Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Filtros aplicados
Base de dados
Intervalo de ano de publicação
1.
Carbohydr Res ; 502: 108272, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33711724

RESUMO

Simple protocols for attaching and detaching carbobenzyloxy (Cbz) groups at the reducing end of sugars was developed. Briefly, lactose was converted into its glycosylamine, which was then acylated with carbobenzyloxy chloride in high overall yield. The obtained lactose Cbz derivative was used in sequential glycosylations using glycosyltransferases and nucleotide sugars in aqueous buffers. Isolation of the reaction products after each step was by simple C-18 solid-phase extraction. The Cbz group was removed by catalytic hydrogenolysis or catalytic transfer hydrogenation followed by in situ glycosylamine hydrolysis. In this way, a trisaccharide (GlcNAc-lactose), a human milk tetrasaccharide (LNnT), and a human milk pentasaccharide (LNFPIII) were prepared in a simple and efficient way.


Assuntos
Derivados de Benzeno/metabolismo , Fucosiltransferases/metabolismo , Oligossacarídeos/biossíntese , Açúcares/metabolismo , Derivados de Benzeno/química , Glucosamina/química , Glucosamina/metabolismo , Helicobacter mustelae/enzimologia , Humanos , Hidrólise , Oligossacarídeos/química , Oligossacarídeos/isolamento & purificação , Açúcares/química
2.
Nat Commun ; 10(1): 1799, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30996301

RESUMO

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.


Assuntos
Proteínas de Bactérias/metabolismo , Glicosiltransferases/metabolismo , Hemaglutininas/imunologia , Interações Hospedeiro-Patógeno/imunologia , Influenza Humana/imunologia , Oligossacarídeos/metabolismo , Animais , Proteínas de Bactérias/genética , Bioensaio/métodos , Células CHO , Cricetulus , Cães , Glicosiltransferases/genética , Voluntários Saudáveis , Helicobacter mustelae/genética , Helicobacter mustelae/metabolismo , Hemaglutininas/metabolismo , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/imunologia , Influenza Humana/virologia , Microscopia Intravital/métodos , Luciferases Bacterianas/genética , Luciferases Bacterianas/metabolismo , Pulmão/patologia , Células Madin Darby de Rim Canino , Engenharia Metabólica/métodos , Microscopia de Fluorescência/métodos , Imagem Molecular/métodos , Oligossacarídeos/imunologia , Pasteurella multocida/genética , Pasteurella multocida/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Antígeno Sialil Lewis X , Coloração e Rotulagem/métodos
3.
J Inorg Biochem ; 111: 195-202, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22196017

RESUMO

The micro aerophilic pathogen Helicobacter mustelae synthesizes an oxygen-labile, iron-containing urease (UreA2B2) in addition to its standard nickel-containing enzyme (UreAB). An apoprotein form of the iron urease was prepared from ureA2B2-expressing recombinant Escherichia coli cells that were grown in minimal medium. Temperature-dependent circular dichroism measurements of holoprotein and apoprotein demonstrate an enhancement of thermal stability associated with the UreA2B2 metallocenter. In parallel to the situation reported for nickel activation of the standard urease apoprotein, incubation of UreA2B2 apoprotein with ferrous ions and bicarbonate generated urease activity in a portion of the nascent active sites. In addition, ferrous ions were shown to be capable of reductively activating the oxidized metallocenter. Resonance Raman spectra of the inactive, aerobically-purified UreA2B2 holoprotein exhibit vibrations at 495cm(-1) and 784cm(-1), consistent with ν(s) and ν(as) modes of an Fe(III)OFe(III) center; these modes undergo downshifts upon binding of urea and were unaffected by changes in pH. The low-frequency mode also exhibits an isotopic shift from 497 to 476cm(-1) upon (16)O/(18)O bulk water isotope substitution. Expression of subunits of the conventional nickel-containing Klebsiella aerogenes urease in cells grown in rich medium without nickel resulted in iron incorporation into a portion of the protein. The inactive iron-loaded species exhibited a UV-visible spectrum similar to oxidized UreA2B2 and was capable of being reductively activated under anoxic conditions. Results from these studies more clearly define the formation and unique properties of the iron urease metallocenter.


Assuntos
Apoproteínas/química , Proteínas de Bactérias/química , Helicobacter mustelae/enzimologia , Ferro/química , Metaloproteínas/química , Urease/química , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bicarbonatos/farmacologia , Domínio Catalítico , Dicroísmo Circular , Ativação Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Compostos Ferrosos/farmacologia , Helicobacter mustelae/genética , Holoenzimas/química , Holoenzimas/metabolismo , Concentração de Íons de Hidrogênio , Ferro/metabolismo , Metaloproteínas/genética , Metaloproteínas/metabolismo , Modelos Moleculares , Estrutura Molecular , Oxirredução , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análise Espectral Raman , Temperatura , Urease/genética , Urease/metabolismo
4.
Proc Natl Acad Sci U S A ; 108(32): 13095-9, 2011 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-21788478

RESUMO

Helicobacter mustelae, a gastric pathogen of ferrets, synthesizes a distinct iron-dependent urease in addition to its archetypical nickel-containing enzyme. The iron-urease is oxygen-labile, with the inactive protein exhibiting a methemerythrin-like electronic spectrum. Significantly, incubation of the oxidized protein with dithionite under anaerobic conditions leads to restoration of activity and bleaching of the spectrum. Structural analysis of the oxidized species reveals a dinuclear iron metallocenter bridged by a lysine carbamate, closely resembling the traditional nickel-urease active site. Although the iron-urease is less active than the nickel-enzyme, its activity allows H. mustelae to survive the carnivore's low-nickel gastric environment.


Assuntos
Helicobacter mustelae/enzimologia , Ferro/metabolismo , Urease/metabolismo , Absorção/efeitos dos fármacos , Cristalografia por Raios X , Meios de Cultura/farmacologia , Elétrons , Helicobacter mustelae/efeitos dos fármacos , Íons , Cinética , Modelos Moleculares , Níquel/metabolismo , Oxigênio/metabolismo , Análise Espectral , Urease/química , Urease/isolamento & purificação
5.
Infect Immun ; 78(10): 4261-7, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20643857

RESUMO

The genomes of Helicobacter species colonizing the mammalian gastric mucosa (like Helicobacter pylori) contain a large number of genes annotated as iron acquisition genes but only few nickel acquisition genes, which contrasts with the central position of nickel in the urease-mediated acid resistance of these gastric pathogens. In this study we have investigated the predicted iron and nickel acquisition systems of the ferret pathogen Helicobacter mustelae. The expression of the outer membrane protein-encoding frpB2 gene was iron and Fur repressed, whereas the expression of the ABC transporter genes fecD and ceuE was iron and Fur independent. The inactivation of the two tonB genes showed that TonB1 is required for heme utilization, whereas the absence of TonB2 only marginally affected iron-dependent growth but led to reduced cellular nickel content and urease activity. The inactivation of the fecD and ceuE ABC transporter genes did not affect iron levels but resulted in significantly reduced urease activity and cellular nickel content. Surprisingly, the inactivation of the nixA nickel transporter gene affected cellular nickel content and urease activity only when combined with the inactivation of other nickel acquisition genes, like fecD or ceuE. The FecDE ABC transporter is not specific for nickel, since an fecD mutant also showed reduced cellular cobalt levels and increased cobalt resistance. We conclude that the H. mustelae fecDE and ceuE genes encode an ABC transporter involved in nickel and cobalt acquisition, which works independently of the nickel transporter NixA, while TonB2 is required primarily for nickel acquisition, with TonB1 being required for heme utilization.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Bactérias/metabolismo , Cobalto/metabolismo , Helicobacter mustelae/metabolismo , Proteínas de Membrana/metabolismo , Níquel/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Proteínas de Bactérias/classificação , Proteínas de Bactérias/genética , Transporte Biológico , Furões , Regulação Bacteriana da Expressão Gênica/fisiologia , Genoma Bacteriano , Helicobacter mustelae/genética , Proteínas de Membrana/classificação , Proteínas de Membrana/genética , Mutação , Urease/metabolismo
6.
BMC Genomics ; 11: 164, 2010 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-20219135

RESUMO

BACKGROUND: Helicobacter mustelae causes gastritis, ulcers and gastric cancer in ferrets and other mustelids. H. mustelae remains the only helicobacter other than H. pylori that causes gastric ulceration and cancer in its natural host. To improve understanding of H. mustelae pathogenesis, and the ulcerogenic and carcinogenic potential of helicobacters in general, we sequenced the H. mustelae genome, and identified 425 expressed proteins in the envelope and cytosolic proteome. RESULTS: The H. mustelae genome lacks orthologs of major H. pylori virulence factors including CagA, VacA, BabA, SabA and OipA. However, it encodes ten autotransporter surface proteins, seven of which were detected in the expressed proteome, and which, except for the Hsr protein, are of unknown function. There are 26 putative outer membrane proteins in H. mustelae, some of which are most similar to the Hof proteins of H. pylori. Although homologs of putative virulence determinants of H. pylori (NapA, plasminogen adhesin, collagenase) and Campylobacter jejuni (CiaB, Peb4a) are present in the H. mustelae genome, it also includes a distinct complement of virulence-related genes including a haemagglutinin/haemolysin protein, and a glycosyl transferase for producing blood group A/B on its lipopolysaccharide. The most highly expressed 264 proteins in the cytosolic proteome included many corresponding proteins from H. pylori, but the rank profile in H. mustelae was distinctive. Of 27 genes shown to be essential for H. pylori colonization of the gerbil, all but three had orthologs in H. mustelae, identifying a shared set of core proteins for gastric persistence. CONCLUSIONS: The determination of the genome sequence and expressed proteome of the ulcerogenic species H mustelae provides a comparative model for H. pylori to investigate bacterial gastric carcinogenesis in mammals, and to suggest ways whereby cag minus H. pylori strains might cause ulceration and cancer. The genome sequence was deposited in EMBL/GenBank/DDBJ under accession number FN555004.


Assuntos
Hibridização Genômica Comparativa , Genoma Bacteriano , Helicobacter mustelae/genética , Proteoma/genética , Sequência de Aminoácidos , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Regulação Bacteriana da Expressão Gênica , Genômica , Helicobacter mustelae/patogenicidade , Helicobacter pylori/genética , Dados de Sequência Molecular , Filogenia , Proteômica , Alinhamento de Sequência , Análise de Sequência de DNA , Virulência
7.
Biometals ; 23(1): 145-59, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19894125

RESUMO

The NikR protein is a nickel-responsive regulator, which in the gastric pathogen Helicobacter pylori controls expression of nickel-transporters and the nickel-cofactored urease acid resistance determinant. Although NikR-DNA interaction has been well studied, the Helicobacter NikR operator site remains poorly defined. In this study we have identified the NikR operators in the promoters of two inversely nickel-regulated urease operons (ureAB and ureA2B2) in the ferret pathogen Helicobacter mustelae, and have used bioinformatic approaches for the prediction of putative NikR operators in the genomes of four urease-positive Helicobacter species. Helicobacter mustelae NikR bound to the ureA2 promoter to a sequence overlapping with the -35 promoter region, leading to repression. In contrast, NikR binding to a site far upstream of the canonical sigma(80) promoter in the H. mustelae ureA promoter resulted in transcriptional induction, similar to the situation in H. pylori. Using H. pylori NikR operators and the newly identified H. mustelae NikR operators a new consensus sequence was generated (TRWYA-N(15)-TRWYA), which was used to screen the genomes of four urease-positive Helicobacter species (H. mustelae, H. pylori, H. acinonychis and H. hepaticus) for putative NikR-regulated promoters. One of these novel putative NikR-regulated promoters in H. mustelae is located upstream of a putative TonB-dependent outer membrane protein designated NikH, which displayed nickel-responsive expression. Insertional inactivation of the nikH gene in H. mustelae resulted in a significant decrease in urease activity, and this phenotype was complemented by nickel-supplementation of the growth medium, suggesting a function for NikH in nickel transport across the outer membrane. In conclusion, the H. mustelae NikR regulator directly controls nickel-responsive regulation of ureases and metal transporters. The improved consensus NikR operator sequence allows the prediction of additional NikR targets in Helicobacter genomes, as demonstrated by the identification of a new nickel-repressed outer membrane protein in H. mustelae.


Assuntos
Proteínas de Transporte de Cátions/genética , Helicobacter mustelae/enzimologia , Helicobacter mustelae/genética , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismo , Urease/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Helicobacter mustelae/metabolismo , Proteínas Repressoras/química , Urease/metabolismo
8.
J Am Chem Soc ; 130(44): 14420-1, 2008 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-18842049

RESUMO

A bacterial version of human blood group A transferase was identified and found to be able to accept five naturally existing H-antigen core structures as good substrates, demonstrating its versatility for synthesis of blood group A antigens. Furthermore, this enzyme was applied in the engineering of bacterial cell surface polysaccharides by remodeling blood group B mimicry into blood group A mimicry.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Fucosil Galactose alfa-N-Acetilgalactosaminiltransferase/química , Fucosil Galactose alfa-N-Acetilgalactosaminiltransferase/metabolismo , Helicobacter mustelae/enzimologia , Sistema ABO de Grupos Sanguíneos/sangue , Sistema ABO de Grupos Sanguíneos/metabolismo , Sequência de Carboidratos , Escherichia coli/metabolismo , Fucosil Galactose alfa-N-Acetilgalactosaminiltransferase/sangue , Humanos , Lipopolissacarídeos/química , Lipopolissacarídeos/metabolismo , Dados de Sequência Molecular , Especificidade por Substrato
9.
Environ Microbiol ; 10(10): 2586-97, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18564183

RESUMO

The acidic gastric environment of mammals can be chronically colonized by pathogenic Helicobacter species, which use the nickel-dependent urea-degrading enzyme urease to confer acid resistance. Nickel availability in the mammal host is low, being mostly restricted to vegetarian dietary sources, and thus Helicobacter species colonizing carnivores may be subjected to episodes of nickel deficiency and associated acid sensitivity. The aim of this study was to investigate how these Helicobacter species have adapted to the nickel-restricted diet of their carnivorous host. Three carnivore-colonizing Helicobacter species express a second functional urea-degrading urease enzyme (UreA2B2), which functions as adaptation to nickel deficiency. UreA2B2 was not detected in seven other Helicobacter species, and is in Helicobacter mustelae only expressed in nickel-restricted conditions, and its expression was higher in iron-rich conditions. In contrast to the standard urease UreAB, UreA2B2 does not require activation by urease or hydrogenase accessory proteins, which mediate nickel incorporation into these enzymes. Activity of either UreAB or UreA2B2 urease allowed survival of a severe acid shock in the presence of urea, demonstrating a functional role for UreA2B2 in acid resistance. Pathogens often express colonization factors which are adapted to their host. The UreA2B2 urease could represent an example of pathogen adaptation to the specifics of the diet of their carnivorous host, rather than to the host itself.


Assuntos
Helicobacter mustelae/enzimologia , Níquel/metabolismo , Urease/biossíntese , Ácidos/farmacologia , Animais , Antibacterianos/farmacologia , Indução Enzimática , Perfilação da Expressão Gênica , Ordem dos Genes , Helicobacter mustelae/efeitos dos fármacos , Viabilidade Microbiana , Óperon
10.
FEMS Immunol Med Microbiol ; 50(2): 257-63, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17316371

RESUMO

Helicobacter mustelae is a gastric pathogen of ferrets, where it causes disorders similar to those caused by Helicobacter pylori in humans. The H. mustelae ferret model therefore has potential for the in vivo study of Helicobacter pathogenesis in general. In this study a library of 500 individual H. mustelae mutants was generated using an in vitro random insertion mutagenesis technique. Mutants were subsequently tested for motility and adherence, and 43 of the 500 mutants tested were found to be nonmotile in a soft agar assay. Of these 43 mutants, seven were subsequently identified as deficient in their ability to adhere to AGS cells. Insertion had taken place in different positions in the H. mustelae genome, and included mutants in or near to genes involved in motility and urease activity (e.g. the chemotaxis gene cheV and the urease accessory gene ureH). The development of a mutant library for a natural animal model of Helicobacter infection provides the opportunity to study in vivo the role of candidate Helicobacter virulence genes.


Assuntos
Infecções por Helicobacter/microbiologia , Helicobacter mustelae/genética , Helicobacter mustelae/patogenicidade , Mutagênese Insercional , Fatores de Virulência/genética , Animais , Aderência Bacteriana/genética , Linhagem Celular , Quimiotaxia/genética , DNA Bacteriano/química , DNA Bacteriano/genética , Furões , Biblioteca Gênica , Genes Bacterianos , Genoma Bacteriano/genética , Helicobacter mustelae/fisiologia , Humanos , Locomoção/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Urease/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...