The 6.6-kb rfb gene cluster from Klebsiella pneumoniae serotype O1 (rfb KpO1 Rfe is an N-acetylglucosamine (GlcpNAc)-1-phosphate transferase which forms lipid I (undecaprenyl pyrophosphoryl GlcpNAc) during the biosynthesis of the cell surface polysaccharide known as enterobacterial common antigen (37). In members of the Enterobacteriaceae, Rfe can also be involved in both pathways for LPS O antigen synthesis. In the Rfc-dependent pathway, Rfe transfers GlcpNAc residues to a lipid intermediate to initiate the formation of each O polysaccharide repeat unit (1,29,58,72). rfb gene products then complete the various O antigen repeat units, and after polymerization, GlcpNAc residues provide the first monomer in each repeat unit. A different role is played by Rfe in the biosynthesis of some homopolymeric O polysaccharides which, like D-galactan I, lack GlcpNAc in their repeating unit structure. In E. coli O8 and O9, Rfe primes the synthesis of the mannose homopolymer O polysaccharide by forming an undecaprenyl pyrophos-

Mycobacterial phosphatidylinositol tetramannosides (PIM4) are agonists for a distinct population of invariant human (Vα24) and mouse (Vα14) NKT cells, when presented by CD1d. We determined the crystal structure at 2.6-Å resolution of mouse CD1d bound to a synthetic dipalmitoyl-PIM2. Natural PIM2, which differs in its fatty acid composition is a biosynthetic precursor of PIM4, PIM6, lipomannan, and lipoarabinomannan. The PIM2 headgroup (inositol-dimannoside) is the most complex to date among all the crystallized CD1d ligands and is remarkably ordered in the CD1d binding groove. A specific hydrogen-bonding network between PIM2 and CD1d orients the headgroup in the center of the binding groove and above the A′ pocket. A central cluster of hydrophilic CD1d residues (Asp153, Thr156, Ser76, Arg79) interacts with the phosphate, inositol, and α1–α6-linked mannose of the headgroup, whereas additional specificity for the α1- and α2-linked mannose is conferred by Thr159. The additional two mannoses in PIM4, relative to PIM2, are located at the distal 6′ carbon of the α1-α6-linked mannose and would project away from the CD1d binding groove for interaction with the TCR. Compared with other CD1d-sphingolipid structures, PIM2 has an increased number of polar interactions between its headgroup and CD1, but reduced specificity for the diacylglycerol backbone. Thus, novel NKT cell agonists can be designed that focus on substitutions of the headgroup rather than on reducing lipid chain length, as in OCH and PBS-25, two potent variants of the highly stimulatory invariant NKT cell agonist α-galactosylceramide.

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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 beta-D-galactofuranose residues; 60% of the available beta-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.

Two classes of mutants with O-antigen-deficient lipopolysaccharides were isolated from the serotype O8 reference strain, belonging to Klebsiella pneumoniae subspecies ozaenae. These mutants were selected by resistance to bacteriophage KO1-2, which recognizes and lyses strains with lipopolysaccharide molecules containing the D-galactan II O antigen. Strain RFK-11 contains a defect in O-antigen synthesis and has a complete core, including the attachment site for O antigen. This mutation is complemented by a plasmid carrying the rfb (O-antigen biosynthesis) gene cluster from the related K. pneumoniae serotype O1. In sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the lipopolysaccharide from strain RFK-9 has a mobility typical of deeprough lipopolysaccharide. RFK-9 lipopolysaccharide lacks the attachment site for O antigen. Lipopolysaccharides from strains RFK-9 and RFK-11 were isolated, and their structures were determined by methylation analyses, nuclear magnetic resonance spectroscopy, and mass spectroscopy. The deduced O8 core oligosaccharide includes the partial core structure reported for the K. pneumoniae subspecies pneumoniae serotype O1 lipopolysaccharide (M. Süsskind, S. Müller-Leonnies, W. Nimmich, H. Brade, and O. Holst, Carbohydr. Res. 269:C1-C7, 1995), consistent with the possibility of a conserved core structure within the species. The core oligosaccharide differs from those of the genera Salmonella and Escherichia by the absence of a hexose-containing outer core, the lack of phosphate residues in the inner core, and the presence of galacturonic acid residues.