Lipopolysaccharide Structure and Biosynthesis in Helicobacter pylori

This review covers the current knowledge and gaps in Helicobacter pylori lipopolysaccharide (LPS) structure and biosynthesis. H. pylori is a Gram‐negative bacterium which colonizes the luminal surface of the human gastric epithelium. Both a constitutive alteration of the lipid A preventing TLR4 elicitation and host mimicry of the Lewis antigen decorated O‐antigen of H. pylori LPS promote immune escape and chronic infection. To date, the complete structure of H. pylori LPS is not available, and the proposed model is a linear arrangement composed of the inner core defined as the hexa‐saccharide (Kdo‐LD‐Hep‐LD‐Hep‐DD‐Hep‐Gal‐Glc), the outer core composed of a conserved trisaccharide (‐GlcNAc‐Fuc‐DD‐Hep‐) linked to the third heptose of the inner core, the glucan, the heptan and a variable O‐antigen, generally consisting of a poly‐LacNAc decorated with Lewis antigens. Although the glycosyltransferases (GTs) responsible for the biosynthesis of the H. pylori O‐antigen chains have been identified and characterized, there are many gaps in regard to the biosynthesis of the core LPS. These limitations warrant additional mutagenesis and structural studies to obtain the complete LPS structure and corresponding biosynthetic pathway of this important gastric bacterium.     

A two‐component Kdo hydrolase in the inner membrane of Francisella novicida

Lipid A coats the outer surface of the outer membrane of Gram‐negative bacteria. In Francisella tularensis subspecies novicida lipid A is present either as the covalently attached anchor of lipopolysaccharide (LPS) or as free lipid A. The lipid A moiety of Francisella LPS is linked to the core domain by a single 2‐keto‐3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residue. F. novicida KdtA is bi‐functional, but F. novicida contains a membrane‐bound Kdo hydrolase that removes the outer Kdo unit. The hydrolase consists of two proteins (KdoH1 and KdoH2), which are expressed from adjacent, co‐transcribed genes. KdoH1 (related to sialidases) has a single predicted N‐terminal transmembrane segment. KdoH2 contains 7 putative transmembrane sequences. Neither protein alone catalyses Kdo cleavage when expressed in E. coli. Activity requires simultaneous expression of both proteins or mixing of membranes from strains expressing the individual proteins under in vitro assay conditions in the presence of non‐ionic detergent. In E. coli expressing KdoH1 and KdoH2, hydrolase activity is localized in the inner membrane. WBB06, a heptose‐deficient E. coli mutant that makes Kdo₂‐lipid A as its sole LPS, accumulates Kdo‐lipid A when expressing the both hydrolase components, and 1‐dephospho‐Kdo‐lipid A when expressing both the hydrolase and the Francisella lipid A 1‐phosphatase (LpxE).     

3-Deoxy-d-manno-octulosonic Acid (Kdo) Hydrolase Identified in Francisella tularensis,Helicobacter pylori,and Legionella pneumophila

3-Deoxy-d-manno-octulosonic acid (Kdo) is an eight-carbon sugar ubiquitous in Gram-negative bacterial lipopolysaccharides (LPS). Although its biosynthesis is well described, no protein has yet been identified as a Kdo hydrolase. However, Kdo hydrolase enzymatic activity has been detected in membranes of Helicobacter pylori and Francisella tularensis and may be responsible for the removal of side-chain Kdo from the LPS core saccharides. We now report the identification of genes encoding a Kdo hydrolase in F. tularensis Schu S4 and live vaccine strain strains, in H. pylori 26695 strain and in Legionella pneumophila Philadelphia 1 strain. We have renamed the genes kdhA for keto-deoxyoctulosonate hydrolase A. Deletion of kdhA abolished Kdo hydrolase activity in membranes of F. tularensis live vaccine strain. The F. tularensis kdhA mutant synthesized a core oligosaccharide containing a Kdo disaccharide with one of the Kdo residues being a terminal side chain. This side-chain Kdo monosaccharide was absent in the wild-type core oligosaccharide. Expression in Escherichia coli of recombinant KdhA from F. tularensis, H. pylori, and L. pneumophila resulted in a reduction of membrane-associated side-chain Kdo. The identification of this previously faceless enzyme will accelerate study of the biosynthetic basis and biologic impact for postbiosynthetic LPS structural modification.     

A novel 3-deoxy-D-manno-octulosonic acid (Kdo) hydrolase that removes the outer Kdo sugar of Helicobacter pylori lipopolysaccharide

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.     

The Lipopolysaccharide from Capnocytophaga canimorsus Reveals an Unexpected Role of the Core-Oligosaccharide in MD-2 Binding

Capnocytophaga canimorsus is a usual member of dog''s mouths flora that causes rare but dramatic human infections after dog bites. We determined the structure of C. canimorsus lipid A. The main features are that it is penta-acylated and composed of a “hybrid backbone” lacking the 4′ phosphate and having a 1 phosphoethanolamine (P-Etn) at 2-amino-2-deoxy-d-glucose (GlcN). C. canimorsus LPS was 100 fold less endotoxic than Escherichia coli LPS. Surprisingly, C. canimorsus lipid A was 20,000 fold less endotoxic than the C. canimorsus lipid A-core. This represents the first example in which the core-oligosaccharide dramatically increases endotoxicity of a low endotoxic lipid A. The binding to human myeloid differentiation factor 2 (MD-2) was dramatically increased upon presence of the LPS core on the lipid A, explaining the difference in endotoxicity. Interaction of MD-2, cluster of differentiation antigen 14 (CD14) or LPS-binding protein (LBP) with the negative charge in the 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) of the core might be needed to form the MD-2 – lipid A complex in case the 4′ phosphate is not present.     

Identification of the lipopolysaccharide O‐antigen biosynthesis priming enzyme and the O‐antigen ligase in Myxococcus xanthus: critical role of LPS O‐antigen in motility and development

Myxococcus xanthus is a model bacterium to study social behavior. At the cellular level, the different social behaviors of M. xanthus involve extensive cell–cell contacts. Here, we used bioinformatics, genetics, heterologous expression and biochemical experiments to identify and characterize the key enzymes in M. xanthus implicated in O‐antigen and lipopolysaccharide (LPS) biosynthesis and examined the role of LPS O‐antigen in M. xanthus social behaviors. We identified WbaP_Mx (MXAN_2922) as the polyisoprenyl‐phosphate hexose‐1‐phosphate transferase responsible for priming O‐antigen synthesis. In heterologous expression experiments, WbaP_Mx complemented a Salmonella enterica mutant lacking the endogenous WbaP that primes O‐antigen synthesis, indicating that WbaP_Mx transfers galactose‐1‐P to undecaprenyl‐phosphate. We also identified WaaL_Mx (MXAN_2919), as the O‐antigen ligase that joins O‐antigen to lipid A‐core. Our data also support the previous suggestion that Wzm_Mx (MXAN_4622) and Wzt_Mx (MXAN_4623) form the Wzm/Wzt ABC transporter. We show that mutations that block different steps in LPS O‐antigen synthesis can cause pleiotropic phenotypes. Also, using a wbaP_Mx deletion mutant, we revisited the role of LPS O‐antigen and demonstrate that it is important for gliding motility, conditionally important for type IV pili‐dependent motility and required to complete the developmental program leading to the formation of spore‐filled fruiting bodies.     

NMR-based Structural Analysis of the Complete Rough-type Lipopolysaccharide Isolated from Capnocytophaga canimorsus

We here describe the NMR analysis of an intact lipopolysaccharide (LPS, endotoxin) in water with 1,2-dihexanoyl-sn-glycero-3-phosphocholine as detergent. When HPLC-purified rough-type LPS of Capnocytophaga canimorsus was prepared, ¹³C,¹⁵N labeling could be avoided. The intact LPS was analyzed by homonuclear (¹H) and heteronuclear (¹H,¹³C, and ¹H,³¹P) correlated one- and two-dimensional NMR techniques as well as by mass spectrometry. It consists of a penta-acylated lipid A with an α-linked phosphoethanolamine attached to C-1 of GlcN (I) in the hybrid backbone, lacking the 4′-phosphate. The hydrophilic core oligosaccharide was found to be a complex hexasaccharide with two mannose (Man) and one each of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo), Gal, GalN, and l-rhamnose residues. Position 4 of Kdo is substituted by phosphoethanolamine, also present in position 6 of the branched Man^I residue. This rough-type LPS is exceptional in that all three negative phosphate residues are “masked” by positively charged ethanolamine substituents, leading to an overall zero net charge, which has so far not been observed for any other LPS. In biological assays, the corresponding isolated lipid A was found to be endotoxically almost inactive. By contrast, the intact rough-type LPS described here expressed a 20,000-fold increased endotoxicity, indicating that the core oligosaccharide significantly contributes to the endotoxic potency of the whole rough-type C. canimorsus LPS molecule. Based on these findings, the strict view that lipid A alone represents the toxic center of LPS needs to be reassessed.     

Helicobacter pylori Lipopolysaccharide Is Synthesized via a Novel Pathway with an Evolutionary Connection to Protein N-Glycosylation

Lipopolysaccharide (LPS) is a major component on the surface of Gram negative bacteria and is composed of lipid A-core and the O antigen polysaccharide. O polysaccharides of the gastric pathogen Helicobacter pylori contain Lewis antigens, mimicking glycan structures produced by human cells. The interaction of Lewis antigens with human dendritic cells induces a modulation of the immune response, contributing to the H. pylori virulence. The amount and position of Lewis antigens in the LPS varies among H. pylori isolates, indicating an adaptation to the host. In contrast to most bacteria, the genes for H. pylori O antigen biosynthesis are spread throughout the chromosome, which likely contributed to the fact that the LPS assembly pathway remained uncharacterized. In this study, two enzymes typically involved in LPS biosynthesis were found encoded in the H. pylori genome; the initiating glycosyltransferase WecA, and the O antigen ligase WaaL. Fluorescence microscopy and analysis of LPS from H. pylori mutants revealed that WecA and WaaL are involved in LPS production. Activity of WecA was additionally demonstrated with complementation experiments in Escherichia coli. WaaL ligase activity was shown in vitro. Analysis of the H. pylori genome failed to detect a flippase typically involved in O antigen synthesis. Instead, we identified a homolog of a flippase involved in protein N-glycosylation in other bacteria, although this pathway is not present in H. pylori. This flippase named Wzk was essential for O antigen display in H. pylori and was able to transport various glycans in E. coli. Whereas the O antigen mutants showed normal swimming motility and injection of the toxin CagA into host cells, the uptake of DNA seemed to be affected. We conclude that H. pylori uses a novel LPS biosynthetic pathway, evolutionarily connected to bacterial protein N-glycosylation.     

Conflicting roles for a cell surface modification in Salmonella

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O‐antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram‐negative bacteria. We determined that activation of the PmrA/PmrB two‐component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA‐activated gene wzz_st, which encodes the protein that determines long O‐antigen chain length and confers resistance to complement‐mediated killing. By contrast, this growth defect did not require the wzz_fepE gene, which controls production of very long O‐antigen, or other PmrA‐activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O‐antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer‐membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O‐antigen in its LPS to respond to the different conditions it faces during infection.     

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide

Background: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6‐glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6‐glucan and characterize their binding properties and functional activity. Materials and Methods: BALB/c mice were injected intraperitoneally with 10⁸ formalin‐fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti‐α1,6‐glucan‐producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole‐cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild‐type and mutant strains of H. pylori. Results: The generated anti‐α1,6‐glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6‐linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD₄₅₀ ≥ 0.2). Bactericidal activity was observed against selective wild‐type and mutant H. pylori strains exhibiting OD₄₅₀ values of ≥0.45 in WCE. Conclusions: Anti‐α1,6‐glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS‐based vaccine against H. pylori infections.     

Identification of a broad family of lipid A late acyltransferases with non‐canonical substrate specificity

Most Gram‐negative organisms produce lipopolysaccharide (LPS), a complex macromolecule anchored to the bacterial membrane by the lipid A moiety. Lipid A is synthesized via the Raetz pathway, a conserved nine‐step enzymatic process first characterized in Escherichia coli. The Epsilonproteobacterium Helicobacter pylori uses the Raetz pathway to synthesize lipid A; however, only eight of nine enzymes in the pathway have been identified in this organism. Here, we identify the missing acyltransferase, Jhp0255, which transfers a secondary acyl chain to the 3′‐linked primary acyl chain of lipid A, an activity similar to that of E. coli LpxM. This enzyme, reannotated as LpxJ due to limited sequence similarity with LpxM, catalyses addition of a C12:0 or C14:0 acyl chain to the 3′‐linked primary acyl chain of lipid A, complementing an E. coli LpxM mutant. Enzymatic assays demonstrate that LpxJ and homologues in Campylobacter jejuni and Wolinella succinogenes can act before the 2′ secondary acyltransferase, LpxL, as well as the 3‐deoxy‐d ‐manno‐octulosonic acid (Kdo) transferase, KdtA. Ultimately, LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homologue, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homologue.     

Oxidative damage of the gastric mucosa in Helicobacter pylori positive chronic atrophic and nonatrophic gastritis, before and after eradication

Background. Helicobacter pylori is the main cause of gastritis and a primary carcinogen. The aim of this study was to assess oxidative damage in mucosal compartments of gastric mucosa in H. pylori positive and negative atrophic and nonatrophic gastritis. Materials and methods. Five groups of 10 patients each were identified according to H. pylori positive or negative chronic atrophic (Hp‐CAG and CAG, respectively) and nonatrophic gastritis (Hp‐CG and CG, respectively), and H. pylori negative normal mucosa (controls). Oxidative damage was evaluated by nitrotyrosine immunohistochemistry in the whole mucosa and in each compartment at baseline and at 2 and 12 months after eradication. Types of intestinal metaplasia were classified by histochemistry. Results. Total nitrotyrosine levels appeared significantly higher in H. pylori positive than in negative patients, and in Hp‐CAG than in Hp‐CG (p < .001); no differences were found between H. pylori negative gastritis and normal mucosa. Nitrotyrosine were found in foveolae and intestinal metaplasia only in Hp‐CAG. At 12 months after H. pylori eradication, total nitrotyrosine levels showed a trend toward a decrease in Hp‐CG and decreased significantly in Hp‐CAG (p = .002), disappearing from the foveolae (p = .002), but remaining unchanged in intestinal metaplasia. Type I and II of intestinal metaplasia were present with the same prevalence in Hp‐CAG and CAG, and did not change after H. pylori eradication. Conclusions. Oxidative damage of the gastric mucosa increases from Hp‐CG to Hp‐CAG, involving the foveolae and intestinal metaplasia. H. pylori eradication induces a complete healing of foveolae but not of intestinal metaplasia, reducing the overall oxidative damage in the mucosa.     

The Origin of 8-Amino-3,8-dideoxy-d-manno-octulosonic Acid (Kdo8N) in the Lipopolysaccharide of Shewanella oneidensis

Lipopolysaccharide (LPS; endotoxin) is an essential component of the outer monolayer of nearly all Gram-negative bacteria. LPS is composed of a hydrophobic anchor, known as lipid A, an inner core oligosaccharide, and a repeating O-antigen polysaccharide. In nearly all species, the first sugar bridging the hydrophobic lipid A and the polysaccharide domain is 3-deoxy-d-manno-octulosonic acid (Kdo), and thus it is critically important for LPS biosynthesis. Modifications to lipid A have been shown to be important for resistance to antimicrobial peptides as well as modulating recognition by the mammalian innate immune system. Therefore, lipid A derivatives have been used for development of vaccine strains and vaccine adjuvants. One derivative that has yet to be studied is 8-amino-3,8-dideoxy-d-manno-octulosonic acid (Kdo8N), which is found exclusively in marine bacteria of the genus Shewanella. Using bioinformatics, a candidate gene cluster for Kdo8N biosynthesis was identified in Shewanella oneidensis. Expression of these genes recombinantly in Escherichia coli resulted in lipid A containing Kdo8N, and in vitro assays confirmed their proposed enzymatic function. Both the in vivo and in vitro data were consistent with direct conversion of Kdo to Kdo8N prior to its incorporation into the Kdo8N-lipid A domain of LPS by a metal-dependent oxidase followed by a glutamate-dependent aminotransferase. To our knowledge, this oxidase is the first enzyme shown to oxidize an alcohol using a metal and molecular oxygen, not NAD(P)⁺. Creation of an S. oneidensis in-frame deletion strain showed increased sensitivity to the cationic antimicrobial peptide polymyxin as well as bile salts, suggesting a role in outer membrane integrity.     

Glycoengineering of host mimicking type‐2 LacNAc polymers and Lewis X antigens on bacterial cell surfaces

Bacterial carbohydrate structures play a central role in mediating a variety of host–pathogen interactions. Glycans can either elicit protective immune response or lead to escape of immune surveillance by mimicking host structures. Lipopolysaccharide (LPS), a major component on the surface of Gram‐negative bacteria, is composed of a lipid A‐core and the O‐antigen polysaccharide. Pathogens like Neisseria meningitidis expose a lipooligosaccharide (LOS), which outermost glycans mimick mammalian epitopes to avoid immune recognition. Lewis X (Galβ1–4(Fucα1–3)GlcNAc) antigens of Helicobacter pylori or of the helminth Schistosoma mansoni modulate the immune response by interacting with receptors on human dendritic cells. In a glycoengineering approach we generate human carbohydrate structures on the surface of recombinant Gram‐negative bacteria, such as Escherichia coli and Salmonella enterica sv. Typhimurium that lack O‐antigen. A ubiquitous building block in mammalian N‐linked protein glycans is Galβ1‐4GlcNAc, referred to as a type‐2 N‐acetyllactosamine, LacNAc, sequence. Strains displaying polymeric LacNAc were generated by introducing a combination of glycosyltransferases that act on modified lipid A‐cores, resulting in efficient expression of the carbohydrate epitope on bacterial cell surfaces. The poly‐LacNAc scaffold was used as an acceptor for fucosylation leading to polymers of Lewis X antigens. We analysed the distribution of the carbohydrate epitopes by FACS, microscopy and ELISA and confirmed engineered LOS containing LacNAc and Lewis X repeats by MALDI‐TOF and NMR analysis. Glycoengineered LOS induced pro‐inflammatory response in murine dendritic cells. These bacterial strains can thus serve as tools to analyse the role of defined carbohydrate structures in different biological processes.     

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.     

Structural diversity of the core oligosaccharide domain of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> lipopolysaccharide

Pseudomonas aeruginosa is a Gram-negative bacterium that is ubiquitous in the environment and generally considered to be a saprophyte, but it is also an important opportunistic human pathogen. Pseudomonas aeruginosa elaborates a variety of virulence factors, one of which is lipopolysaccharide (LPS). LPS of P. aeruginosa is composed of three distinct regions: lipid A, core oligosaccharide (OS), and the long-chain O antigen. The core OS of P. aeruginosa is composed of L-glycero-D-manno-heptose, 3-deoxy-D-manno-oct-2-ulosonic acid, D-galactosamine, D-glucose, and L-rhamnose. Noncarbohydrate substituents are also found in the core OS including phosphate, 2-aminoethyl (di)phosphate, acetyl, alanyl and carbamoyl groups. Pseudomonas aeruginosa simultaneously synthesizes two core glycoforms, namely, capped and uncapped core. The capped core is covalently attached to an O antigen, whereas the uncapped core is devoid of O antigen. Although the core of P. aeruginosa LPS is relatively conserved, strain-to-strain variability of its structure exists. This includes phosphorylation pattern, the level of O-acetylation, and the presence or absence of a fourth glucose residue at the distal end of the uncapped core. A number of studies have been reported on the structures of unique truncated core OS with unusual modifications. This mini-review summarizes the diversity of P. aeruginosa complete and truncated core OS structures published over the past fifteen years     

Structure of the lipid A-inner core region and biological activity of Plesiomonas shigelloides O54 (strain CNCTC 113/92) lipopolysaccharide

Plesiomonas shigelloides is a Gram-negative rod associated with episodes of intestinal infections and outbreaks of diarrhea in humans. The extraintestinal infections caused by this bacterium, for example, endopthalmitis, meningitidis, bacteremia, and septicemia, usually have gastrointestinal origin and serious course. The lipopolysaccharide (LPS, endotoxin) as virulence factor is important in enteropathogenicity of this bacterium. LPSs of P. shigelloides and especially their lipid A part, that is, the immunomodulatory center of LPS, have not been extensively investigated. The structure of P. shigelloides O54 lipid A was determined by chemical analysis combined with MALDI-TOF mass spectrometry, and the intact Kdo-containing core region was investigated by NMR spectroscopy on deacylated LPS. Products from alkaline deacylation of LPS, containing 4-substituted uronic acids, are usually very complex and difficult to separate. Since Kdo residues, like sialic acids, form complexes with serotonin, we used immobilized serotonin for one-step isolation of oligosaccharide containing the intact Kdo region from the reaction mixture by affinity chromatography. The major form of lipid A was built of beta-d-GlcpN4PPEtn-(1-->6)-alpha-d-GlcpN1P disaccharide substituted with 14:0(3-OH), 12:0(3-OH), 14:0(3-O-14:0), and 12:0(3-O-12:0) acyl groups at N-2, O-3, N-2', and O-3', respectively. This is a novel structure among known lipid A molecules. Analysis of intact Kdo-lipid A region, lipid A and its linkage with the core oligosaccharide completes the structural investigation of P. shigelloides O54 LPS, resolving the entire molecule. Biological activities and observed discrepancy between in vitro and in vivo activity of P. shigelloides and Escherichia coli LPS are discussed.     

A deletion in the wapB promoter in many serotypes of Pseudomonas aeruginosa accounts for the lack of a terminal glucose residue in the core oligosaccharide and resistance to killing by R3‐pyocin

Pseudomonas aeruginosa is an opportunistic human pathogen producing a variety of virulence factors. One of them is lipopolysaccharide, consisting of endotoxic lipid A and long‐chain O‐antigen polysaccharide, which are connected together through a short linker region, called core oligosaccharide. Chemical structures of the core oligosaccharide are well conserved, with one exception, in that certain strains of P. aeruginosa add a terminal glucose residue (Glc^IV) to core by a transferase reaction, due to the activity of a glucosyltransferase, WapB. Here, we investigated the regulation of wapB expression. Our results showed that while the majority of analysed genomes of P. aeruginosa contain wapB, many of these have a conserved identical 5‐nucleotide deletion in the upstream region that inactivated the promoter. This deletion is within the ?10 hexamer that is recognized by a principle sigma factor (RpoD, or σ70) as proven by data from an electromobility shift assay. These results provide the molecular basis of why LPS core of many P. aeruginosa strains is lacking Glc^IV. In addition, we show that absence of Glc^IV due to an inactive wapB promoter confers resistance to killing by R3‐pyocin, a phage tail‐like bacteriocin of P. aeruginosa.     

WaaA of the Hyperthermophilic Bacterium Aquifex aeolicus Is a Monofunctional 3-Deoxy-d-manno-oct-2-ulosonic Acid Transferase Involved in Lipopolysaccharide Biosynthesis

The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli ΔwaaA suppressor strain resulted in synthesis of lipid IV_A precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.Lipopolysaccharide (LPS)⁷ is the major constituent of the outer leaflet of the outer membrane (OM) of virtually all Gram-negative bacteria. LPS is a unique amphiphilic molecule composed of a hydrophilic heteropolysaccharide and a covalently bound lipid moiety, lipid A, which anchors the molecule in the OM. The polysaccharide component of many wild-type bacteria can be subdivided into a highly variable O-specific polysaccharide and a structurally less heterogeneous outer and inner core oligosaccharide (1). The 8-carbon sugar 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) links the lipid A to the carbohydrate domain of LPS and is the only conserved structural element found in all inner core regions investigated to date (2).The ubiquitous nature of Kdo within LPS structures and its essential role in maintaining OM integrity and viability of the majority of Gram-negative bacteria has prompted detailed studies into its biosynthesis. The Kdo pathway is initiated by the enzyme d-arabinose-5-phosphate isomerase, which catalyzes the interconversion of d-ribulose 5-phosphate and d-arabinose 5-phosphate (3). The Kdo-8-phosphate synthase KdsA subsequently condenses d-arabinose 5-phosphate with phosphoenolpyruvate to form Kdo 8-phosphate (4), followed by hydrolysis of Kdo 8-phosphate to Kdo and inorganic phosphate by the Kdo-8-phosphate phosphatase KdsC (5) and activation of Kdo to CMP-Kdo by the CMP-Kdo synthetase KdsB, before finally Kdo is transferred from CMP-Kdo to the lipid A moiety by the glycosyltransferase WaaA (6). In Escherichia coli, the Kdo-dependent late acyltransferases LpxL and LpxM subsequently transfer the fatty acids laurate and myristate, respectively, to Kdo₂-lipid IV_A to generate the characteristic acyloxyacyl units of hexaacylated Kdo₂-lipid A (7).It has long been recognized that Kdo transferases are unusual glycosyltransferases. WaaA is bifunctional in bacteria with LPS that contains an α-(2→4)-linked Kdo disaccharide in the inner core region, such as E. coli (6), Klebsiella pneumoniae (8), Legionella pneumophila (9), Acinetobacter baumannii, and Acinetobacter haemolyticus (10). In E. coli, CMP-Kdo is utilized for the transfer of Kdo to the tetraacylated lipid A precursor lipid IV_A, resulting in an α-(2→6)-linkage between the distal glucosamine (GlcN) of the lipid A backbone and the first Kdo residue and an α-(2→4)-linkage between a second Kdo residue and the first one. Thus, WaaA is capable of catalyzing the formation of two different glycosidic bonds, tolerating acceptor molecules with varying extents of acylation but strictly depending on the 4′-phosphate group of the tetraacyldisaccharide 1,4′-bisphosphate intermediate (6). In chlamydiae, however, which express an LPS composed of a Kdo trisaccharide with an unusual α-(2→8)-linkage between the second and a third Kdo residue (11), the Kdo transferases were shown to display at least trifunctional activity (12). The LPS of Chlamydophila psittaci consists of up to four Kdo residues of the structure α-Kdo-(2→4)-[α-Kdo-(2→8)]-α-Kdo-(2→4)-α-Kdo (13), and heterologous expression of the waaA gene in E. coli was found to be sufficient for synthesis of the complete chlamydial Kdo structure (12). Finally, the Kdo transferases of Haemophilus influenzae and Bordetella pertussis were shown to be monofunctional (14, 15), consistent with the presence of a single phosphorylated Kdo residue in their respective LPS (16, 17).On the basis of phylogenetic analyses of 16 S ribosomal RNA sequences, members of the family Aquificaceae with growth-temperature maxima near 95 °C are thought to represent the deepest branching species of the kingdom Bacteria (18). The cells are Gram-negative with a rather complex cell envelope of a surface protein layer, murein, and an OM (19). Previous studies provided the first direct evidence for the presence of smooth form LPS in Aquifex pyrophilus (20). Furthermore, KdsA and the UDP-(3-O-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacetylase (LpxC) of A. aeolicus, a close relative of A. pyrophilus, have been characterized in detail, and it was demonstrated that these enzymes catalyze the first committed steps in Kdo and lipid A formation, respectively (21–23). Moreover, a number of genes presumably encoding different steps of LPS biosynthesis have been identified on the A. aeolicus genome, including putative kdsB and waaA orthologues for Kdo activation and subsequent incorporation of the sugar into LPS (24). However, the number of Kdo residues transferred by WaaA of A. aeolicus remains unknown. We herein provide evidence that the A. aeolicus enzyme is a strictly monofunctional Kdo transferase through the characterization of its enzymatic activity and the chemical analysis of the native A. aeolicus LPS.     

Molecular basis of lipopolysaccharide heterogeneity in Escherichia coli: envelope stress-responsive regulators control the incorporation of glycoforms with a third 3-deoxy-α-D-manno-oct-2-ulosonic acid and rhamnose

Mass spectrometric analyses of lipopolysaccharide (LPS) from isogenic Escherichia coli strains with nonpolar mutations in the waa locus or overexpression of their cognate genes revealed that waaZ and waaS are the structural genes required for the incorporation of the third 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) linked to Kdo disaccharide and rhamnose, respectively. The incorporation of rhamnose requires prior sequential incorporation of the Kdo trisaccharide. The minimal in vivo lipid A-anchored core structure Kdo(2)Hep(2)Hex(2)P(1) in the LPS from ΔwaaO (lacking α-1,3-glucosyltransferase) could incorporate Kdo(3)Rha, without the overexpression of the waaZ and waaS genes. Examination of LPS heterogeneity revealed overlapping control by RpoE σ factor, two-component systems (BasS/R and PhoB/R), and ppGpp. Deletion of RpoE-specific anti-σ factor rseA led to near-exclusive incorporation of glycoforms with the third Kdo linked to Kdo disaccharide. This was accompanied by concomitant incorporation of rhamnose, linked to either the terminal third Kdo or to the second Kdo, depending upon the presence or absence of phosphoethanolamine on the second Kdo with truncation of the outer core. This truncation in ΔrseA was ascribed to decreased levels of WaaR glycosyltransferase, which was restored to wild-type levels, including overall LPS composition, upon the introduction of rybB sRNA deletion. Thus, ΔwaaR contained LPS primarily with Kdo(3) without any requirement for lipid A modifications. Accumulation of a glycoform with Kdo(3) and 4-amino-4-deoxy-l-arabinose in lipid A in ΔrseA required ppGpp, being abolished in a Δ(ppGpp(0) rseA). Furthermore, Δ(waaZ lpxLMP) synthesizing tetraacylated lipid A exhibited synthetic lethality at 21-23°C pointing to the significance of the incorporation of the third Kdo.