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2.
mSphere ; 5(2)2020 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-32269150

RESUMO

We are exposed daily to many glycans from bacteria and food plants. Bacterial glycans are generally antigenic and elicit antibody responses. It is unclear if food glycans' sharing of antigens with bacterial glycans influences our immune responses to bacteria. We studied 14 different plant foods for cross-reactivity with monoclonal antibodies (MAbs) against 24 pneumococcal serotypes which commonly cause infections and are included in pneumococcal vaccines. Serotype 15B-specific MAb cross-reacts with fruit peels, and serotype 10A MAb cross-reacts with many natural and processed plant foods. The serotype 10A cross-reactive epitope is terminal 1,6-linked ß-galactose [ßGal(1-6)], present in the rhamno-galacturonan I (RG-I) domain of pectin. Despite wide consumption of pectin, the immune response to 10A is comparable to the responses to other serotypes. An antipectin antibody can opsonize serotype 10A pneumococci, and the shared ßGal(1-6) may be useful as a simple vaccine against 10A. Impact of food glycans should be considered in host-pathogen interactions and future vaccine designs.IMPORTANCE The impact of food consumption on vaccine responses is unknown. Streptococcus pneumoniae (the pneumococcus) is an important human pathogen, and its polysaccharide capsule is used as a vaccine. We show that capsule type 10A in a pneumococcal vaccine shares an antigenic epitope, ßGal(1-6), with pectin, which is in many plant foods and is widely consumed. Immune response to 10A is comparable to that seen with other capsule types, and pectin ingestion may have little impact on vaccine responses. However, antibody to pectin can kill serotype 10A pneumococci and this shared epitope may be considered in pneumococcal vaccine designs.


Assuntos
Antígenos de Bactérias/imunologia , Cápsulas Bacterianas/imunologia , Reações Cruzadas , Pectinas/imunologia , Streptococcus pneumoniae/imunologia , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Frutas , Humanos , Fagocitose , Sorogrupo , Verduras
3.
PLoS One ; 13(10): e0206187, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30335828

RESUMO

The rare branched-chain sugar apiose, once thought to only be present in the plant kingdom, was found in two bacterial species: Geminicoccus roseus and Xanthomonas pisi. Glycans with apiose residues were detected in aqueous methanol-soluble fractions as well as in the insoluble pellet fraction of X. pisi. Genes encoding bacterial uridine diphosphate apiose (UDP-apiose) synthases (bUASs) were characterized in these bacterial species, but the enzyme(s) involved in the incorporation of the apiose into glycans remained unknown. In the X. pisi genome two genes flanking the XpUAS were annotated as hypothetical glycosyltransferase (GT) proteins. The first GT (here on named XpApiT) belongs to GT family 90 and has a Leloir type B fold and a putative lipopolysaccharide-modifying (LPS) domain. The second GT (here on XpXylT) belongs to GT family 2 and has a type A fold. The XpXylT and XpApiT genes were cloned and heterologously expressed in E. coli. Analysis of nucleotide sugar extracts from E. coli expressing XpXylT or XpApiT with UAS showed that recombinant XpApiT utilized UDP-apiose and XpXylT utilized UDP-xylose as substrate. Indirect activity assay (UDP-Glo) revealed that XpApiT is an apiosyltransferase (ApiT) able to specifically use UDP-apiose. Further support for the apiosyltransferase activity was demonstrated by in microbe co-expression of UAS and XpApiT in E. coli showing the utilization of UDP-apiose to generate an apioside detectable in the pellet fraction. This work provides evidence that X. pisi developed the ability to synthesize an apioside of indeterminate function; however, the evolution of the bacterial ApiT remains to be determined. From genetic and evolutionary perspectives, the apiose operon may provide a unique opportunity to examine how genomic changes reflect ecological adaptation during the divergence of a bacterial group.


Assuntos
Pentoses/metabolismo , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Xanthomonas/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Óperon , Pentosiltransferases/química , Filogenia , Plantas/microbiologia , Domínios Proteicos , Xanthomonas/genética
4.
mBio ; 8(6)2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29162710

RESUMO

Can accumulation of a normally transient metabolite affect fungal biology? UDP-4-keto-6-deoxyglucose (UDP-KDG) represents an intermediate stage in conversion of UDP-glucose to UDP-rhamnose. Normally, UDP-KDG is not detected in living cells, because it is quickly converted to UDP-rhamnose by the enzyme UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase (ER). We previously found that deletion of the er gene in Botrytis cinerea resulted in accumulation of UDP-KDG to levels that were toxic to the fungus due to destabilization of the cell wall. Here we show that these negative effects are at least partly due to inhibition by UDP-KDG of the enzyme UDP-galactopyranose mutase (UGM), which reversibly converts UDP-galactopyranose (UDP-Galp) to UDP-galactofuranose (UDP-Galf). An enzymatic activity assay showed that UDP-KDG inhibits the B. cinerea UGM enzyme with a Ki of 221.9 µM. Deletion of the ugm gene resulted in strains with weakened cell walls and phenotypes that were similar to those of the er deletion strain, which accumulates UDP-KDG. Galf residue levels were completely abolished in the Δugm strain and reduced in the Δer strain, while overexpression of the ugm gene in the background of a Δer strain restored Galf levels and alleviated the phenotypes. Collectively, our results show that the antifungal activity of UDP-KDG is due to inhibition of UGM and possibly other nucleotide sugar-modifying enzymes and that the rhamnose metabolic pathway serves as a shunt that prevents accumulation of UDP-KDG to toxic levels. These findings, together with the fact that there is no Galf in mammals, support the possibility of developing UDP-KDG or its derivatives as antifungal drugs.IMPORTANCE Nucleotide sugars are donors for the sugars in fungal wall polymers. We showed that production of the minor sugar rhamnose is used primarily to neutralize the toxic intermediate compound UDP-KDG. This surprising finding highlights a completely new role for minor sugars and other secondary metabolites with undetermined function. Furthermore, the toxic potential of predicted transition metabolites that never accumulate in cells under natural conditions are highlighted. We demonstrate that UDP-KDG inhibits the UDP-galactopyranose mutase enzyme, thereby affecting production of Galf, which is one of the components of cell wall glycans. Given the structural similarity, UDP-KDG likely inhibits additional nucleotide sugar-utilizing enzymes, a hypothesis that is also supported by our findings. Our results suggest that UDP-KDG could serve as a template to develop antifungal drugs.


Assuntos
Antifúngicos/metabolismo , Botrytis/metabolismo , Glucose/análogos & derivados , Transferases Intramoleculares/antagonistas & inibidores , Difosfato de Uridina/análogos & derivados , Parede Celular/metabolismo , Cristalografia por Raios X , Glucose/metabolismo , Cinética , Redes e Vias Metabólicas , Phaseolus/microbiologia , Folhas de Planta/microbiologia , Difosfato de Uridina/metabolismo , Açúcares de Uridina Difosfato/metabolismo
5.
PLoS One ; 12(9): e0184953, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28931093

RESUMO

The branched-chain sugar apiose was widely assumed to be synthesized only by plant species. In plants, apiose-containing polysaccharides are found in vascularized plant cell walls as the pectic polymers rhamnogalacturonan II and apiogalacturonan. Apiosylated secondary metabolites are also common in many plant species including ancestral avascular bryophytes and green algae. Apiosyl-residues have not been documented in bacteria. In a screen for new bacterial glycan structures, we detected small amounts of apiose in methanolic extracts of the aerobic phototroph Geminicoccus roseus and the pathogenic soil-dwelling bacteria Xanthomonas pisi. Apiose was also present in the cell pellet of X. pisi. Examination of these bacterial genomes uncovered genes with relatively low protein homology to plant UDP-apiose/UDP-xylose synthase (UAS). Phylogenetic analysis revealed that these bacterial UAS-like homologs belong in a clade distinct to UAS and separated from other nucleotide sugar biosynthetic enzymes. Recombinant expression of three bacterial UAS-like proteins demonstrates that they actively convert UDP-glucuronic acid to UDP-apiose and UDP-xylose. Both UDP-apiose and UDP-xylose were detectable in cell cultures of G. roseus and X. pisi. We could not, however, definitively identify the apiosides made by these bacteria, but the detection of apiosides coupled with the in vivo transcription of bUAS and production of UDP-apiose clearly demonstrate that these microbes have evolved the ability to incorporate apiose into glycans during their lifecycles. While this is the first report to describe enzymes for the formation of activated apiose in bacteria, the advantage of synthesizing apiose-containing glycans in bacteria remains unknown. The characteristics of bUAS and its products are discussed.


Assuntos
Alphaproteobacteria/metabolismo , Carboxiliases/metabolismo , Pisum sativum/microbiologia , Açúcares de Uridina Difosfato/biossíntese , Xanthomonas/metabolismo , Alphaproteobacteria/crescimento & desenvolvimento , Filogenia , Xanthomonas/crescimento & desenvolvimento
6.
Plant Physiol ; 175(1): 438-456, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28710128

RESUMO

In search of Botrytis cinerea cell death-inducing proteins, we found a xyloglucanase (BcXYG1) that induced strong necrosis and a resistance response in dicot plants. Expression of the BcXYG1 gene was strongly induced during the first 12 h post inoculation, and analysis of disease dynamics using PathTrack showed that a B. cinerea strain overexpressing BcXYG1 produced early local necrosis, supporting a role of BcXYG1 as an early cell death-inducing factor. The xyloglucanase activity of BcXYG1 was not necessary for the induction of necrosis and plant resistance, as a mutant of BcXYG1 lacking the xyloglucanase enzymatic activity retained both functions. Residues in two exposed loops on the surface of BcXYG1 were found to be necessary for the induction of cell death but not to induce plant resistance. Further analyses showed that BcXYG1 is apoplastic and possibly interacts with the proteins of the plant cell membrane and also that the BcXYG1 cell death-promoting signal is mediated by the leucine-rich repeat receptor-like kinases BAK1 and SOBIR1. Our findings support the role of cell death-inducing proteins in establishing the infection of necrotrophic pathogens and highlight the recognition of fungal apoplastic proteins by the plant immune system as an important mechanism of resistance against this class of pathogens.


Assuntos
Botrytis/enzimologia , Glicosídeo Hidrolases/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal , Transdução de Sinais , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Botrytis/genética , Glicosídeo Hidrolases/genética , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Phaseolus/imunologia , Phaseolus/microbiologia , Doenças das Plantas/imunologia , Folhas de Planta/imunologia , Folhas de Planta/microbiologia , Nicotiana/genética , Nicotiana/imunologia , Nicotiana/microbiologia , Triticum/imunologia , Triticum/microbiologia
7.
J Biol Chem ; 292(18): 7636-7650, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28298443

RESUMO

Bacterial glycan structures on cell surfaces are critical for cell-cell recognition and adhesion and in host-pathogen interactions. Accordingly, unraveling the sugar composition of bacterial cell surfaces can shed light on bacterial growth and pathogenesis. Here, we found that two rare sugars with a 3-C-methyl-6-deoxyhexose structure were linked to spore glycans in Bacillus cereus ATCC 14579 and ATCC 10876. Moreover, we identified a four-gene operon in B. cereus ATCC 14579 that encodes proteins with the following sequential enzyme activities as determined by mass spectrometry and one- and two-dimensional NMR methods: CTP:glucose-1-phosphate cytidylyltransferase, CDP-Glc 4,6-dehydratase, NADH-dependent SAM:C-methyltransferase, and NADPH-dependent CDP-3-C-methyl-6-deoxyhexose 4-reductase. The last enzyme predominantly yielded CDP-3-C-methyl-6-deoxygulose (CDP-cereose) and likely generated a 4-epimer CDP-3-C-methyl-6-deoxyallose (CDP-cillose). Some members of the B. cereus sensu lato group produce CDP-3-C-methyl-6-deoxy sugars for the formation of cereose-containing glycans on spores, whereas others such as Bacillus anthracis do not. Gene knockouts of the Bacillus C-methyltransferase and the 4-reductase confirmed their involvement in the formation of cereose-containing glycan on B. cereus spores. We also found that cereose represented 0.2-1% spore dry weight. Moreover, mutants lacking cereose germinated faster than the wild type, yet the mutants exhibited no changes in sporulation or spore resistance to heat. The findings reported here may provide new insights into the roles of the uncommon 3-C-methyl-6-deoxy sugars in cell-surface recognition and host-pathogen interactions of the genus Bacillus.


Assuntos
Bacillus cereus/metabolismo , Óperon/fisiologia , Polissacarídeos Bacterianos/biossíntese , Esporos Bacterianos/metabolismo , Bacillus cereus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Técnicas de Silenciamento de Genes , Metiltransferases/genética , Metiltransferases/metabolismo , Oxirredutases/genética , Oxirredutases/metabolismo , Polissacarídeos Bacterianos/genética , Esporos Bacterianos/genética
8.
Mol Plant Pathol ; 18(2): 263-275, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-26991954

RESUMO

Botrytis cinerea is a model plant-pathogenic fungus that causes grey mould and rot diseases in a wide range of agriculturally important crops. A previous study has identified two enzymes and corresponding genes (bcdh, bcer) that are involved in the biochemical transformation of uridine diphosphate (UDP)-glucose, the major fungal wall nucleotide sugar precursor, to UDP-rhamnose. We report here that deletion of bcdh, the first biosynthetic gene in the metabolic pathway, or of bcer, the second gene in the pathway, abolishes the production of rhamnose-containing glycans in these mutant strains. Deletion of bcdh or double deletion of both bcdh and bcer has no apparent effect on fungal development or pathogenicity. Interestingly, deletion of the bcer gene alone adversely affects fungal development, giving rise to altered hyphal growth and morphology, as well as reduced sporulation, sclerotia production and virulence. Treatments with wall stressors suggest the alteration of cell wall integrity. Analysis of nucleotide sugars reveals the accumulation of the UDP-rhamnose pathway intermediate UDP-4-keto-6-deoxy-glucose (UDP-KDG) in hyphae of the Δbcer strain. UDP-KDG could not be detected in hyphae of the wild-type strain, indicating fast conversion to UDP-rhamnose by the BcEr enzyme. The correlation between high UDP-KDG and modified cell wall and developmental defects raises the possibility that high levels of UDP-KDG result in deleterious effects on cell wall composition, and hence on virulence. This is the first report demonstrating that the accumulation of a minor nucleotide sugar intermediate has such a profound and adverse effect on a fungus. The ability to identify molecules that inhibit Er (also known as NRS/ER) enzymes or mimic UDP-KDG may lead to the development of new antifungal drugs.


Assuntos
Botrytis/genética , Botrytis/patogenicidade , Deleção de Genes , Redes e Vias Metabólicas/genética , Ramnose/metabolismo , Açúcares de Uridina Difosfato/metabolismo , Difosfato de Uridina/metabolismo , Botrytis/crescimento & desenvolvimento , Botrytis/metabolismo , Carbono/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Fabaceae/efeitos dos fármacos , Fabaceae/imunologia , Fabaceae/microbiologia , Genes Fúngicos , Redes e Vias Metabólicas/efeitos dos fármacos , Micélio/efeitos dos fármacos , Micélio/metabolismo , Fenótipo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Estresse Fisiológico/efeitos dos fármacos
9.
Mol Plant Pathol ; 18(3): 347-362, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-26996832

RESUMO

The initial interaction of a pathogenic fungus with its host is complex and involves numerous metabolic pathways and regulatory proteins. Considerable attention has been devoted to proteins that play a crucial role in these interactions, with an emphasis on so-called effector molecules that are secreted by the invading microbe to establish the symbiosis. However, the contribution of other types of molecules, such as glycans, is less well appreciated. Here, we present a random genetic screen that enabled us to identify 58 novel candidate genes that are involved in the pathogenic potential of the fungal pathogen Verticillium dahliae, which causes vascular wilt diseases in over 200 dicotyledonous plant species, including economically important crops. One of the candidate genes that was identified concerns a putative biosynthetic gene involved in nucleotide sugar precursor formation, as it encodes a putative nucleotide-rhamnose synthase/epimerase-reductase (NRS/ER). This enzyme has homology to bacterial enzymes involved in the biosynthesis of the nucleotide sugar deoxy-thymidine diphosphate (dTDP)-rhamnose, a precursor of L-rhamnose, which has been shown to be required for virulence in several human pathogenic bacteria. Rhamnose is known to be a minor cell wall glycan in fungi and has therefore not been suspected as a crucial molecule in fungal-host interactions. Nevertheless, our study shows that deletion of the VdNRS/ER gene from the V. dahliae genome results in complete loss of pathogenicity on tomato and Nicotiana benthamiana plants, whereas vegetative growth and sporulation are not affected. We demonstrate that VdNRS/ER is a functional enzyme in the biosynthesis of uridine diphosphate (UDP)-rhamnose, and further analysis has revealed that VdNRS/ER deletion strains are impaired in the colonization of tomato roots. Collectively, our results demonstrate that rhamnose, although only a minor cell wall component, is essential for the pathogenicity of V. dahliae.


Assuntos
Carboidratos Epimerases/metabolismo , Proteínas Fúngicas/metabolismo , Doenças das Plantas/microbiologia , Feixe Vascular de Plantas/microbiologia , Verticillium/enzimologia , Verticillium/patogenicidade , Parede Celular/metabolismo , DNA Bacteriano/genética , DNA Intergênico/genética , Deleção de Genes , Interações Hospedeiro-Patógeno/genética , Solanum lycopersicum/microbiologia , Mutagênese Insercional/genética , Raízes de Plantas/microbiologia , Ramnose/metabolismo , Esporos Fúngicos/fisiologia , Nicotiana/microbiologia , Transformação Genética , Difosfato de Uridina/metabolismo , Verticillium/genética , Virulência
10.
Front Plant Sci ; 7: 1580, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27833622

RESUMO

Background: Switchgrass (Panicum virgatum L.) is a C4 perennial prairie grass and a dedicated feedstock for lignocellulosic biofuels. Saccharification and biofuel yields are inhibited by the plant cell wall's natural recalcitrance against enzymatic degradation. Plant hemicellulose polysaccharides such as arabinoxylans structurally support and cross-link other cell wall polymers. Grasses predominately have Type II cell walls that are abundant in arabinoxylan, which comprise nearly 25% of aboveground biomass. A primary component of arabinoxylan synthesis is uridine diphosphate (UDP) linked to arabinofuranose (Araf). A family of UDP-arabinopyranose mutase (UAM)/reversible glycosylated polypeptides catalyze the interconversion between UDP-arabinopyranose (UDP-Arap) and UDP-Araf. Results: The expression of a switchgrass arabinoxylan biosynthesis pathway gene, PvUAM1, was decreased via RNAi to investigate its role in cell wall recalcitrance in the feedstock. PvUAM1 encodes a switchgrass homolog of UDP-arabinose mutase, which converts UDP-Arap to UDP-Araf. Southern blot analysis revealed each transgenic line contained between one to at least seven T-DNA insertions, resulting in some cases, a 95% reduction of native PvUAM1 transcript in stem internodes. Transgenic plants had increased pigmentation in vascular tissues at nodes, but were otherwise similar in morphology to the non-transgenic control. Cell wall-associated arabinose was decreased in leaves and stems by over 50%, but there was an increase in cellulose. In addition, there was a commensurate change in arabinose side chain extension. Cell wall lignin composition was altered with a concurrent increase in lignin content and transcript abundance of lignin biosynthetic genes in mature tillers. Enzymatic saccharification efficiency was unchanged in the transgenic plants relative to the control. Conclusion: Plants with attenuated PvUAM1 transcript had increased cellulose and lignin in cell walls. A decrease in cell wall-associated arabinose was expected, which was likely caused by fewer Araf residues in the arabinoxylan. The decrease in arabinoxylan may cause a compensation response to maintain cell wall integrity by increasing cellulose and lignin biosynthesis. In cases in which increased lignin is desired, e.g., feedstocks for carbon fiber production, downregulated UAM1 coupled with altered expression of other arabinoxylan biosynthesis genes might result in even higher production of lignin in biomass.

11.
J Biol Chem ; 291(41): 21434-21447, 2016 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-27551039

RESUMO

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDP-apiose as the donor. UDP-apiose (UDP-Api) together with UDP-xylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans.


Assuntos
Bryopsida/metabolismo , Carboxiliases/metabolismo , Clorófitas/metabolismo , Evolução Molecular , Proteínas de Plantas/metabolismo , Açúcares de Uridina Difosfato/biossíntese , Bryopsida/genética , Carboxiliases/genética , Parede Celular/genética , Parede Celular/metabolismo , Clorófitas/genética , Proteínas de Plantas/genética , Polissacarídeos/biossíntese , Polissacarídeos/genética , Açúcares de Uridina Difosfato/genética
12.
J Biol Chem ; 291(36): 19051-67, 2016 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-27402849

RESUMO

An exopolysaccharide, produced during the late stage of stationary growth phase, was discovered and purified from the culture medium of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis when strains were grown in a defined nutrient medium that induces biofilm. Two-dimensional NMR structural characterization of the polysaccharide, named pzX, revealed that it is composed of an unusual three amino-sugar sequence repeat of [-3)XylNAc4OAc(α1-3)GlcNAcA4OAc(α1-3)XylNAc(α1-]n The sugar residue XylNAc had never been described previously in any glycan structure. The XNAC operon that contains the genes for the assembly of pzX is also unique and so far has been identified only in members of the Bacillus cereus sensu lato group. Microscopic and biochemical analyses indicate that pzX co-forms during sporulation, so that upon the release of the spore to the extracellular milieu it becomes surrounded by pzX. The relative amounts of pzX produced can be manipulated by specific nutrients in the medium, but rich medium appears to suppress pzX formation. pzX has the following unique characteristics: a surfactant property that lowers surface tension, a cell/spore antiaggregant, and an adherence property that increases spores binding to surfaces. pzX in Bacillus could represent a trait shared by many spore-producing microorganisms. It suggests pzX is an active player in spore physiology and may provide new insights to the successful survival of the B. cereus species in natural environments or in the hosts.


Assuntos
Bacillus anthracis/metabolismo , Bacillus cereus/metabolismo , Bacillus thuringiensis/metabolismo , Polissacarídeos Bacterianos/biossíntese , Bacillus anthracis/química , Bacillus cereus/química , Bacillus thuringiensis/química , Esporos Bacterianos/química , Esporos Bacterianos/metabolismo
13.
PLoS One ; 10(7): e0133790, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26207987

RESUMO

N-acetylquinovosamine (2-acetamido-2,6-di-deoxy-D-glucose, QuiNAc) is a relatively rare amino sugar residue found in glycans of few pathogenic gram-negative bacteria where it can play a role in infection. However, little is known about QuiNAc-related polysaccharides in gram-positive bacteria. In a routine screen for bacillus glycan grown at defined medium, it was surprising to identify a QuiNAc residue in polysaccharides isolated from this gram-positive bacterium. To gain insight into the biosynthesis of these glycans, we report the identification of an operon in Bacillus cereus ATCC 14579 that contains two genes encoding activities not previously described in gram-positive bacteria. One gene encodes a UDP-N-acetylglucosamine C4,6-dehydratase, (abbreviated Pdeg) that converts UDP-GlcNAc to UDP-4-keto-4,6-D-deoxy-GlcNAc (UDP-2-acetamido-2,6-dideoxy-α-D-xylo-4-hexulose); and the second encodes a UDP-4-reductase (abbr. Preq) that converts UDP-4-keto-4,6-D-deoxy-GlcNAc to UDP-N-acetyl-quinovosamine in the presence of NADPH. Biochemical studies established that the sequential Pdeg and Preq reaction product is UDP-D-QuiNAc as determined by mass spectrometry and one- and two-dimensional NMR experiments. Also, unambiguous evidence for the conversions of the dehydratase product, UDP-α-D-4-keto-4,6-deoxy-GlcNAc, to UDP-α-D-QuiNAc was obtained using real-time 1H-NMR spectroscopy and mass spectrometry. The two genes overlap by 4 nucleotides and similar operon organization and identical gene sequences were also identified in a few other Bacillus species suggesting they may have similar roles in the lifecycle of this class of bacteria important to human health. Our results provide new information about the ability of Bacilli to form UDP-QuiNAc and will provide insight to evaluate their role in the biology of Bacillus.


Assuntos
Acetilglucosamina/análogos & derivados , Bacillus cereus/metabolismo , Acetilglucosamina/biossíntese , Proteínas de Bactérias/metabolismo , Oxirredutases/metabolismo , Uridina Difosfato N-Acetilglicosamina/metabolismo
14.
Anal Bioanal Chem ; 407(14): 4053-63, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25804729

RESUMO

Quantification of drug metabolites in biological samples has been of great interest in current pharmaceutical research, since metabolite concentrations and pharmacokinetics can contribute to a better understanding of the toxicity of drug candidates. Two major categories of Phase II metabolites, glucuronide conjugates and glutathione conjugates, may cause significant drug toxicity and therefore require close monitoring at early stages of drug development. In order to achieve high precision, accuracy, and robustness, stable isotope-labeled (SIL) internal standards (IS) are widely used in quantitative bioanalytical methods using liquid chromatography and tandem mass spectrometry (LC-MS/MS), due to their capability of compensating for matrix effects, extraction variations and instrument response fluctuations. However, chemical synthesis of SIL analogues of Phase II metabolites can often be very difficult and require extensive exploratory research, leading to higher cost and significant delays in drug research and development. To overcome these challenges, we have developed a generic method which can synthesize SIL analogues of Phase II metabolites from more available SIL parent drugs or SIL conjugation co-factors, using in vitro biotransformation. This methodology was successfully applied to the bio-generation of SIL glucuronide conjugates and glutathione conjugates. The method demonstrated satisfactory performance in both absolute quantitation and assessment of relative exposure coverage across species in safety tests of drug metabolites (MIST). This generic technique can be utilized as an alternative to chemical synthesis and potentially save time and cost for drug research and development.


Assuntos
Acetaminofen/sangue , Benzimidazóis/sangue , Benzoatos/sangue , Cromatografia Líquida/métodos , Genfibrozila/sangue , Espectrometria de Massas em Tandem/métodos , Acetaminofen/química , Acetaminofen/metabolismo , Analgésicos não Narcóticos/sangue , Analgésicos não Narcóticos/química , Analgésicos não Narcóticos/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/sangue , Bloqueadores do Receptor Tipo 1 de Angiotensina II/química , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Animais , Benzimidazóis/química , Benzimidazóis/metabolismo , Benzoatos/química , Benzoatos/metabolismo , Cromatografia Líquida/economia , Genfibrozila/química , Genfibrozila/metabolismo , Humanos , Hipolipemiantes/sangue , Hipolipemiantes/química , Hipolipemiantes/metabolismo , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Massas em Tandem/economia , Telmisartan
15.
J Biol Chem ; 290(2): 691-704, 2015 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-25414257

RESUMO

CMP-pseudaminic acid is a precursor required for the O-glycosylation of flagellin in some pathogenic Gram-negative bacteria, a process known to be critical in bacterial motility and infection. However, little is known about flagellin glycosylation in Gram-positive bacteria. Here, we identified and functionally characterized an operon, named Bti_pse, in Bacillus thuringiensis israelensis ATCC 35646, which encodes seven different enzymes that together convert UDP-GlcNAc to CMP-pseudaminic acid. In contrast, Gram-negative bacteria complete this reaction with six enzymes. The first enzyme, which we named Pen, converts UDP-d-GlcNAc to an uncommon UDP-sugar, UDP-6-deoxy-D-GlcNAc-5,6-ene. Pen contains strongly bound NADP(+) and has distinct UDP-GlcNAc 4-oxidase, 5,6-dehydratase, and 4-reductase activities. The second enzyme, which we named Pal, converts UDP-6-deoxy-D-GlcNAc-5,6-ene to UDP-4-keto-6-deoxy-L-AltNAc. Pal is NAD(+)-dependent and has distinct UDP-6-deoxy-d-GlcNAc-5,6-ene 4-oxidase, 5,6-reductase, and 5-epimerase activities. We also show here using NMR spectroscopy and mass spectrometry that in B. thuringiensis, the enzymatic product of Pen and Pal, UDP-4-keto-6-deoxy-L-AltNAc, is converted to CMP-pseudaminic acid by the sequential activities of a C4″-transaminase (Pam), a 4-N-acetyltransferase (Pdi), a UDP-hydrolase (Phy), an enzyme (Ppa) that adds phosphoenolpyruvate to form pseudaminic acid, and finally a cytidylyltransferase that condenses CTP to generate CMP-pseudaminic acid. Knowledge of the distinct dehydratase-like enzymes Pen and Pal and their role in CMP-pseudaminic acid biosynthesis in Gram-positive bacteria provides a foundation to investigate the role of pseudaminic acid and flagellin glycosylation in Bacillus and their involvement in bacterial motility and pathogenicity.


Assuntos
Bacillus thuringiensis/genética , Proteínas de Bactérias/genética , Flagelina/metabolismo , Hidroliases/genética , Açúcares Ácidos/metabolismo , Bacillus thuringiensis/enzimologia , Bacillus thuringiensis/patogenicidade , Monofosfato de Citidina/metabolismo , Glucosamina/análogos & derivados , Glucosamina/metabolismo , Glicosilação , Hidroliases/metabolismo , Dados de Sequência Molecular , Açúcares Ácidos/química
16.
J Biol Chem ; 289(51): 35620-32, 2014 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-25368324

RESUMO

Surface glycan switching is often observed when micro-organisms transition between different biotic and abiotic niches, including biofilms, although the advantages of this switching to the organism are not well understood. Bacillus cereus grown in a biofilm-inducing medium has been shown to synthesize an unusual cell wall polysaccharide composed of the repeating subunit →6)Gal(α1-2)(2-R-hydroxyglutar-5-ylamido)Fuc2NAc4N(α1-6)GlcNAc(ß1→, where galactose is linked to the hydroxyglutarate moiety of FucNAc-4-amido-(2)-hydroxyglutarate. The molecular mechanism involved in attaching 2-hydroxyglutarate to 4-amino-FucNAc has not been determined. Here, we show two genes in B. cereus ATCC 14579 encoding enzymes involved in the synthesis of UDP-FucNAc-4-amido-(2)-oxoglutarate (UDP-Yelosamine), a modified UDP-sugar not previously reported to exist. Using mass spectrometry and real time NMR spectroscopy, we show that Bc5273 encodes a C4″-aminotransferase (herein referred to as Pat) that, in the presence of pyridoxal phosphate, transfers the primary amino group of l-Glu to C-4″ of UDP-4-keto-6-deoxy-d-GlcNAc to form UDP-4-amino-FucNAc and 2-oxoglutarate. Pat also converts 4-keto-xylose, 4-keto-glucose, and 4-keto-2-acetamido-altrose to their corresponding UDP-4-amino-sugars. Bc5272 encodes a carboxylate-amine ligase (herein referred as Pyl) that, in the presence of ATP and Mg(II), adds 2-oxoglutarate to the 4-amino moiety of UDP-4-amino-FucNAc to form UDP-Yelosamine and ADP. Pyl is also able to ligate 2-oxoglutarate to other 4-amino-sugar derivatives to form UDP-Yelose, UDP-Solosamine, and UDP-Aravonose. Characterizing the metabolic pathways involved in the formation of modified nucleotide sugars provides a basis for understanding some of the mechanisms used by bacteria to modify or alter their cell surface polysaccharides in response to changing growth and environmental challenges.


Assuntos
Bacillus cereus/metabolismo , Proteínas de Bactérias/metabolismo , Carbamoil-Fosfato Sintase (Amônia)/metabolismo , Transaminases/metabolismo , Açúcares de Uridina Difosfato/biossíntese , Bacillus cereus/genética , Proteínas de Bactérias/genética , Carbamoil-Fosfato Sintase (Amônia)/genética , Sequência de Carboidratos , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Concentração de Íons de Hidrogênio , Cinética , Espectrometria de Massas/métodos , Dados de Sequência Molecular , Espectroscopia de Prótons por Ressonância Magnética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Transaminases/genética
17.
Front Plant Sci ; 3: 92, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22639675

RESUMO

There is compelling evidence showing that the structurally complex pectic polysaccharide rhamnogalacturonan II (RG-II) exists in the primary cell wall as a borate cross-linked dimer and that this dimer is required for the assembly of a functional wall and for normal plant growth and development. The results of several studies have also established that RG-II structure and cross-linking is conserved in vascular plants and that RG-II likely appeared early in the evolution of land plants. Two features that distinguish RG-II from other plant polysaccharides are that RG-II is composed of 13 different glycoses linked to each other by up to 22 different glycosidic linkages and that RG-II is the only polysaccharide known to contain both apiose and aceric acid. Thus, one key event in land plant evolution was the emergence of genes encoding nucleotide sugar biosynthetic enzymes that generate the activated forms of apiose and aceric acid required for RG-II synthesis. Many of the genes involved in the generation of the nucleotide sugars used for RG-II synthesis have been functionally characterized. By contrast, only one glycosyltransferase involved in the assembly of RG-II has been identified. Here we provide an overview of the formation of the activated sugars required for RG-II synthesis and point to the possible cellular and metabolic processes that could be involved in assembling and controlling the formation of a borate cross-linked RG-II molecule. We discuss how nucleotide sugar synthesis is compartmentalized and how this may control the flux of precursors to facilitate and regulate the formation of RG-II.

18.
Database (Oxford) ; 2012: bas013, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22465851

RESUMO

Understanding how cellular metabolism works and is regulated requires that the underlying biochemical pathways be adequately represented and integrated with large metabolomic data sets to establish a robust network model. Genetically engineering energy crops to be less recalcitrant to saccharification requires detailed knowledge of plant polysaccharide structures and a thorough understanding of the metabolic pathways involved in forming and regulating cell-wall synthesis. Nucleotide-sugars are building blocks for synthesis of cell wall polysaccharides. The biosynthesis of nucleotide-sugars is catalyzed by a multitude of enzymes that reside in different subcellular organelles, and precise representation of these pathways requires accurate capture of this biological compartmentalization. The lack of simple localization cues in genomic sequence data and annotations however leads to missing compartmentalization information for eukaryotes in automatically generated databases, such as the Pathway-Genome Databases (PGDBs) of the SRI Pathway Tools software that drives much biochemical knowledge representation on the internet. In this report, we provide an informal mechanism using the existing Pathway Tools framework to integrate protein and metabolite sub-cellular localization data with the existing representation of the nucleotide-sugar metabolic pathways in a prototype PGDB for Populus trichocarpa. The enhanced pathway representations have been successfully used to map SNP abundance data to individual nucleotide-sugar biosynthetic genes in the PGDB. The manually curated pathway representations are more conducive to the construction of a computational platform that will allow the simulation of natural and engineered nucleotide-sugar precursor fluxes into specific recalcitrant polysaccharide(s). Database URL: The curated Populus PGDB is available in the BESC public portal at http://cricket.ornl.gov/cgi-bin/beocyc_home.cgi and the nucleotide-sugar biosynthetic pathways can be directly accessed at http://cricket.ornl.gov:1555/PTR/new-image?object=SUGAR-NUCLEOTIDES.


Assuntos
Bases de Dados Genéticas , Açúcares de Nucleosídeo Difosfato/genética , Açúcares de Nucleosídeo Difosfato/metabolismo , Populus/genética , Populus/metabolismo , Genes de Plantas , Genoma de Planta , Genômica , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Espaço Intracelular/metabolismo , Redes e Vias Metabólicas , Populus/enzimologia
19.
Anal Biochem ; 421(2): 691-8, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22244806

RESUMO

Numerous different nucleotide sugars are used as sugar donors for the biosynthesis of glycans by bacteria, humans, fungi, and plants. However, many of these nucleotide sugars are not available either in their native form or with the sugar portion labeled with a stable or radioactive isotope. Here we demonstrate the use of Escherichia coli metabolically engineered to contain genes that encode proteins that convert monosaccharides into their respective monosaccharide-1-phosphates and subsequently into the corresponding nucleotide sugars. In this system, which we designated "in-microbe", reactions occur within 2 to 4 h and can be used to generate nucleotide sugars in amounts ranging from 5 to 12.5 µg/ml cell culture. We show that the E. coli can be engineered to produce the seldom observed nucleotide sugars UDP-2-acetamido-2-deoxy-glucuronic acid (UDP-GlcNAcA) and UDP-2-acetamido-2-deoxy-xylose (UDP-XylNAc). Using similar strategies, we also engineered E. coli to synthesize UDP-galacturonic acid (UDP-GalA) and UDP-galactose (UDP-Gal). ¹³C- and ¹5N-labeled NDP-sugars are formed using [¹³C] glucose as the carbon source and with [¹5N]NH4Cl as the nitrogen source.


Assuntos
Metabolismo dos Carboidratos , Escherichia coli/metabolismo , Nucleotídeos/metabolismo , Açúcares de Uridina Difosfato/metabolismo , Sequência de Bases , Primers do DNA , Escherichia coli/genética , Engenharia Genética , Açúcares de Uridina Difosfato/genética
20.
J Biol Chem ; 287(2): 879-92, 2012 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-22102281

RESUMO

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.


Assuntos
Genes Fúngicos/fisiologia , Glucose/análogos & derivados , Magnaporthe/metabolismo , Açúcares de Uridina Difosfato/biossíntese , Difosfato de Uridina/análogos & derivados , Bactérias/genética , Bactérias/metabolismo , Sequência de Bases , Glucose/biossíntese , Glucose/genética , Magnaporthe/genética , Magnaporthe/patogenicidade , Dados de Sequência Molecular , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Difosfato de Uridina/biossíntese , Difosfato de Uridina/genética , Açúcares de Uridina Difosfato/genética
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