RESUMEN

Phosphoethanolamine (pEtN) decoration of E. coli Lipopolysaccharide (LPS) provides resistance to the antimicrobial Polymyxin B (PolB). While EptA and EptB enzymes catalyze the addition of pEtN to the Lipid A and Kdo (pEtN-Kdo-Lipid A), EptC catalyzes the pEtN addition to the Heptose I (pEtN-HeptI). In this study, we investigated the contribution of pEtN-HeptI to PolB resistance using eptA/eptB and eptC deficient E. coli K12 and its wild-type parent strains. These mutations were shown to decrease the antimicrobial activity of PolB on cells grown under pEtN-addition inducing conditions. Furthermore, the 1-N-phenylnapthylamine uptake assay revealed that in vivo PolB has a reduced OM-permeabilizing activity on the ΔeptA/eptB strain compared with the ΔeptC strain. In vitro, the changes in size and zeta potential of LPS-vesicles indicate that pEtN-HeptI reduce the PolB binding, but in a minor extent than pEtN-Kdo-Lipid A. Molecular dynamics analysis revealed the structural basis of the PolB resistance promoted by pEtN-HeptI, which generate a new hydrogen-bonding networks and a denser inner core region. Altogether, the experimental and theoretical assays shown herein indicate that pEtN-HeptI addition promote an LPS conformational rearrangement, that could act as a shield by hindering the accession of PolB to inner LPS-targets moieties.

Asunto(s)

Membrana Celular/metabolismo , Escherichia coli/metabolismo , Etanolaminas/metabolismo , Heptosas/metabolismo , Lípido A/metabolismo , Polimixina B/química , Membrana Celular/química , Membrana Celular/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Etanolaminas/química , Eliminación de Gen , Heptosas/química , Heptosas/genética , Lípido A/química , Lípido A/genética , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo

RESUMEN

The deletion of PHO13 (pho13Δ) in Saccharomyces cerevisiae, encoding a phosphatase enzyme of unknown specificity, results in the transcriptional activation of genes related to the pentose phosphate pathway (PPP) such as TAL1 encoding transaldolase. It has been also reported that the pho13Δ mutant of S. cerevisiae expressing a heterologous xylose pathway can metabolize xylose efficiently compared to its parental strain. However, the interaction between the pho13Δ-induced transcriptional changes and the phenotypes of xylose fermentation was not understood. Thus we investigated the global metabolic changes in response to pho13Δ when cells were exponentially growing on xylose. Among the 134 intracellular metabolites that we identified, the 98% reduction of sedoheptulose was found to be the most significant change in the pho13Δ mutant as compared to its parental strain. Because sedoheptulose-7-phosphate (S7P), a substrate of transaldolase, reduced significantly in the pho13Δ mutant as well, we hypothesized that limited transaldolase activity in the parental strain might cause dephosphorylation of S7P, leading to carbon loss and inefficient xylose metabolism. Mutants overexpressing TAL1 at different degrees were constructed, and their TAL1 expression levels and xylose consumption rates were positively correlated. Moreover, as TAL1 expression levels increased, intracellular sedoheptulose concentration dropped significantly. Therefore, we concluded that TAL1 upregulation, preventing the accumulation of sedoheptulose, is the most critical mechanism for the improved xylose metabolism by the pho13Δ mutant of engineered S. cerevisiae.

Asunto(s)

Heptosas/metabolismo , Ingeniería Metabólica/métodos , Monoéster Fosfórico Hidrolasas/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/fisiología , Activación Transcripcional/fisiología , Xilosa/metabolismo , Activación Enzimática , Silenciador del Gen , Mejoramiento Genético/métodos , Heptosas/genética

RESUMEN

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.

Asunto(s)

Cápsulas Bacterianas/metabolismo , Campylobacter jejuni/patogenicidad , Heptosas/metabolismo , Polisacáridos Bacterianos/metabolismo , Animales , Cápsulas Bacterianas/genética , Ácidos y Sales Biliares/metabolismo , Células CACO-2 , Infecciones por Campylobacter/microbiología , Campylobacter jejuni/enzimología , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/metabolismo , Secuencia de Carbohidratos , Pollos , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Tracto Gastrointestinal/microbiología , Técnicas de Inactivación de Genes , Heptosas/genética , Humanos , Cetona Oxidorreductasas/genética , Cetona Oxidorreductasas/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Ratones , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Células RAW 264.7 , Virulencia

RESUMEN

Clinical and epidemiological synergy exists between the globally important sexually transmitted infections, gonorrhea and HIV. Neisseria gonorrhoeae, which causes gonorrhea, is particularly adept at driving HIV-1 expression, but the molecular determinants of this relationship remain undefined. N. gonorrhoeae liberates a soluble factor that potently induces expression from the HIV-1 LTR in coinfected cluster of differentiation 4-positive (CD4(+)) T lymphocytes, but this factor is not a previously described innate effector. A genome-wide mutagenesis approach was undertaken to reveal which component(s) of N. gonorrhoeae induce HIV-1 expression in CD4(+) T lymphocytes. A mutation in the ADP-heptose biosynthesis gene, hldA, rendered the bacteria unable to induce HIV-1 expression. The hldA mutant has a truncated lipooligosaccharide structure, contains lipid A in its outer membrane, and remains bioactive in a TLR4 reporter-based assay but did not induce HIV-1 expression. Mass spectrometry analysis of extensively fractionated N. gonorrhoeae-derived supernatants revealed that the LTR-inducing fraction contained a compound having a mass consistent with heptose-monophosphate (HMP). Heptose is a carbohydrate common in microbes but is absent from the mammalian glycome. Although ADP-heptose biosynthesis is common among Gram-negative bacteria, and heptose is a core component of most lipopolysaccharides, N. gonorrhoeae is peculiar in that it effectively liberates HMP during growth. This N. gonorrhoeae-derived HMP activates CD4(+) T cells to invoke an NF-κB-dependent transcriptional response that drives HIV-1 expression and viral production. Our study thereby shows that heptose is a microbial-specific product that is sensed as an innate immune agonist and unveils the molecular link between N. gonorrhoeae and HIV-1.

Asunto(s)

Coinfección/inmunología , Gonorrea , Infecciones por VIH , VIH-1/enzimología , Heptosas/inmunología , Neisseria gonorrhoeae/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Proteínas Bacterianas/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/microbiología , Linfocitos T CD4-Positivos/virología , Femenino , Gonorrea/inmunología , Gonorrea/microbiología , Gonorrea/virología , Infecciones por VIH/inmunología , Infecciones por VIH/microbiología , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH/genética , VIH-1/inmunología , Heptosas/genética , Heptosas/metabolismo , Humanos , Células Jurkat , Masculino , Neisseria gonorrhoeae/inmunología , Receptor Toll-Like 5/inmunología

RESUMEN

Uniquely modified heptoses found in surface carbohydrates of bacterial pathogens are potential therapeutic targets against such pathogens. Our recent biochemical characterization of the GDP-6-deoxy-D-manno- and GDP-6-deoxy-D-altro-heptose biosynthesis pathways has provided the foundation for elucidation of the more complex L-gluco-heptose synthesis pathway of Campylobacter jejuni strain NCTC 11168. In this work we use GDP-4-keto,6-deoxy-D-lyxo-heptose as a surrogate substrate to characterize three enzymes predicted to be involved in this pathway: WcaGNCTC (also known as Cj1427), MlghB (Cj1430), and MlghC (Cj1428). We compare them with homologues involved in d-altro-heptose production: WcaG81176 (formerly WcaG), DdahB (Cjj1430), and DdahC (Cjj1427). We show that despite high levels of similarity, the enzymes have pathway-specific catalytic activities and substrate specificities. MlghB forms three products via C3 and C5 epimerization activities, whereas its DdahB homologue only had C3 epimerase activity along its cognate pathway. MlghC is specific for the double C3/C5 epimer generated by MlghB and produces L-gluco-heptose via stereospecific C4 reductase activity. In contrast, its homologue DdahC only uses the C3 epimer to yield d-altro-heptose via C4 reduction. Finally, we show that WcaGNCTC is not necessary for L-gluco-heptose synthesis and does not affect its production by MlghB and MlghC, in contrast to its homologue WcaG81176, that has regulatory activity on d-altro-heptose synthesis. These studies expand our fundamental understanding of heptose modification, provide new glycobiology tools to synthesize novel heptose derivatives with biomedical applications, and provide a foundation for the structure function analysis of these enzymes.

Asunto(s)

Proteínas Bacterianas/metabolismo , Campylobacter jejuni/enzimología , Carbohidrato Epimerasas/metabolismo , Heptosas/biosíntesis , Proteínas Bacterianas/genética , Campylobacter jejuni/genética , Carbohidrato Epimerasas/genética , Heptosas/genética , Homología de Secuencia de Aminoácido

RESUMEN

The Campylobacter jejuni capsule is important for colonization and virulence in various infection models. In most strains, the capsule includes a modified heptose whose biological role and biosynthetic pathway are unknown. To decipher the biosynthesis pathway for the 6-deoxy-D-altro-heptose of strain 81-176, we previously showed that the 4,6-dehydratase WcbK and the reductase WcaG generated GDP-6-deoxy-D-manno-heptose, but the C3 epimerase necessary to form GDP-6-deoxy-D-altro-heptose was not identified. Herein, we characterized the putative C3/C5 epimerase Cjj1430 and C3/C5 epimerase/C4 reductase Cjj1427 from the capsular cluster. We demonstrate that GDP-6-deoxy-D-altro-heptose biosynthesis is more complex than anticipated and requires the sequential action of WcbK, Cjj1430, and Cjj1427. We show that Cjj1430 serves as C3 epimerase devoid of C5 epimerization activity and that Cjj1427 has no epimerization activity and only serves as a reductase to produce GDP-6-deoxy-D-altro-heptose. Cjj1430 and Cjj1427 are the only members of the C3/C5 epimerases and C3/C5 epimerase/C4 reductase families shown to have activity on a heptose substrate and to exhibit only one of their two to three potential activities, respectively. Furthermore, we show that although the reductase WcaG is not part of the main pathway, its presence and its product affect the outcome of the pathway in a complex regulatory loop involving Cjj1427. This work provides the grounds for the elucidation of similar pathways found in other C. jejuni strains and other pathogens. It provides new molecular tools for the synthesis of carbohydrate antigens useful for vaccination and for the screening of enzymatic inhibitors that may have antibacterial effects.

Asunto(s)

Proteínas Bacterianas/metabolismo , Campylobacter jejuni/metabolismo , Metabolismo de los Hidratos de Carbono/fisiología , Heptosas/biosíntesis , Proteínas Bacterianas/genética , Campylobacter jejuni/genética , Heptosas/genética

RESUMEN

Pasteurella multocida is a capsulated, gram-negative cocco-bacillus that can cause serious disease in a wide range of mammals and birds. P. multocida strains are classified into 16 serovars based on lipopolysaccharide (LPS) antigens. LPS is an essential virulence factor of P. multocida; mutants expressing severely truncated LPS are completely attenuated in chickens. LPS is also a major immunogen of P. multocida and protection against infections caused by P. multocida is generally considered to be serovar specific. In this review we summarize current knowledge of the structure and genetics of LPS assembly of P. multocida strains belonging to five different serovars. These include strains belonging to serovars 1 and 3, the most common serovars found in the poultry industry, and strains belonging serovars 2 and 5, the serovars associated with bovine haemorrhagic septicaemia outbreaks. A number of the serovars are genetically related; serovars 1 and 14 share the same LPS outer core biosynthesis locus, but due to a mutation within the phosphocholine biosynthesis gene, pcgA, the serovar 14 strain produces a truncated LPS structure. Similarly serovars 2 and 5 share an identical LPS outer core locus and express near-identical LPS structures. However, due to a single point mutation in the phosphoethanolamine (PEtn) transferase gene, lpt_3, the serovar 2 strain does not elaborate a PEtn residue on heptose II. Knowledge of the genetic basis for the LPS structures expressed by P. multocida will facilitate the development of rapid molecular methods for typing and diagnosis and will be essential for a rational approach to vaccine formulation.

Asunto(s)

Lipopolisacáridos/química , Infecciones por Pasteurella/veterinaria , Pasteurella multocida/química , Animales , Heptosas/genética , Heptosas/inmunología , Humanos , Lipopolisacáridos/inmunología , Infecciones por Pasteurella/genética , Infecciones por Pasteurella/inmunología , Pasteurella multocida/genética , Pasteurella multocida/inmunología , Pasteurella multocida/patogenicidad , Factores de Virulencia/genética , Factores de Virulencia/inmunología

RESUMEN

D-Glycero-d-manno-heptose-1,7-bisphosphate phosphatase (GmhB) is a member of the histidinol-phosphate phosphatase (HisB) subfamily of the haloalkanoic acid dehalogenase (HAD) enzyme superfamily. GmhB supports two divergent biochemical pathways in bacteria: the d-glycero-d-manno-heptose-1alpha-GDP pathway (in S-layer glycoprotein biosynthesis) and the l-glycero-d-manno-heptose-1beta-ADP pathway (in lipid A biosynthesis). Herein, we report the comparative analysis of substrate recognition in selected GmhB orthologs. The substrate specificity of the l-glycero-d-manno-heptose-1beta-ADP pathway GmhB from Escherichia coli K-12 was evaluated using hexose and heptose bisphosphates, histidinol phosphate, and common organophosphate metabolites. Only d-glycero-d-manno-heptose 1beta,7-bisphosphate (k(cat)/K(m) = 7 x 10(6) M(-1) s(-1)) and d-glycero-d-manno-heptose 1alpha,7-bisphosphate (k(cat)/K(m) = 7 x 10(4) M(-1) s(-1)) displayed physiologically significant substrate activity. (31)P NMR analysis demonstrated that E. coli GmhB selectively removes the C(7) phosphate. Steady-state kinetic inhibition studies showed that d-glycero-d-manno-heptose 1beta-phosphate (K(is) = 60 microM, and K(ii) = 150 microM) and histidinol phosphate (K(is) = 1 mM, and K(ii) = 6 mM), while not hydrolyzed, do in fact bind to E. coli GmhB, which leads to the conclusion that nonproductive binding contributes to substrate discrimination. High catalytic efficiency and a narrow substrate range are characteristic of a well-evolved metabolic enzyme, and as such, E. coli GmhB is set apart from most HAD phosphatases (which are typically inefficient and promiscuous). The specialization of the biochemical function of GmhB was examined by measuring the kinetic constants for hydrolysis of the alpha- and beta-anomers of d-glycero-d-manno-heptose 1beta,7-bisphosphate catalyzed by the GmhB orthologs of the l-glycero-d-manno-heptose 1beta-ADP pathways operative in Bordetella bronchiseptica and Mesorhizobium loti and by the GmhB of the d-glycero-d-manno-heptose 1alpha-GDP pathway operative in Bacteroides thetaiotaomicron. The results show that although each of these representatives possesses physiologically significant catalytic activity toward both anomers, each displays substantial anomeric specificity. Like E. coli GmhB, B. bronchiseptica GmhB and M. loti GmhB prefer the beta-anomer, whereas B. thetaiotaomicron GmhB is selective for the alpha-anomer. By determining the anomeric configuration of the physiological substrate (d-glycero-d-manno-heptose 1,7-bisphosphate) for each of the four GmhB orthologs, we discovered that the anomeric specificity of GmhB correlates with that of the pathway kinase. The conclusion drawn from this finding is that the evolution of the ancestor to GmhB in the HisB subfamily provided for specialization toward two distinct biochemical functions.

Asunto(s)

Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Hidrolasas/química , Familia de Multigenes , Monoéster Fosfórico Hidrolasas/química , Alphaproteobacteria/enzimología , Bacteroides/enzimología , Bordetella bronchiseptica/enzimología , Catálisis , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Heptosas/química , Heptosas/genética , Histidinol-Fosfatasa/química , Histidinol-Fosfatasa/genética , Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/genética , Especificidad por Sustrato/genética

RESUMEN

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.

Asunto(s)

Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Lipopolisacáridos/biosíntesis , Monoéster Fosfórico Hidrolasas/química , Monoéster Fosfórico Hidrolasas/metabolismo , Secuencias de Aminoácidos/genética , Ácido Aspártico/química , Ácido Aspártico/metabolismo , Catálisis , Permeabilidad de la Membrana Celular/genética , Secuencia Conservada/genética , Cristalografía por Rayos X , Análisis Mutacional de ADN , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Heptosas/biosíntesis , Heptosas/deficiencia , Heptosas/genética , Lipopolisacáridos/química , Lipopolisacáridos/genética , Monoéster Fosfórico Hidrolasas/genética , Fosforilación/genética , Relación Estructura-Actividad

RESUMEN

Soluble CD14 (sCD14) is a serum glycoprotein that binds to the Lipid A moiety of lipopolysaccharides (LPS) with high affinity as part of the innate immune response to bacterial endotoxins. In order to investigate structural interactions of Lipid A with sCD14, we have prepared an isotopically labeled form of a fully active and chemically defined endotoxin, Kdo(2)-Lipid A, which allowed us to carry out detailed NMR spectral mapping of this agonist ligand bound to sCD14 and identify for the first time structural regions that are strongly affected during complex formation with sCD14. These map to two adjacent areas comprising the lower portions of the sugar headgroup and upper half of the acyl chains I, III, and V, which are spatially proximal to the 1- and 4'-phosphate ends. Additionally, we have detected for the first time, presence of differential dynamic behavior for the affected resonances, suggesting a likely role for dynamics in the mechanism of Lipid A pattern recognition by sCD14.

Asunto(s)

Lípido A/inmunología , Receptores de Lipopolisacáridos/inmunología , Lipopolisacáridos/inmunología , Escherichia coli/genética , Heptosas/genética , Marcaje Isotópico , Ligandos , Lípido A/química , Lípido A/genética , Receptores de Lipopolisacáridos/química , Lipopolisacáridos/química , Lipopolisacáridos/genética , Resonancia Magnética Nuclear Biomolecular

RESUMEN

The full structure of the long- and short-chain O-antigen of Yersinia pseudotuberculosis O:2a containing two uncommon deoxy sugars, abequose and 6-deoxy-d-manno-heptose (6dmanHep), was established, for the first time, by sugar analysis, NMR spectroscopy, and high-resolution ESIMS. Similar structural studies were also performed on two O:2a mutants with single disruption of 6dmanHep synthesis pathway genes each, which synthesize modified long-chain (dmhA mutant) and short-chain (both dmhA and dmhB mutants) O-antigens with 6dmanHep replaced by its putative biosynthetic precursor, D-glycero-D-manno-heptose.

Asunto(s)

Heptosas/química , Mutación , Antígenos O/química , Yersinia pseudotuberculosis/química , Secuencia de Carbohidratos , Heptosas/biosíntesis , Heptosas/genética , Lipopolisacáridos/aislamiento & purificación , Lipopolisacáridos/metabolismo , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Antígenos O/genética , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Yersinia pseudotuberculosis/genética , Yersinia pseudotuberculosis/metabolismo

RESUMEN

We recently demonstrated that Campylobacter jejuni produces a capsular polysaccharide (CPS) that is the major antigenic component of the classical Penner serotyping system distinguishing Campylobacter into >60 groups. Although the wide variety of C. jejuni serotypes are suggestive of structural differences in CPS, the genetic mechanisms of such differences are unknown. In this study we sequenced biosynthetic cps regions, ranging in size from 15 to 34 kb, from selected C. jejuni strains of HS:1, HS:19, HS:23, HS:36, HS:23/36 and HS:41 serotypes. Comparison of the determined cps sequences of the HS:1, HS:19 and HS:41 strains with the sequenced strain, NCTC11168 (HS:2), provides evidence for multiple mechanisms of structural variation including exchange of capsular genes and entire clusters by horizontal transfer, gene duplication, deletion, fusion and contingency gene variation. In contrast, the HS:23, HS:36 and HS:23/36 cps sequences were highly conserved. We report the first detailed structural analysis of 81-176 (HS:23/36) and G1 (HS:1) and refine the previous structural interpretations of the HS:19, HS:23, HS:36 and HS:41 serostrains. For the first time, we demonstrate the commonality and function of a second heptose biosynthetic pathway for Campylobacter CPS independent of the pathway for lipooligosaccharide (LOS) biosynthesis and identify a novel heptosyltransferase utilized by this alternate pathway. Furthermore, we show the retention of two functional heptose isomerases in Campylobacter and the sharing of a phosphatase for both LOS and CPS heptose biosynthesis.

Asunto(s)

Cápsulas Bacterianas/genética , Campylobacter jejuni/genética , Variación Genética , Cápsulas Bacterianas/química , Cápsulas Bacterianas/metabolismo , Campylobacter jejuni/metabolismo , Carbohidrato Epimerasas/genética , Carbohidrato Epimerasas/fisiología , Secuencia de Carbohidratos , ADN Bacteriano , Duplicación de Gen , Transferencia de Gen Horizontal , Genes Bacterianos , Glicosiltransferasas/genética , Glicosiltransferasas/fisiología , Heptosas/química , Heptosas/genética , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Familia de Multigenes , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/fisiología , Recombinación Genética , Análisis de Secuencia de ADN , Eliminación de Secuencia

RESUMEN

Lipooligosaccharide (LOS) is a critical virulence factor of Neisseria meningitidis. A Tn916 insertion mutant, designated 469, was found to exhibit a markedly truncated LOS of 2.9 kDa when compared by Tricine/SDS-PAGE to the parental LOS (4.6 kDa). Electrospray mass spectrometry analysis of 469 LOS revealed that it consisted of the deep rough, heptose-deficient structure, Kdo(2)-lipid A. Sequencing of chromosomal DNA flanking the Tn916 insertion in mutant 469 revealed that the transposon had inserted into an ORF predicted to encode a 187 aa protein with sequence homology to the histidinol-phosphate phosphatase domain of Escherichia coli HisB and to a family of genes of unknown function. The gene, designated gmhX, is part of a polycistronic operon (ice-2) containing two other genes, nlaB and orfC. nlaB encodes a lysophosphatidic-acid acyltransferase and orfC is predicted to encode a N-acetyltransferase. Specific polar and non-polar gmhX mutations in the parental strain, NMB, exhibited the truncated LOS structure of mutant 469, and repair of gmhX mutants by homologous recombination with the wild-type gmhX restored the LOS parental phenotype. GmhX mutants demonstrated increased sensitivity to polymyxin B. GmhX mutants and other Kdo(2)-lipid A mutants also demonstrated increased sensitivity to killing by normal human serum but were not as sensitive as inner-core mutants containing heptose. In the genomes of Helicobacter pylori and Synechocystis, gmhX homologues are associated with heptose biosynthesis genes; however, in N. meningitidis, gmhX was found in a location distinct from that of gmhA, rfaD, rfaE, aut and rfaC. GmhX is a novel enzyme required for the incorporation of L-glycero-D-manno-heptose into meningococcal LOS, and is a candidate for the 2-D-glycero-manno-heptose phosphatase of the heptose biosynthesis pathway.

Asunto(s)

Proteínas Bacterianas/genética , Heptosas/metabolismo , Lípido A/análogos & derivados , Lipopolisacáridos/metabolismo , Neisseria meningitidis/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Genes Bacterianos , Heptosas/genética , Lípido A/química , Lípido A/genética , Lípido A/metabolismo , Lipopolisacáridos/química , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Neisseria meningitidis/genética , Neisseria meningitidis/crecimiento & desarrollo , Operón , Monoéster Fosfórico Hidrolasas/metabolismo , Análisis de Secuencia de ADN

RESUMEN

A new novobiocin-supersensitive mutant of Escherichia coli K-12 has been characterized biochemically and genetically. Lipopolysaccharide prepared from this mutant strain is truncated and contains 2-keto-3-deoxyoctulosonic acid as its only core sugar. This new core-defective mutation, designated rfa-2, results in increased sensitivity to several hydrophobic and some hydrophilic agents. Genetic analysis of the rfa mutant indicated that the rfa-2 locus is located at 81 min on the chromosome. The order of the genes in this region based on transduction analysis is xyl cysE rfa-2 rfaD70 pyrE. P1 transduction analyses indicate that the rfa-2 marker is nonallelic with the recently described cysE-pyrE-linked rfaD70 locus. Plasmids carrying the wild-type rfaD70+ allele failed to abolish the rfa-2 phenotypes. Further, the rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, was detected in crude extracts of a rfa-2 mutant strain, CL609, and was absent in the rfaD70 mutant. The wild-type rfa-2 allele codes either for a specific heptose biosynthetic enzyme (different from the rfaD gene product) or an enzymatic activity required for the addition of heptose to the lipid A-2-keto-3-deoxyoctulosonic acid acceptor.

Asunto(s)

Escherichia coli/genética , Lipopolisacáridos/genética , Polisacáridos Bacterianos/genética , Mapeo Cromosómico , Escherichia coli/efectos de los fármacos , Heptosas/genética , Mutación

RESUMEN

The structure and metal-binding properties of lipopolysaccharides (LPS) from heptoseless mutants of Escherichia coli were studied by 13C and 31P NMR techniques. Carbon-13 NMR spectra were used to determine the linkages and configurations of the saccharide backbone and the types and locations of fatty acyl groups in E. coli LPS. Resonance assignments for native LPS were made by chemical shift correlation with model compounds, deacylated LPS, lipid A, deacylated lipid A, and fatty acids released from LPS by mild alkaline hydrolysis. The 3-deoxy-D-manno-octulosonate (KDO) disaccharide was tentatively assigned the structure KDO alpha 2 leads to 5KDO alpha 2 leads to. The presence of amide- and ester-linked 3-hydroxy and 3-acyloxy fatty acids in native LPS was confirmed directly from the 13C spectrum and evidence is presented for a labile acyl ester at C-3' (GlcNII) of the lipid A moiety. A significant finding was that the KDO disaccharide is linked to the C-6' position of the lipid A moiety, rather than C-3', as previously reported. The effects of binding Ca2+, Cd2+, Yb3+, Gd3+, and La3+ on the 31P NMR spectrum of LPS indicated that the glycosidic diphosphate moiety participates in a high affinity metal-binding site.

Asunto(s)

Escherichia coli/genética , Heptosas/genética , Lipopolisacáridos , Metales/metabolismo , Mutación , Cadmio , Calcio , Conformación de Carbohidratos , Secuencia de Carbohidratos , Lipopolisacáridos/genética , Espectroscopía de Resonancia Magnética , Metales de Tierras Raras

RESUMEN

A highly purified monophosphoryl lipid A, TLC-3 fraction obtained from the lipopolysaccharides of the heptoseless mutant Salmonella typhimurium G30/C21 was converted to the dimethyl pentatrimethylsilyl derivative and analyzed by proton NMR spectroscopy at 400 MHz. Substantial downfield shifts of the resonances for protons at the 3- and 3'-carbons of the glucosamine disaccharide to 5.06 and 5.15 ppm, respectively, occurred from the normal range of 3.5-4.1 ppm, indicating that these two positions on the sugar rings were acylated. Significant downfield shift of the resonances for protons at the 4- and 6'-carbons did not occur, indicating the absence of acyl groups at these two positions. Since positive ion fast atom bombardment mass spectrometry previously established the presence of hydroxymyristoyl and myristoxymyristoyl esters at the reducing end and distal subunits, respectively, these acyl groups must be attached to the oxygen of the corresponding 3- and 3'-carbons of lipid A. With these results, we can now describe the complete structure of the monophosphoryl lipid A, TLC-3 from S. typhimurium.

Asunto(s)

Lípido A/aislamiento & purificación , Lipopolisacáridos/genética , Mutación , Salmonella typhimurium/genética , Heptosas/genética , Espectroscopía de Resonancia Magnética , Conformación Molecular

RESUMEN

Lipopolysaccharide (LPS) isolated from Escherichia coli D31m4, a heptoseless mutant, was studied by 13C and 31P NMR spectroscopy. Modified isolation and purification procedures are described which permitted high resolution NMR spectra to be obtained from samples of intact LPS. 31P NMR was used to monitor the purity and native heterogeneity of LPS samples. The anomeric carbon region of the 13C NMR spectrum taken at pH 7 contained five resonances that were assigned on the basis of chemical shift correlation, 13C-1H couplings, and T1 relaxation times. Two resonances, at 99.9 and 100.8 ppm, were attributed to two residues of 3-deoxy-D-manno-octulosonate (KDO) of which both were tentatively assigned to the alpha configuration. The Lipid A moiety gave rise to resonances at 94.0 and 94.9 ppm, both assigned to GlcNI, and a resonance at 103.1 ppm, assigned to GlcNII. The two anomeric carbon resonances observed for GlcNI reflected the variable substitution of C-1 with monophosphate or diphosphate groups. GlcNI and GlcNII were ascertained to be of the alpha and beta anomeric configuration, respectively, through chemical shift comparisons with model saccharides. The accepted KDO linkage site at C-3' of GlcNII appears not to be supported by the 13C chemical shift data.

Asunto(s)

Escherichia coli/genética , Heptosas/genética , Lipopolisacáridos/aislamiento & purificación , Mutación , Carbohidratos/análisis , Espectroscopía de Resonancia Magnética

Asunto(s)

Neoplasias Hepáticas Experimentales/terapia , Mycobacterium bovis/inmunología , Salmonella typhimurium/inmunología , Animales , Vacuna BCG/administración & dosificación , Pared Celular/inmunología , Emulsiones , Femenino , Glucolípidos/inmunología , Cobayas , Heptosas/genética , Inmunoterapia , Mutación , Remisión Espontánea , Salmonella typhimurium/genética