistance have been observed in both hvKp and cKp, further underscoring the need for preventive and effective immunotherapies. Two distinct surface polysaccharides have gained traction as vaccine candidates against K. pneumoniae: capsular polysaccharide and the O-antigen of lipopolysaccharide. While both targets have practical advantages and disadvantages, it remains unclear which of these antigens included in a vaccine would provide superior protection against matched K. pneumoniae strains. Here, we report the production of two bioconjugate vaccines, one targeting the K2 capsular serotype and the other targeting the O1 O-antigen. Using murine models, we investigated whether these vaccines induced specific antibody responses that recognize K2:O1 K. pneumoniae strains. While each vaccine was immunogenic in mice, both cKp and hvKp strains exhibited decreased O-antibody binding in the presence of capsule. Further, O1 antibodies demonstrated decreased killing in serum bactericidal assays with encapsulated strains, suggesting that the presence of K. pneumoniae capsule blocks O1-antibody binding and function. Finally, the K2 vaccine outperformed the O1 vaccine against both cKp and hvKp in two different murine infection models. These data suggest that capsule-based vaccines may be superior to O-antigen vaccines for targeting hvKp and some cKp strains, due to capsule blocking the O-antigen.

  O-antigen, capsular polysaccharide, Klebsiella pneumoniae

  NCBI PubMed ID: 37146068
  Publication DOI: 10.1371/journal.ppat.1011367
  Journal NLM ID: 101238921
  Publisher: San Francisco, CA: Public Library of Science
  Correspondence: C.M. Harding ; D.A. Rosen
  Institutions: Department of Pediatrics, Division of Infectious Diseases, Washington University School of Medicine, Saint Louis, Missouri, United States of America, Omniose, Saint Louis, Missouri, United States of America, National Research Council Canada, Human Health Therapeutics Centre, Ottawa, Ontario, Canada, Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria, Australia, Department of Molecular Microbiology, Washington University School of Medicine, Saint Louis, Missouri, United States of America
  Methods: 13C NMR, 1H NMR, NMR-2D, PCR, ELISA, ESI-MS, anion-exchange chromatography, Western blotting, serological methods, genetic methods, serum bactericidal assays, SEC, immunization, genome sequencing, bioconjugation, SRC
  
   
  
  Klebsiella pneumoniae O2a, Klebsiella pneumoniae O1v1
  CSDB ID 22335 (all data & tools)