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Paul Kosma - One of the best experts on this subject based on the ideXlab platform.
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ADP heptose, a novel pathogen‐associated molecular pattern identified in Helicobacter pylori
The FASEB Journal, 2019Co-Authors: Lennart Pfannkuch, Robert Hurwitz, Jan Traulsen, Janine Sigulla, Marcella Poeschke, Laura Matzner, Paul Kosma, Monika Schmid, Thomas F. MeyerAbstract:The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-β-D-manno-heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the β-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.
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adp heptose a novel pathogen associated molecular pattern identified in helicobacter pylori
The FASEB Journal, 2019Co-Authors: Lennart Pfannkuch, Robert Hurwitz, Jan Traulsen, Janine Sigulla, Marcella Poeschke, Laura Matzner, Paul Kosma, Monika Schmid, Thomas F. MeyerAbstract:The gastric pathogen Helicobacter pylori activates the NF-κB pathway in human epithelial cells via the recently discovered α-kinase 1 TRAF-interacting protein with forkhead-associated domain (TIFA) axis. We and others showed that this pathway can be triggered by heptose 1,7-bisphosphate (HBP), an LPS intermediate produced in gram-negative bacteria that represents a new pathogen-associated molecular pattern (PAMP). Here, we report that our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts, failing to explain NF-κB activation. Instead, we identified ADP-glycero-β-D-manno-heptose (ADP heptose), a derivative of HBP, as the predominant PAMP in lysates of H. pylori and other gram-negative bacteria. ADP heptose exhibits significantly higher activity than HBP, and cells specifically sensed the presence of the β-form, even when the compound was added extracellularly. The data lead us to conclude that ADP heptose not only constitutes the key PAMP responsible for H. pylori-induced NF-κB activation in epithelial cells, but it acts as a general gram-negative bacterial PAMP.-Pfannkuch, L., Hurwitz, R., Traulsen, J., Sigulla, J., Poeschke, M., Matzner, L., Kosma, P., Schmid, M., Meyer, T. F. ADP heptose, a novel pathogen-associated molecular pattern identified in Helicobacter pylori.
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adp heptose a novel pathogen associated molecular pattern associated with helicobacter pylori type 4 secretion
bioRxiv, 2018Co-Authors: Lennart Pfannkuch, Robert Hurwitz, Jan Traulsen, Paul Kosma, Monika Schmid, Thomas F. MeyerAbstract:The gastric pathogen Helicobacter pylori activates the NF-kappaB pathway in human epithelial cells via the alpha-kinase 1 (Alpk1) TIFA axis. We and others have previously shown that heptose 1,7-bisphosphate (HBP) acts as a pathogen-associated molecular pattern (PAMP). HBP is an intermediate of lipopolysaccharide (LPS) synthesis in H. pylori and other gram-negative bacteria. Deletion of the hldE (rfaE) gene encoding the enzyme responsible for HBP synthesis, as well as deletion of further upstream genes, causes loss of NF-κB stimulation by H. pylori, while deletion of the downstream phosphatase encoding gene gmhB does not. This has led to the conclusion that HBP is the PAMP responsible for NF-κB induction. Here, our attempts to identify HBP in lysates of H. pylori revealed surprisingly low amounts that fail to explain NF-κB activation. Instead, we identified ADP heptose, a major downstream metabolite of HdlE, as the predominant PAMP in H. pylori lysates, exhibiting around 100-fold stronger activity compared to HBP. It therefore appears that synthesis of ADP heptose from HBP in H. pylori occurs independently of GmhB. The data lead us to conclude that ADP heptose constitutes the key PAMP, secreted via the pathogens cagPAI encoded type 4 secretion (T4SS).
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Chemical synthesis of the innate immune modulator – bacterial d-glycero-β-d-manno-heptose-1,7-bisphosphate (HBP)
Tetrahedron Letters, 2017Co-Authors: Alessio Borio, Andreas Hofinger, Paul Kosma, Alla ZamyatinaAbstract:Abstract The bacterial metabolite and potent innate immune modulator d - glycero -β- d - manno -heptose-1,7-bisphosphate (HBP) and its α-configured counterpart d - glycero -α- d - manno -heptose-1,7-bisphosphate were synthesized via stereoselective anomeric phosphorylation of the peracetylated d , d -heptose 7-dibenzylphosphate by exploiting different nucleophilicity of equatorial and axial lactols in the d - manno -series. We also report a novel approach for anomeric phosphorylation using modified Mitsunobu reaction conditions and provide the first full structural characterization of HBP. The first chemical synthesis of HBP offers access to an anomerically pure structurally defined probe for biological studies and to a lead compound operating as a powerful stimulator of intracellular signaling for possible therapeutic immunomodulation.
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Synthesis of a deoxy analogue of ADP L-glycero-D-manno-heptose
Carbohydrate research, 2007Co-Authors: Edit Balla, Alla Zamyatina, Andreas Hofinger, Paul KosmaAbstract:Abstract Starting from l -lyxose, indium-mediated chain elongation with allyl bromide followed by acetylation and oxidative cleavage of the double bond and deprotection afforded 2-deoxy- l - galacto -heptose as a 2-deoxy analogue of the bacterial carbohydrate l - glycero - d - manno -heptose in good overall yield. For the synthesis of the ADP-activated derivative, the 2-deoxy-heptose was O-acetylated and transformed into the anomeric bromide derivative, which was then converted into the acetylated heptopyranosyl phosphate by reaction with tetrabutylammonium phosphate. Deprotection and separation of the anomeric phosphates furnished 2-deoxy-β- l - galacto -heptopyranosyl phosphate. Coupling of the acetylated heptosyl phosphate with AMP morpholidate afforded the acetylated ADP derivative in good yield. Removal of the acetyl groups gave the target compound ADP 2-deoxy- l - galacto -heptopyranose, which may serve as substrate analogue of bacterial ADP heptosyl transferases for biochemical and crystallographic studies.
Alla Zamyatina - One of the best experts on this subject based on the ideXlab platform.
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ADP‐heptose is a newly identified pathogen‐associated molecular pattern of Shigella flexneri
EMBO Reports, 2018Co-Authors: Diego García-weber, Anne-sophie Dangeard, Johan Cornil, Linda Thai, Héloïse Rytter, Alla Zamyatina, Laurence Mulard, Cécile ArrieumerlouAbstract:During an infection, the detection of pathogens is mediated through the interactions between pathogen-associated molecular patterns (PAMPs) and pathogen recognition receptors. β-Heptose 1,7-bisphosphate (βHBP), an intermediate of the lipopolysaccharide (LPS) biosynthesis pathway, was recently identified as a bacterial PAMP. It was reported that βHBP sensing leads to oligomerization of TIFA proteins, a mechanism controlling NF-κB activation and pro-inflammatory gene expression. Here, we compare the ability of chemically synthesized βHBP and Shigella flexneri lysate to induce TIFA oligomerization in epithelial cells. We find that, unlike bacterial lysate, βHBP fails to initiate rapid TIFA oligomerization. It only induces delayed signaling, suggesting that βHBP must be processed intracellularly to trigger inflammation. Gene deletion and complementation analysis of the LPS biosynthesis pathway revealed that ADP-heptose is the bacterial metabolite responsible for rapid TIFA oligomerization. ADP-heptose sensing occurs down to 10-10 M. During S. flexneri infection, it results in cytokine production, a process dependent on the kinase ALPK1. Altogether, our results rule out a major role of βHBP in S. flexneri infection and identify ADP-heptose as a new bacterial PAMP.
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Chemical synthesis of the innate immune modulator – bacterial d-glycero-β-d-manno-heptose-1,7-bisphosphate (HBP)
Tetrahedron Letters, 2017Co-Authors: Alessio Borio, Andreas Hofinger, Paul Kosma, Alla ZamyatinaAbstract:Abstract The bacterial metabolite and potent innate immune modulator d - glycero -β- d - manno -heptose-1,7-bisphosphate (HBP) and its α-configured counterpart d - glycero -α- d - manno -heptose-1,7-bisphosphate were synthesized via stereoselective anomeric phosphorylation of the peracetylated d , d -heptose 7-dibenzylphosphate by exploiting different nucleophilicity of equatorial and axial lactols in the d - manno -series. We also report a novel approach for anomeric phosphorylation using modified Mitsunobu reaction conditions and provide the first full structural characterization of HBP. The first chemical synthesis of HBP offers access to an anomerically pure structurally defined probe for biological studies and to a lead compound operating as a powerful stimulator of intracellular signaling for possible therapeutic immunomodulation.
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Synthesis of a deoxy analogue of ADP L-glycero-D-manno-heptose
Carbohydrate research, 2007Co-Authors: Edit Balla, Alla Zamyatina, Andreas Hofinger, Paul KosmaAbstract:Abstract Starting from l -lyxose, indium-mediated chain elongation with allyl bromide followed by acetylation and oxidative cleavage of the double bond and deprotection afforded 2-deoxy- l - galacto -heptose as a 2-deoxy analogue of the bacterial carbohydrate l - glycero - d - manno -heptose in good overall yield. For the synthesis of the ADP-activated derivative, the 2-deoxy-heptose was O-acetylated and transformed into the anomeric bromide derivative, which was then converted into the acetylated heptopyranosyl phosphate by reaction with tetrabutylammonium phosphate. Deprotection and separation of the anomeric phosphates furnished 2-deoxy-β- l - galacto -heptopyranosyl phosphate. Coupling of the acetylated heptosyl phosphate with AMP morpholidate afforded the acetylated ADP derivative in good yield. Removal of the acetyl groups gave the target compound ADP 2-deoxy- l - galacto -heptopyranose, which may serve as substrate analogue of bacterial ADP heptosyl transferases for biochemical and crystallographic studies.
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Synthesis of C-glycosidically linked ADP glycero-β-d-manno-heptose analogues
Tetrahedron: Asymmetry, 2007Co-Authors: Andrea Graziani, Alla Zamyatina, Hassan Amer, Andreas Hofinger, Paul KosmaAbstract:Abstract C-Glycosides of l - glycero - d - manno - and d - glycero - d - manno -heptose containing either ( S )- or ( R )-2-hydroxypropyl aglycons are easily accessible compounds via condensation of reducing Heptoses with pentane-2,4-dione. 2′,3′-Di- O -acetyl adenosine was transformed into the corresponding 5′- O -cyanoethyl N , N -diisopropylaminophosphoramidite derivative, which was coupled in fair yields to the O-acetylated diastereoisomeric C-glycosidic alcohols. Oxidation of the phosphite triesters followed by deprotection furnished four ADP-heptose analogues, wherein the heptosyl phosphate moiety had been replaced by a three carbon-skeleton. The compounds serving as substrate analogues will be used for co-crystallization experiments with ADP heptosyl transferases.
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Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli
Journal of bacteriology, 2002Co-Authors: Bernd Kneidinger, Alla Zamyatina, Paul Kosma, Cristina L. Marolda, Michael Graninger, Fiona Mcarthur, Miguel A. Valvano, Paul MessnerAbstract: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.
Miguel A. Valvano - One of the best experts on this subject based on the ideXlab platform.
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Role of capsular modified heptose in the virulence of Campylobacter jejuni
Molecular microbiology, 2015Co-Authors: Anthony Wong, Miguel A. Valvano, Dirk Lange, Sébastien Houle, Nikolay P. Arbatsky, Yuriy A. Knirel, Charles M. Dozois, Carole CreuzenetAbstract:The Campylobacter jejuni capsular polysaccharide is important for virulence and often contains a modified heptose. In strain ATCC 700819 (a.k.a. NCTC 11168), the modified heptose branches off from the capsular backbone and is directly exposed to the environment. We reported previously that the enzymes encoded by wcaG, mlghB and mlghC are involved in heptose modification. Here, we show that inactivation of any of these genes leads to production of capsule lacking modified heptose and alters the transcription of other capsule modification genes differentially. Inactivation of mlghB or mlghC, but not of wcaG, decreased susceptibility to bile salts and abrogated invasion of intestinal cells. All mutants showed increased sensitivity to serum killing, especially wcaG::cat, and had defects in colonization and persistence in chicken intestine, but did not show significant differences in adhesion, phagocytosis and intracellular survival in murine macrophages. Together, our findings suggest that the capsular heptose modification pathway contributes to bacterial resistance against gastrointestinal host defenses and supports bacterial persistence via its role in serum resistance and invasion of intestinal cells. Our data further suggest a dynamic regulation of expression of this pathway in the gastrointestinal tract.
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Structural and Kinetic Characterization of the LPS Biosynthetic Enzyme D-alpha,beta-D-heptose-1,7-bisphosphate Phosphatase (GmhB) from Escherichia coli
Biochemistry, 2010Co-Authors: Patricia L. Taylor, Miguel A. Valvano, Seiji Sugiman-marangos, Kun Zhang, Gerard D. Wright, Murray S. JunopAbstract:Lipopolysaccharide is a major component of the outer membrane of Gram-negative bacteria and provides a permeability barrier to many commonly used antibiotics. ADP-heptose residues are an integral part of the LPS inner core, and mutants deficient in heptose biosynthesis demonstrate increased membrane permeability. The heptose biosynthesis pathway involves phosphorylation and dephosphorylation steps not found in other pathways for the synthesis of nucleotide sugar precursors. Consequently, the heptose biosynthetic pathway has been marked as a novel target for antibiotic adjuvants, which are compounds that facilitate and potentiate antibiotic activity. D-{alpha},{beta}-D-Heptose-1,7-bisphosphate phosphatase (GmhB) catalyzes the third essential step of LPS heptose biosynthesis. This study describes the first crystal structure of GmhB and enzymatic analysis of the protein. Structure-guided mutations followed by steady state kinetic analysis, together with established precedent for HAD phosphatases, suggest that GmhB functions through a phosphoaspartate intermediate. This study provides insight into the structure-function relationship of GmhB, a new target for combatting Gram-negative bacterial infection.
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Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli
Journal of bacteriology, 2002Co-Authors: Bernd Kneidinger, Alla Zamyatina, Paul Kosma, Cristina L. Marolda, Michael Graninger, Fiona Mcarthur, Miguel A. Valvano, Paul MessnerAbstract: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.
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Biosynthesis and genetics of ADP-heptose
Journal of Endotoxin Research, 1999Co-Authors: Miguel A. ValvanoAbstract:Glycero-manno-heptose is a common component in the lipopolysaccharide (LPS) of many Gramnegative bacteria. Mutants deficient in the synthesis of glycero-manno-heptose are highly sensitive to hydrophobic compounds, and display reduced virulence, making these genes and their products potential targets for developing novel antimicrobials. To date, the biosynthesis of the heptosyl precursors for the inner core oligosaccharide of the LPS molecule is not completely characterized. In this work, the genes and enzyme functions involved in the various steps of the biosynthesis of ADP- Lglycero-D-manno-heptose are discussed, especially those involved in the intermediate steps.
Paul Messner - One of the best experts on this subject based on the ideXlab platform.
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Biosynthesis Pathway of ADP-l-glycero-β-d-manno-Heptose in Escherichia coli
Journal of bacteriology, 2002Co-Authors: Bernd Kneidinger, Alla Zamyatina, Paul Kosma, Cristina L. Marolda, Michael Graninger, Fiona Mcarthur, Miguel A. Valvano, Paul MessnerAbstract: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.
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Biosynthesis of nucleotide-activated D-glycero-D-manno-heptose.
The Journal of biological chemistry, 2001Co-Authors: Bernd Kneidinger, Paul Kosma, Michael Graninger, Michael Puchberger, Paul MessnerAbstract:The glycan chain repeats of the S-layer glycoprotein of Aneurinibacillus thermoaerophilus DSM 10155 contain d-glycero-d-manno-heptose, which has also been described as constituent of lipopolysaccharide cores of Gram-negative bacteria. The four genes required for biosynthesis of the nucleotide-activated form GDP-d-glycero-d-manno-heptose were cloned, sequenced, and overexpressed in Escherichia coli, and the corresponding enzymes GmhA, GmhB, GmhC, and GmhD were purified to homogeneity. The isomerase GmhA catalyzed the conversion of d-sedoheptulose 7-phosphate to d-glycero-d-manno-heptose 7-phosphate, and the phosphokinase GmhB added a phosphate group to form d-glycero-d-manno-heptose 1,7-bisphosphate. The phosphatase GmhC removed the phosphate in the C-7 position, and the intermediate d-glycero-alpha-d-manno-heptose 1-phosphate was eventually activated with GTP by the pyrophosphorylase GmhD to yield the final product GDP-d-glycero-alpha-d-manno-heptose. The intermediate and end products were analyzed by high performance liquid chromatography. Nuclear magnetic resonance spectroscopy was used to confirm the structure of these substances. This is the first report of the biosynthesis of GDP-d-glycero-alpha-d-manno-heptose in Gram-positive organisms. In addition, we propose a pathway for biosynthesis of the nucleotide-activated form of l-glycero-d-manno-heptose.
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Structural analysis of the lipooligosaccharide from the commensal Haemophilus somnus genome strain 129Pt.
Carbohydrate research, 2004Co-Authors: Frank St. Michael, Michael D Howard, A Jane Duncan, Jianjun Li, Thomas J. InzanaAbstract:The structure for the carbohydrate moiety of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 129Pt was elucidated. The structure of the core oligosaccharide and O-deacylated LOS was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the major fully extended carbohydrate glycoform of the LOS was determined on the basis of the combined data from these experiments. [Carbohydrate structure: see text]. In the structure Kdo is 3-deoxy-D-manno-octulosonic acid, Hep is L-glycero-D-manno-heptose and PEtn is phosphoethanolamine. Minor amounts of glycoforms containing nonstoichiometric substituents glycine and phosphate at the distal heptose residue were also identified.
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structural analysis of the lipooligosaccharide from the commensal haemophilus somnus genome strain 129pt
Carbohydrate Research, 2003Co-Authors: Michael D Howard, Thomas J. InzanaAbstract:Abstract The structure for the carbohydrate moiety of the lipooligosaccharide (LOS) from the commensal Haemophilus somnus strain 129Pt was elucidated. The structure of the core oligosaccharide and O-deacylated LOS was established by monosaccharide and methylation analyses, NMR spectroscopy and mass spectrometry. The following structure for the major fully extended carbohydrate glycoform of the LOS was determined on the basis of the combined data from these experiments. In the structure Kdo is 3-deoxy- d - manno -octulosonic acid, Hep is l - glycero - d - manno -heptose and PEtn is phosphoethanolamine. Minor amounts of glycoforms containing nonstoichiometric substituents glycine and phosphate at the distal heptose residue were also identified.