Alla Zamyatina

Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli

Lipopolysaccharide (LPS) is a major component of the outer membrane of gram-negative bacteria (28). It has a tripartite structural organization consisting of lipid A, a conserved core oligosaccharide region, and an O-specific polysaccharide chain or O antigen. In the majority of gram-negative bacteria, the core oligosaccharide can be subdivided into an outer core, generally composed of hexoses and hexosamines, and an inner core made of 3-deoxy-d-manno-oct-2-ulosonic acid and l,d-heptose units. LPS plays an important role in maintaining the structural integrity of the bacterial outer membrane by interacting with outer membrane proteins and divalent cations (15), thereby providing a barrier against the entry of toxic hydrophobic compounds into the bacterial cell (27). Escherichia coli mutants defective in the biosynthesis of 3-deoxy-d-manno-oct-2-ulosonic acid are nonviable, whereas those impaired in l,d-heptose synthesis survive in vitro, although they display a pleiotropic phenotype referred to as “deep rough” (17). This phenotype is characterized by an extreme sensitivity to very low concentrations of novobiocin, detergents, and bile salts (32). Deep rough mutants also have defects in F plasmid conjugation and generalized transduction by the bacteriophage P1 (6, 16). Haemophilus influenzae heptose-deficient mutants were found to be serum sensitive and displayed a reduced virulence in vivo (18, 36). The complete biosynthesis pathway of the l,d-heptose precursor has not been elucidated. Eidels and Osborn (11) proposed a four-step pathway for the synthesis of NDP-l,d-heptose, which is still widely accepted in the literature (see reference 13 for a review). It includes (i) conversion of d-sedoheptulose 7-phosphate to d,d-heptose 7-phosphate by a phosphoheptose isomerase; (ii) formation of d,d-heptose 1-phosphate by a phosphoheptose mutase; (iii) activation of the d,d-heptose 1-phosphate intermediate to NDP-d,d-heptose by an NDP-heptose synthetase; and (iv) epimerization of the NDP-heptose to form the final product, NDP-l,d-heptose. Subsequent studies involving the isolation of ADP-d,d-heptose and ADP-l,d-heptose from Shigella sonnei and Salmonella enterica serovar Typhimurium indicated that ADP is the activating nucleotide (20–22). In the absence of purified ADP-heptose, Kadrmas and Raetz (19) used ADP-mannose as a substrate for the E. coli heptosyltransferase I (WaaC). More recently, it has been clearly demonstrated that heptosyltransferases I and II (WaaF) from E. coli accept ADP-l-β-d-heptose and ADP-d-β-d-heptose as substrates, although the efficiency of the transfer reactions with the d-β-d isomer is markedly reduced (14, 35). In gram-negative bacteria, functional studies have only been performed for the isomerization reaction and the epimerization step (3, 9, 26), while the conversion of d,d-heptose 7-phosphate to d,d-heptose 1-phosphate and a functional proof of the activating step have not been demonstrated. The d-sedoheptulose 7-phosphate isomerase activity was described in S. enterica serovar Typhimurium (12), and the corresponding gene, gmhA, has been cloned both from E. coli and from H. influenzae (3, 4). The amino acid sequence of the GmhA polypeptide is highly conserved in different gram-negative bacteria (33). The epimerization step is catalyzed by the WaaD (formerly RfaD) protein (5), which has also been crystallized (8). We have recently shown that the E. coli rfaE gene product consists of two distinct domains that may be involved in the biosynthesis of d,d-heptose 1-phosphate, as well as the activating step (34). It was demonstrated that one of the RfaE domains shares structural features with members of the ribokinase family, while the other domain has conserved features present in nucleotidyltransferases (34). The demonstration of a protein domain corresponding to a putative sugar kinase suggested that the original pathway for NDP-heptose biosynthesis as proposed by Eidels and Osborn may not be accurate and, at the same time, predicted the existence of an additional phosphatase step (33). The complete biosynthesis pathway of GDP-d-α-d-heptose from d-sedoheptulose 7-phosphate in the gram-positive bacterium Aneurinibacillus thermoaerophilus DSM 10155 was recently characterized (20). We demonstrated that two independent enzymes catalyze the originally proposed mutase step. A d,d-heptose 7-phosphate kinase adds a phosphate group at the C-1 position, and subsequently a d,d-heptose 1,7-bisphosphate phosphatase removes the phosphate group at the C-7 position. The GDP-activated d,d-isomer serves as a precursor for the incorporation of the heptose into the glycan moiety of a surface layer (S-layer) glycoprotein produced by A. thermoaerophilus (20). Amino acid sequence analysis of completely sequenced genomes revealed that the A. thermoaerophilus phosphatase is highly conserved among different gram-negative bacteria (20), in agreement with a previous suggestion that a phosphatase reaction is also required for the synthesis of ADP-d,d-heptose and ADP-l,d-heptose in these microorganisms (34). In the present study, we report the reconstruction in vitro with purified enzyme components of the complete biosynthesis pathway for ADP-d-β-d-heptose in E. coli. We also provide genetic evidence demonstrating that the function of a novel phosphatase gene in E. coli K-12, now designated gmhB (formerly yaeD), is required for the synthesis of ADP-d-β-d-heptose. Furthermore, we propose a new gene nomenclature to account for the differences and similarities between the components of the pathways leading to the formation of ADP-l-β-d-heptose and GDP-d-α-d-heptose in gram-negative and gram-positive bacteria, respectively.

Chemistry of Lipid A: At the Heart of Innate Immunity

In many Gram-negative bacteria, lipopolysaccharide (LPS) and its lipid A moiety are pivotal for bacterial survival. Depending on its structure, lipid A carries the toxic properties of the LPS and acts as a potent elicitor of the host innate immune system via the Toll-like receptor 4/myeloid differentiation factor 2 (TLR4/MD-2) receptor complex. It often causes a wide variety of biological effects ranging from a remarkable enhancement of the resistance to the infection to an uncontrolled and massive immune response resulting in sepsis and septic shock. Since the bioactivity of lipid A is strongly influenced by its primary structure, a broad range of chemical syntheses of lipid A derivatives have made an enormous contribution to the characterization of lipid A bioactivity, providing novel pharmacological targets for the development of new biomedical therapies. Here, we describe and discuss the chemical aspects regarding lipid A and its role in innate immunity, from the (bio)synthesis, isolation and characterization to the molecular recognition at the atomic level.

Efficient chemical synthesis of both anomers of ADP L-glycero- and D-glycero-D-manno-heptopyranose.

A series of anomeric phosphates and ADP-activated L-glycero- and D-glycero-D-manno-heptopyranoses has been prepared in high overall yields, which provided model compounds and substrates in the elucidation of biosynthetic pathways and glycosyl transfer reactions of nucleotide-activated bacterial heptoses. The alpha-anomers of the heptosyl phosphates were obtained in high yield and selectivity using the phosphoramidite procedure, whereas the beta-phosphates were formed preferentially employing acylation of reducing heptoses with diphenyl phosphorochloridate. An efficient route to the formation of the nucleotide diphosphate sugars was elaborated by coupling of the O-acetylated phosphates with AMP-morpholidate followed by alkaline deprotection to furnish ADP-L- and D-glycero-alpha-D-manno-heptose in 84 and 89% yield, respectively. Deacetylation of the O-acetylated beta-configured ADP heptoses was conducted at strictly controlled conditions (-28 degrees C at pH 10.5) to suppress formation of cyclic heptose-1,2-phosphodiesters with concomitant release of AMP. Isolation of the unstable beta-configured ADP-heptoses by anion-exchange chromatography and gel-filtration afforded ADP L- and D-glycero-beta-D-manno-heptose in high yields.

Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II

L-Glycero-D- manno-heptopyranose is a characteristic compound of many lipopolysaccharide (LPS) core structures of Gram-negative bacteria. In Escherichia coli two heptosyltransferases, namely WaaC and WaaF, are known to transfer L- glycero-D-manno-heptopyranose to Re-LPS and Rd 2-LPS, respectively. It had been proposed that both reactions involve ADPL- glycero-D-manno-heptose as a sugar donor; however, the structure of this nucleotide sugar had never been completely elucidated. In the present study, ADPL-glycero-D-manno-heptose was isolated from a heptosyltransferase-deficient E. coli mutant, and its structure was determined by nuclear magnetic resonance spectroscopy and matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry as ADPL-glycero-β-D-manno-heptopyranose. This compound represented the sole constituent of the bacterial extract that was accepted as a sugar donor by heptosyltransferases I and II in vitro .

Conformationally Constrained Lipid A Mimetics for Exploration of Structural Basis of TLR4/MD-2 Activation by Lipopolysaccharide

Recognition of the lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, by the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD-2) complex is essential for the control of bacterial infection. A pro-inflammatory signaling cascade is initiated upon binding of membrane-associated portion of LPS, a glycophospholipid Lipid A, by a coreceptor protein MD-2, which results in a protective host innate immune response. However, activation of TLR4 signaling by LPS may lead to the dysregulated immune response resulting in a variety of inflammatory conditions including sepsis syndrome. Understanding of structural requirements for Lipid A endotoxicity would ensure the development of effective anti-inflammatory medications. Herein, we report on design, synthesis, and biological activities of a series of conformationally confined Lipid A mimetics based on β,α-trehalose-type scaffold. Replacement of the flexible three-bond β(1→6) linkage in diglucosamine backbone of Lipid A by a two-bond β,α(1↔1) glycosidic linkage afforded novel potent TLR4 antagonists. Synthetic tetraacylated bisphosphorylated Lipid A mimetics based on a β–GlcN(1↔1)α–GlcN scaffold selectively block the LPS binding site on both human and murine MD-2 and completely abolish lipopolysaccharide-induced pro-inflammatory signaling, thereby serving as antisepsis drug candidates. In contrast to their natural counterpart lipid IVa, conformationally constrained Lipid A mimetics do not activate mouse TLR4. The structural basis for high antagonistic activity of novel Lipid A mimetics was confirmed by molecular dynamics simulation. Our findings suggest that besides the chemical structure, also the three-dimensional arrangement of the diglucosamine backbone of MD-2-bound Lipid A determines endotoxic effects on TLR4.

Investigation on the agonistic and antagonistic biological activities of synthetic Chlamydia lipid A and its use in in vitro enzymatic assays

The synthetic 1,4′-bisphosphorylated penta-acyl and tetra-acyl lipid A structures representing the major molecular species of natural chlamydial lipid A were tested for their endotoxic activities as measured by interleukin-8 release from human embryonic kidney (HEK) 293 cells expressing Toll-like receptor (TLR) 2 or TLR4. Both compounds were unable to activate HEK293 cells transiently transfected with TLR2. The penta-acyl lipid A was a weak activator of HEK293 cells expressing TLR4/MD-2/CD14 whereas tetra-acyl lipid A was inactive even at high concentrations. The weak activity of the penta-acyl lipid A could be antagonized by the tetra-acyl derivative of Escherichia coli lipid A (compound 406) or the anti-CD14 monoclonal antibody MEM-18. Both, tetra- and pentaacyl lipid A were unable to antagonize the activity of synthetic E. coli-type lipid A (compound 506) or smooth lipopolysaccharide of Salmonella enterica serovar Friedenau. Tetra- and penta-acyl lipid A served as acceptors for Kdo transferases from E. coli, Chlamydia trachomatis and Chlamydophila psittaci as shown by in vitro assays and detection of the products by thin layer chromatography and immune staining with monoclonal antibody.

A structural mechanism for bacterial autotransporter glycosylation by a dodecameric heptosyltransferase family

A large group of bacterial virulence autotransporters including AIDA-I from diffusely adhering E. coli (DAEC) and TibA from enterotoxigenic E. coli (ETEC) require hyperglycosylation for functioning. Here we demonstrate that TibC from ETEC harbors a heptosyltransferase activity on TibA and AIDA-I, defining a large family of bacterial autotransporter heptosyltransferases (BAHTs). The crystal structure of TibC reveals a characteristic ring-shape dodecamer. The protomer features an N-terminal β-barrel, a catalytic domain, a β-hairpin thumb, and a unique iron-finger motif. The iron-finger motif contributes to back-to-back dimerization; six dimers form the ring through β-hairpin thumb-mediated hand-in-hand contact. The structure of ADP-D-glycero-β-D-manno-heptose (ADP-D,D-heptose)-bound TibC reveals a sugar transfer mechanism and also the ligand stereoselectivity determinant. Electron-cryomicroscopy analyses uncover a TibC–TibA dodecamer/hexamer assembly with two enzyme molecules binding to one TibA substrate. The complex structure also highlights a high efficient hyperglycosylation of six autotransporter substrates simultaneously by the dodecamer enzyme complex. DOI: http://dx.doi.org/10.7554/eLife.03714.001

Hemoglobin Enhances the Biological Activity of Synthetic and Natural Bacterial (Endotoxic) Virulence Factors: A General Principle

Although hemoglobin (Hb) is mainly present in the cytoplasm of erythrocytes (red blood cells), lower concentrations of pure, cell-free Hb are released permanently into the circulation due to an inherent intravascular hemolytic disruption of erythrocytes. Previously it was shown that the interaction of Hb with bacterial endotoxins (lipopolysaccharides, LPS) results in a significant increase of the biological activity of LPS. There is clear evidence that the enhancement of the biological activity of LPS by Hb is connected with a disaggregation of LPS. From these findings one questions whether the property to enhance the biological activity of endotoxin, in most cases proven by the ability to increase the cytokine (tumor-necrosis-factor-alpha, interleukins) production in human mononuclear cells, is restricted to bacterial endotoxin or is a more general principle in nature. To elucidate this question, we investigated the interaction of various synthetic and natural virulence (pathogenicity) factors with hemoglobin of human or sheep origin. In addition to enterobacterial R-type LPS a synthetic bacterial lipopeptide and synthetic phospholipid-like structures mimicking the lipid A portion of LPS were analysed. Furthermore, we also tested endotoxically inactive LPS and lipid A compounds such as those from Chlamydia trachomatis. We found that the observations made for endotoxically active form of LPS can be generalized for the other synthetic and natural virulence factors: In every case, the cytokine-production induced by them is increased by the addition of Hb. This biological property of Hb is connected with its physical property to convert the aggregate structures of the virulence factors into one with cubic symmetry, accompanied with a considerable reduction of the size and number of the original aggregates.

Crystal and molecular structure of methyl l-glycero-α-d-manno-heptopyranoside, and synthesis of 1→7 linked l-glycero-d-manno-heptobiose and its methyl α-glycoside

Graphical abstract

Development of αGlcN(1↔1)αMan-based lipid A mimetics as a novel class of potent Toll-like receptor 4 agonists.

The endotoxic portion of lipopolysaccharide (LPS), a glycophospholipid Lipid A, initiates the activation of the Toll-like Receptor 4 (TLR4)–myeloid differentiation factor 2 (MD-2) complex, which results in pro-inflammatory immune signaling. To unveil the structural requirements for TLR4·MD-2-specific ligands, we have developed conformationally restricted Lipid A mimetics wherein the flexible βGlcN(1→6)GlcN backbone of Lipid A is exchanged for a rigid trehalose-like αGlcN(1↔1)αMan scaffold resembling the molecular shape of TLR4·MD-2-bound E. coli Lipid A disclosed in the X-ray structure. A convergent synthetic route toward orthogonally protected αGlcN(1↔1)αMan disaccharide has been elaborated. The α,α-(1↔1) linkage was attained by the glycosylation of 2-N-carbamate-protected α-GlcN-lactol with N-phenyl-trifluoroacetimidate of 2-O-methylated mannose. Regioselective acylation with (R)-3-acyloxyacyl fatty acids and successive phosphorylation followed by global deprotection afforded bis- and monophosphorylated hexaacylated Lipid A mimetics. αGlcN(1↔1)αMan-based Lipid A mimetics (α,α-GM-LAM) induced potent activation of NF-κB signaling in hTLR4/hMD-2/CD14-transfected HEK293 cells and robust LPS-like cytokines expression in macrophages and dendritic cells. Thus, restricting the conformational flexibility of Lipid A by fixing the molecular shape of its carbohydrate backbone in the “agonistic” conformation attained by a rigid αGlcN(1↔1)αMan scaffold represents an efficient approach toward powerful and adjustable TLR4 activation.