Structural Analysis of the Core Region of O-Lipopolysaccharide of Porphyromonas gingivalis from Mutants Defective in O-Antigen Ligase and O-Antigen Polymerase

Porphyromonas gingivalis synthesizes two lipopolysaccharides (LPSs), O-LPS and A-LPS. Here, we elucidate the structure of the core oligosaccharide (OS) of O-LPS from two mutants of P. gingivalis W50, ΔPG1051 (WaaL, O-antigen ligase) and ΔPG1142 (O-antigen polymerase), which synthesize R-type LPS (core devoid of O antigen) and SR-type LPS (core plus one repeating unit of O antigen), respectively. Structural analyses were performed using one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy in combination with composition and methylation analysis. The outer core OS of O-LPS occurs in two glycoforms: an “uncapped core,” which is devoid of O polysaccharide (O-PS), and a “capped core,” which contains the site of O-PS attachment. The inner core region lacks l(d)-glycero-d(l)-manno-heptosyl residues and is linked to the outer core via 3-deoxy-d-manno-octulosonic acid, which is attached to a glycerol residue in the outer core via a monophosphodiester bridge. The outer region of the “uncapped core” is attached to the glycerol and is composed of a linear α-(1→3)-linked d-Man OS containing four or five mannopyranosyl residues, one-half of which are modified by phosphoethanolamine at position 6. An amino sugar, α-d-allosamine, is attached to the glycerol at position 3. In the “capped core,” there is a three- to five-residue extension of α-(1→3)-linked Man residues glycosylating the outer core at the nonreducing terminal residue. β-d-GalNAc from the O-PS repeating unit is attached to the nonreducing terminal Man at position 3. The core OS of P. gingivalis O-LPS is therefore a highly unusual structure, and it is the basis for further investigation of the mechanism of assembly of the outer membrane of this important periodontal bacterium.Porphyromonas gingivalis is a gram-negative anaerobe which is strongly implicated in the etiology of periodontal disease. Several putative virulence factors are produced by this organism. These virulence factors include the cysteine proteases Arg-gingipains (Rgps) and Lys-gingipain (Kgp) specific for Arg-X and Lys-X peptide bonds, respectively, which are capable of degrading several host proteins (56), and lipopolysaccharide (LPS), which has the potential to cause an inflammatory response in the periodontal tissues of the host. These factors are important antigens in patients with periodontal disease and may account for a considerable proportion of the immune response directed against P. gingivalis (58).LPS is a major constituent of the outer membrane of gram-negative bacteria and facilitates interactions with the external environment. It consists of three regions: a hydrophobic lipid A embedded in the outer leaflet of the outer membrane, a core oligosaccharide (OS), and the O-polysaccharide (O-PS) side chain composed of several repeating units. The hydrophobic lipid A serves as an anchor for the LPS and consists of β-1,6-linked d-glucosamine disaccharide, which is usually phosphorylated at the 1 and/or 4′ positions and N and/or O acylated at positions 2, 3, 2′, and 3′ with various amounts of fatty acids. The rest of the LPS molecule projects from the surface. The core region is attached to lipid A and is composed of ∼10 sugars in most bacteria studied to date and can be further subdivided into an inner core and an outer core. The inner core usually contains l(d)-glycero-d-(l)-manno-heptose and 3-deoxy-d-manno-octulosonic acid (Kdo) residues, whereas the outer core is usually composed of hexoses. Attached to the outer core are the repeating units of O antigen (O-PS), which vary in composition, stereochemistry, and the sequence of O-glycosidic linkages between bacterial strains and thereby give rise to O-serotype specificity within bacterial species. Attachment of O antigen to core lipid A results in “smooth” LPS (S-type LPS), whereas LPS lacking O antigen is “rough” LPS (R-type LPS). Attachment of one repeating unit of O-PS to core lipid A results in SR-LPS (core-plus-one repeating unit) (41, 47, 48). In addition, the outer core OS region can be either “uncapped” or “capped.” The “uncapped” core OS is devoid of O-PS repeating units, whereas the “capped” core OS contains attached O-PS repeating units (47, 53) due to modifications in the outer core region.P. gingivalis W50 was originally thought to synthesize a single LPS composed of a tetrasaccharide repeating unit in the O-PS, [→6)-α-d-Glcp-(1→4)-α-l-Rhap-(1→3)-β-d-GalNAc-(1→3)-α-d-Galp-(1→], which is modified by phosphoethanolamine (PEA) at position 2 of Rha in a nonstoichiometric manner (43). However, a second LPS in this organism, namely A-LPS (49), which has a phosphorylated mannan-containing anionic polysaccharide (A-PS), was identified in our laboratory. The A-PS repeating unit is built up of a phosphorylated branched d-Man-containing oligomer composed of an α1→6-linked d-mannose backbone to which α1→2-linked d-Man side chains of different lengths (one or two residues) are attached at position 2. One of the side chains contains Manα1→2-Manα-1-phosphate linked via phosphorus to a backbone Man residue at position O-2. Although A-LPS is predominantly composed of α-d-mannose residues, it cannot be referred to as a homopolymer due to the presence of Manα1→2Manα1-phosphate-containing OS side chains forming a nonglycosidic linkage between the backbone α-mannose and side chains. Hence, it is likely that the synthesis of A-PS (A-LPS) occurs via a “wzy-dependent” pathway in which repeating units formed on the cytoplasmic face of the inner membrane are polymerized at the periplasmic face following transport or flipping across the cytoplasmic membrane. A-LPS cross-reacts with monoclonal antibody (MAb) 1B5 raised against one of the isoforms of Arg-gingipains, a family of differentially glycosylated cysteine proteases (14, 19). Deglycosylation of the cross-reacting Rgps with anhydrous trifluoromethane sulfonic acid abolishes their immunoreactivity to MAb 1B5, indicating that this antibody recognizes a carbohydrate-containing epitope also present in A-LPS (14, 44). Hence, there appear to be common elements in the biosynthesis of A-LPS and the Arg-gingipains of this organism.Inactivation of P. gingivalis waaL (PG1051, O-antigen ligase) abolishes the synthesis of both O-LPS and A-LPS (49). Hence, the WaaL O-antigen ligase appears to have dual specificity and is capable of ligating both O-PS and A-PS chains to core lipid A. The dual specificity of WaaL in the final step of LPS biosynthesis has also been demonstrated in the synthesis of Escherichia coli O-LPS and M_LPS (38) and for Pseudomonas aeruginosa A-band and B-band LPSs (1).However, the linkage between O-PS and A-PS and core OS has not been identified in P. gingivalis. In this paper, we describe a structural investigation of the core OS of O-LPS in which we used R-LPS prepared from ΔPG1051 (49) and ΔPG1142 (putative O-antigen polymerase), which we hypothesized would synthesize an SR-LPS (core plus one repeating unit) (60). The putative O-antigen polymerase encoded at PG1142 (42) is a phenylalanine-rich membrane protein consisting of 347 amino acids which shows 46% similarity over 297 amino acids to EpsK of Lactobacillus delbrueckii subsp. bulgaricus. EpsK is proposed to be a polymerase on the basis of homology and topological similarity to the O-antigen polymerase (Wzy) of E. coli and is required for the synthesis of an exopolysaccharide composed of Gal, Glc, and Rha (5:1:1) containing repeating units in L. delbrueckii (32). Application of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) spectroscopy and methylation and monosaccharide analyses using gas chromatography-mass spectrometry (GC-MS) to purified core-containing OSs isolated from LPS from ΔPG1051 and ΔPG1142 mutants enabled us to solve the LPS core structure of an oral gram-negative bacterium for the first time.     

Characterization of the α- and β-Mannosidases of Porphyromonas gingivalis

Mannose is an important sugar in the biology of the Gram-negative bacterium Porphyromonas gingivalis. It is a major component of the oligosaccharides attached to the Arg-gingipain cysteine proteases, the repeating units of an acidic lipopolysaccharide (A-LPS), and the core regions of both types of LPS produced by the organism (O-LPS and A-LPS) and a reported extracellular polysaccharide (EPS) isolated from spent culture medium. The organism occurs at inflamed sites in periodontal tissues, where it is exposed to host glycoproteins rich in mannose, which may be substrates for the acquisition of mannose by P. gingivalis. Five potential mannosidases were identified in the P. gingivalis W83 genome that may play a role in mannose acquisition. Four mannosidases were characterized in this study: PG0032 was a β-mannosidase, whereas PG0902 and PG1712 were capable of hydrolyzing p-nitrophenyl α-d-mannopyranoside. PG1711 and PG1712 were α-1→3 and α-1→2 mannosidases, respectively. No enzyme function could be assigned to PG0973. α-1→6 mannobiose was not hydrolyzed by P. gingivalis W50. EPS present in the culture supernatant was shown to be identical to yeast mannan and a component of the medium used for culturing P. gingivalis and was resistant to hydrolysis by mannosidases. Synthesis of O-LPS and A-LPS and glycosylation of the gingipains appeared to be unaffected in all mutants. Thus, α- and β-mannosidases of P. gingivalis are not involved in the harnessing of mannan/mannose from the growth medium for these biosynthetic processes. P. gingivalis grown in chemically defined medium devoid of carbohydrate showed reduced α-mannosidase activity (25%), suggesting these enzymes are environmentally regulated.     

Molecular Dynamics and NMR Spectroscopy Studies of E.?coli Lipopolysaccharide Structure and Dynamics

Lipopolysaccharide (LPS), a component of Gram-negative bacterial outer membranes, comprises three regions: lipid A, core oligosaccharide, and O-antigen polysaccharide. Using the CHARMM36 lipid and carbohydrate force fields, we have constructed a model of an Escherichia coli R1 (core) O6 (antigen) LPS molecule. Several all-atom bilayers are built and simulated with lipid A only (LIPA) and varying lengths of 0 (LPS0), 5 (LPS5), and 10 (LPS10) O6 antigen repeating units; a single unit of O6 antigen contains five sugar residues. From ¹H,¹H-NOESY experiments, cross-relaxation rates are obtained from an O-antigen polysaccharide sample. Although some experimental deviations are due to spin-diffusion, the remaining effective proton-proton distances show generally very good agreement between NMR experiments and molecular dynamics simulations. The simulation results show that increasing the LPS molecular length has an impact on LPS structure and dynamics and also on LPS bilayer properties. Terminal residues in a LPS bilayer are more flexible and extended along the membrane normal. As the core and O-antigen are added, per-lipid area increases and lipid bilayer order decreases. In addition, results from mixed LPS0/5 and LPS0/10 bilayer simulations show that the LPS O-antigen conformations at a higher concentration of LPS5 and LPS10 are more orthogonal to the membrane and less flexible. The O-antigen concentration of mixed LPS bilayers does not have a significant effect on per-lipid area and hydrophobic thickness. Analysis of ion and water penetration shows that water molecules can penetrate inside the inner core region, and hydration is critical to maintain the integrity of the bilayer structure.     

Molecular Dynamics and NMR Spectroscopy Studies of E. coli Lipopolysaccharide Structure and Dynamics

Lipopolysaccharide (LPS), a component of Gram-negative bacterial outer membranes, comprises three regions: lipid A, core oligosaccharide, and O-antigen polysaccharide. Using the CHARMM36 lipid and carbohydrate force fields, we have constructed a model of an Escherichia coli R1 (core) O6 (antigen) LPS molecule. Several all-atom bilayers are built and simulated with lipid A only (LIPA) and varying lengths of 0 (LPS0), 5 (LPS5), and 10 (LPS10) O6 antigen repeating units; a single unit of O6 antigen contains five sugar residues. From ¹H,¹H-NOESY experiments, cross-relaxation rates are obtained from an O-antigen polysaccharide sample. Although some experimental deviations are due to spin-diffusion, the remaining effective proton-proton distances show generally very good agreement between NMR experiments and molecular dynamics simulations. The simulation results show that increasing the LPS molecular length has an impact on LPS structure and dynamics and also on LPS bilayer properties. Terminal residues in a LPS bilayer are more flexible and extended along the membrane normal. As the core and O-antigen are added, per-lipid area increases and lipid bilayer order decreases. In addition, results from mixed LPS0/5 and LPS0/10 bilayer simulations show that the LPS O-antigen conformations at a higher concentration of LPS5 and LPS10 are more orthogonal to the membrane and less flexible. The O-antigen concentration of mixed LPS bilayers does not have a significant effect on per-lipid area and hydrophobic thickness. Analysis of ion and water penetration shows that water molecules can penetrate inside the inner core region, and hydration is critical to maintain the integrity of the bilayer structure.     

A single nucleotide exchange in the wzy gene is responsible for the semirough O6 lipopolysaccharide phenotype and serum sensitivity of Escherichia coli strain Nissle 1917

Structural analysis of lipopolysaccharide (LPS) isolated from semirough, serum-sensitive Escherichia coli strain Nissle 1917 (DSM 6601, serotype O6:K5:H1) revealed that this strain's LPS contains a bisphosphorylated hexaacyl lipid A and a tetradecasaccharide consisting of one E. coli O6 antigen repeating unit attached to the R1-type core. Configuration of the GlcNAc glycosidic linkage between O-antigen oligosaccharide and core (beta) differs from that interlinking the repeating units in the E. coli O6 antigen polysaccharide (alpha). The wa(*) and wb(*) gene clusters of strain Nissle 1917, required for LPS core and O6 repeating unit biosyntheses, were subcloned and sequenced. The DNA sequence of the wa(*) determinant (11.8 kb) shows 97% identity to other R1 core type-specific wa(*) gene clusters. The DNA sequence of the wb(*) gene cluster (11 kb) exhibits no homology to known DNA sequences except manC and manB. Comparison of the genetic structures of the wb(*)(O6) (wb(*) from serotype O6) determinants of strain Nissle 1917 and of smooth and serum-resistant uropathogenic E. coli O6 strain 536 demonstrated that the putative open reading frame encoding the O-antigen polymerase Wzy of strain Nissle 1917 was truncated due to a point mutation. Complementation with a functional wzy copy of E. coli strain 536 confirmed that the semirough phenotype of strain Nissle 1917 is due to the nonfunctional wzy gene. Expression of a functional wzy gene in E. coli strain Nissle 1917 increased its ability to withstand antibacterial defense mechanisms of blood serum. These results underline the importance of LPS for serum resistance or sensitivity of E. coli.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

Functional characterization of WaaL, a ligase associated with linking O-antigen polysaccharide to the core of Pseudomonas aeruginosa lipopolysaccharide

The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of D-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.     

The outer membrane protein LptO is essential for the O-deacylation of LPS and the co-ordinated secretion and attachment of A-LPS and CTD proteins in Porphyromonas gingivalis

Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) essential for secretion and attachment to the cell surface. Inactivation of lptO (PG0027) or porT produced mutants that lacked surface protease activity and an electron-dense surface layer. Both mutants showed co-accumulation of A-LPS and unmodified CTD proteins in the periplasm. Lipid profiling by mass spectrometry showed the presence of both tetra- and penta-acylated forms of mono-phosphorylated lipid A in the wild-type and porT mutant, while only the penta-acylated forms of mono-phosphorylated lipid A were found in the lptO mutant, indicating a specific role of LptO in the O-deacylation of mono-phosphorylated lipid A. Increased levels of non-phosphorylated lipid A and the presence of novel phospholipids in the lptO mutant were also observed that may compensate for the missing mono-phosphorylated tetra-acylated lipid A in the outer membrane (OM). Molecular modelling predicted LptO to adopt a β-barrel structure characteristic of an OM protein, supported by the enrichment of LptO in OM vesicles. The results suggest that LPS deacylation by LptO is linked to the co-ordinated secretion of A-LPS and CTD proteins by a novel secretion and attachment system to form a structured surface layer.     

The complete structure of the core of the LPS from Plesiomonas shigelloides 302-73 and the identification of its O-antigen biological repeating unit

Plesiomonas shigelloides is a Gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal infections, which especially invades immunocompromised patients and neonates. The lipopolysaccharides are one of the major virulence determinants in Gram-negative bacteria and are structurally composed of three different domains: the lipid A, the core oligosaccharide and the O-antigen polysaccharide.In the last few years we elucidated the structures of the O-chain and the core oligosaccharide from the P. shigelloides strain 302-73. In this paper we now report the characterization of the linkage between the core and the O-chain. The LPS obtained after PCP extraction contained a small number of O-chain repeating units. The product obtained by hydrazinolysis was analysed by FTICR-ESIMS and suggested the presence of an additional Kdo in the core oligosaccharide. Furthermore, the LPS was hydrolysed under mild acid conditions and a fraction that contained one O-chain repeating unit linked to a Kdo residue was isolated and characterized by FTICR-ESIMS and NMR spectroscopy. Moreover, after an alkaline reductive hydrolysis, a disaccharide α-Kdo-(2→6)-GlcNol was isolated and characterized. The data obtained proved the presence of an α-Kdo in the outer core and allowed the identification of the O-antigen biological repeating unit as well as its linkage with the core oligosaccharide.     

Physicochemical surface properties ofKlebsiella pneumoniae

The following cell surface physicochemical characteristics were investigated inKlebsiella pneumoniae: surface charge, surface hydrophobicity by different methods, and accessibility of the lipid fraction of the outer membrane. The capsular polysaccharide, as well as the O-antigen repeating units of the lipopolysaccharide (LPS), conferred a hydrophilic, negatively charged surface to the bacterium, and a barrier to the dye congo red, which binds sites within the lipid fraction of the outer membrane (OM).     

A mannosyl transferase required for lipopolysaccharide inner core assembly in Rhizobium leguminosarum. Purification,substrate specificity,and expression in Salmonella waaC mutants

The lipopolysaccharide (LPS) core domain of Gram-negative bacteria plays an important role in outer membrane stability and host interactions. Little is known about the biochemical properties of the glycosyltransferases that assemble the LPS core. We now report the purification and characterization of the Rhizobium leguminosarum mannosyl transferase LpcC, which adds a mannose unit to the inner 3-deoxy-d-manno-octulosonic acid (Kdo) moiety of the LPS precursor, Kdo(2)-lipid IV(A). LpcC containing an N-terminal His(6) tag was assayed using GDP-mannose as the donor and Kdo(2)-[4'-(32)P]lipid IV(A) as the acceptor and was purified to near homogeneity. Sequencing of the N terminus confirmed that the purified enzyme is the lpcC gene product. Mild acid hydrolysis of the glycolipid generated in vitro by pure LpcC showed that the mannosylation occurs on the inner Kdo residue of Kdo(2)-[4'-(32)P]lipid IV(A). A lipid acceptor substrate containing two Kdo moieties is required by LpcC, since no activity is seen with lipid IV(A) or Kdo-lipid IV(A). The purified enzyme can use GDP-mannose or, to a lesser extent, ADP-mannose (both of which have the alpha-anomeric configuration) for the glycosylation of Kdo(2)-[4'-(32)P]lipid IV(A). Little or no activity is seen with ADP-glucose, UDP-glucose, UDP-GlcNAc, or UDP-galactose. A Salmonella typhimurium waaC mutant, which lacks the enzyme for incorporating the inner l-glycero-d-manno-heptose moiety of LPS, regains LPS with O-antigen when complemented with lpcC. An Escherichia coli heptose-less waaC-waaF deletion mutant expressing the R. leguminosarum lpcC gene likewise generates a hybrid LPS species consisting of Kdo(2)-lipid A plus a single mannose residue. Our results demonstrate that heterologous lpcC expression can be used to modify the structure of the Salmonella and E. coli LPS cores in living cells.     

Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity

The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O-antigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.     

Molecular characterization of the Pseudomonas aeruginosa serotype O5 (PAO1) B-band lipopolysaccharide gene cluster

Pseudomonas aeruginosa co-expresses A-band lipopolysaccharide (LPS), a homopolymer of rhamnose, and B-band LPS, a heteropolymer with a repeating unit of 2–5 sugars which is the serotype-specific antigen. The gene clusters for A- and B-band biosynthesis in P. aeruginosa O5 (strain PAO1) have been cloned previously. Here we report the DNA sequence and molecular analysis of the B-band O-antigen biosynthetic cluster. Sixteen open reading frames (ORFs) thought to be involved in synthesis of the O5 O antigen were identified, including wzz ( rol ), wzy ( rfc ), and wbpA – wbpN . A further 3 ORFs not thought to be involved with LPS synthesis were identified ( hisH , hisF , and uvrB ). Most of the wbp genes are found only in serotypes O2, O5, O16, O18, and O20, which form a chemically and structurally related O-antigen serogroup. In contrast, wbpM and wbpN are common to all 20 serotypes of P. aeruginosa. Although wbpM is not serogroup-specific, knockout mutations confirmed it is necessary for O5 O-antigen biosynthesis. A novel insertion sequence, IS 1209 , is present at the junction between the serogroup-specific and non-specific regions. We have predicted the functions of the proteins encoded in the wbp cluster based on their homologies to those in the databases, and provide a proposed pathway of P. aeruginosa O5 O-antigen biosynthesis.     

Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion

In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization.     

Human anti-scrub typhus rickettsia and rabbit anti-Proteus antibodies recognize similar epitope in the O-polysaccharide part of Proteus mirabilis OXK lipopolysaccharide

In the Weil-Felix test, sera from patients infected with Orientia tsutsugamushi reacted with lipopolysaccharide (LPS) from Proteus mirabilis OXK strains. The O-polysaccharide of P. mirabilis OXK LPS consisted of pentasaccharide repeating units, with amidically-linked lysine residues. The lysine, linked to galacturonic residues, which plays an important role in the reaction with rabbit anti-OXK antibodies, was revealed with the aid of synthetic antigens. Using ELISA, immunoglobulin M antibodies from scrub typhus patients reacted with the O-specific polysaccharide of strain OXK LPS only. This reaction was inhibited by rabbit antibodies specific to the O-antigen of strain OXK LPS. Both human and rabbit antibodies may bind to similar epitopes on the O-polysaccharide part of P. mirabilis OXK LPS.     

Separation of two lipopolysaccharide populations with different contents of O-antigen factor 122 in Salmonella enterica serovar Typhimurium

Lipopolysaccharide (LPS) extraction from smooth-type Salmonella enterica sv. Typhimurium was carried out with the modified phenol/chloroform/petroleum ether method (volume ratio 5:5:8). In this procedure, LPS was precipitated from 90% phenol sequentially with water and acetone to yield LPS-H2O (minute amounts) and LPS-Ac (major amounts), respectively. Chemical analyses of the LPS fractions revealed that in the O antigen of LPS-H2O position C4 of the D-galactose was extensively glucosylated, corresponding corresponding to the O-antigen factor 122. In LPS-Ac, this glucosylation was negligible. Inspection of the LPS fractions by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and silver staining suggested that the glucosylation in LPS-H2O was present only in LPS species with a chain length higher than six repeating units.     

Characterization of lipopolysaccharide fractions and their interactions with cells and model membranes.

The role of the length of the O-antigen polysaccharide side chain of bacterial lipopolysaccharide (LPS) in biological and model membrane systems was investigated. LPS from Salmonella typhimurium ATCC 14028 was chromatographed on a Sephadex G-200 column in the presence of sodium deoxycholate and separated into three fractions on the basis of molecular size. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot (immunoblot), and chemical analyses indicated that these fractions differed from each other primarily in the number of repeating units in the O-antigen polysaccharide side chain. In a biological system fractions 2 and 3 had the same effects to induce mitogenesis in murine lymphocytes, but fraction 1 was less effective than the other two fractions. In a model membrane system, LPS induced changes in small unilamellar vesicles (SUVs) which were measured by changes in the behavior of a fluorescent probe, 1,6-diphenylhexa-1,3,5-triene (DPH), and interaction of increasing amounts of all LPS fractions with SUVs gradually increased DPH anisotropy. Fractions 2 and 3 had similar effects on the SUVs as detected by changes in DPH anisotropy, while fraction 1 had almost twice as much activity as the other two fractions. These results suggest that the polysaccharide side chain of LPS may modulate the ability of biologically active lipid A to interact with cells and model membranes. In addition, factors other than changes in membrane fluidity may play a role in mediating LPS-induced cell activation.     

Mechanism of O-antigen distribution in lipopolysaccharide.

O-antigen units are nonuniformly distributed among lipid A-core molecules in lipopolysaccharide (LPS) from gram-negative bacteria, as revealed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; the actual distribution patterns are complex, multimodal, and strain specific. Although the basic biochemical steps involved in synthesis and polymerization of O-antigen monomers and their subsequent attachment to lipid A-core are known, the mechanism by which specific multimodal distribution patterns are attained in mature LPS has not been previously considered theoretically or experimentally. We have developed probability equations which completely describe O-antigen distribution among lipid A-core molecules in terms of the probability of finding a nascent polymer (O antigen linked to carrier lipid) of length k (Tk) and the probability that a nascent polymer of length k will be extended to k + 1 by polymerase (pk) or transferred to lipid A-core by ligase (qk). These equations were used to show that multimodal distribution patterns in mature LPS cannot be produced if all pk are equal to p and all qk are equal to q, conditions which indicate a lack of selectivity of polymerase and ligase, respectively, for nascent O-antigen chain lengths. A completely stochastic model (pk = p, qk = q) of O-antigen polymerization and transfer to lipid A-core was also inconsistent with observed effects of mutations which resulted in partial inhibition of O-antigen monomer synthesis, lipid A-core synthesis, or ligase activity. The simplest explanation compatible with experimental observations is that polymerase or ligase, or perhaps both, have specificity for certain O-antigen chain lengths during biosynthesis of LPS. Our mathematical model indicates selectively probably was associated with the polymerase reaction. Although one may argue for a multimodal distribution pattern based on a kinetic mechanism i.e., varying reaction parameters in space or in time during cell growth, such a model requires complex sensory and regulatory mechanisms to explain the mutant data and mechanisms for sequestering specific components of LPS biosynthesis to explain the distribution pattern in normal cells. We favor the simple alternative of enzyme specificity and present generalized equations which should be useful in analysis of other analogous biochemical systems.     

Differential induction of IL-1β and IL-6 production by the nontoxic lipid A from Porphyromonas gingivalis in comparison with synthetic Escherichia coli lipid A in human peripheral blood mononuclear cells

Abstract Porphyromonas gingivalis 381 lipid A possesses 1-phospho β(1–6)-linked glucosamine disaccharide with 3-hydroxy-15-methylhexadecanoyl and 3-hexadecanoyloxy-15-methylhexadecanoyl groups at the 2- and 2′-positions, respectively. P. gingivalis lipid A indicated lower activities in inducing interleukin-1β (IL-1β) mRNA expression, pro-IL-1β protein synthesis and IL-1β production than those of synthetic Escherichia coli lipid A (compound 506) in human peripheral blood mononuclear cells (PBMC). The induction of IL-6 mRNA and IL-6 synthesis by P. gingivalis lipid A were comparable to those of compound 506. Herbimycin A, H-7 and H-8, inhibitors of tyrosine kinase, protein kinase C and cyclic nucleotide-dependent protein kinase, inhibited P. gingivalis lipid A- and compound 506-induced IL-1β and IL-6 synthesis. W-7, an inhibitor of calmodulin (CaM) kinase, inhibited only P. gingivalis lipid A-induced IL-1β production. The result suggests that the CaM kinase-dependent cascade is involved in the down-regulation of IL-1β production by P. gingivalis lipid A. P. gingivalis lipid A and compound 506 also functioned in the induction of tyrosine and serine/threonine phosphorylation of several proteins in PBMC. P. gingivalis lipid A inhibited specific binding of fluorescein-labelled E. coli LPS to the PBMC. The nontoxic lipid A of P. gingivalis , having a chemical structure different from toxic compound 506, appears to induce the up- and down-regulation of the differential cytokine-producing activities following the activation of various intracellular enzymes including the CaM kinase through the common receptor sites of LPS.     

Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50.

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro-inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg-gingipains of P. gingivalis are post-translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O-polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF-COSY, TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-31P HXTOCSY) and GC-MS data. These data allowed us to conclude that the O-polysaccharide is built up of the tetrasaccharide repeating sequence: -->6)-alpha-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Galp-(1--> and carries a monophosphoethanolamine residue at position C-2 of the alpha-rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O-polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.