RESUMEN
The Gram-negative bacterium Pseudomonas aeruginosa simultaneously expresses two O-antigenic glycoforms. While the O-specific antigen (OSA) is variable in composition, the common polysaccharide antigen (CPA) is highly conserved and is composed of a homopolymer of D-rhamnose (D-Rha) in trisaccharide repeating units [D-Rhaα1-2-D-Rhaα1-3-D-RhaÉ1-3]n. We have previously reported that α3-D-Rha-transferase WbpZ transfers a D-Rha residue from GDP-D-Rha to D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl. Genes encoding two more D-Rha-transferases are found in the O antigen gene cluster (wbpX and wbpY). In this study we showed that WbpX and WbpY recombinantly expressed in E. coli differ in their donor and acceptor specificities and have properties of GT-B folded enzymes of the GT4 glycosyltransferase family. NMR spectroscopic analysis of the WbpY reaction product showed that WbpY transferred one D-Rha residue in α1-3 linkage to synthetic D-Rhaα1-3-D-GlcNAcα-O-PO3-PO3-(CH2)11-O-phenyl acceptor. WbpX synthesized several products that contained D-Rha in both α1-2 and α1-3 linkages. Mass spectrometry indicated that the mixture of WbpX and WbpY efficiently catalyzed the synthesis of D-Rha oligomers in a non-processive mechanism. Since O antigens are virulence factors, these findings open the door to advancing technology for antibacterial drug discovery and vaccine development.
Asunto(s)
Antígenos O , Pseudomonas aeruginosa , Escherichia coli/genética , Glicosiltransferasas/genética , Lipopolisacáridos/química , Antígenos O/química , Pseudomonas aeruginosa/genética , RamnosaRESUMEN
The O antigen of enterotoxigenic Escherichia coli serotype O117 consists of repeating units with the structure [-D-GalNAcß1-3-L-Rhaα1-4-D-Glcα1-4-D-Galß1-3-D-GalNAcα1-4]n. A related structure is found in E. coli O107 where Glc is replaced by a GlcNAc residue. The O117 and O107 antigen biosynthesis gene clusters are homologous and reveal the presence of four putative glycosyltransferase (GT) genes, wclW, wclX, wclY and wclZ, but the enzymes have not yet been biochemically characterized. We show here that the His6-tagged WclY protein expressed in E. coli Lemo21(DE3) cells is an α1,4-Glc-transferase that transfers Glc to the Gal moiety of Galß1-3GalNAcα-OPO3-PO3-phenoxyundecyl as a specific acceptor and that the diphosphate moiety of this acceptor is required. WclY utilized UDP-Glc, TDP-Glc, ADP-Glc, as well as UDP-GlcNAc, UDP-Gal or UDP-GalNAc as donor substrates, suggesting an unusual broad donor specificity. Activity using GDP-Man suggested the presence of a novel Man-transferase in Lemo21(DE3) cells. Mutations of WclY revealed that both Glu residues of the Ex7E motif within the predicted GT domain are essential for activity. High GlcNAc-transferase (GlcNAc-T) activities of WclY were created by mutating Arg194 to Cys. A triple mutant identical to WclY in E. coli O107 was identified as an α1,4 GlcNAc-T. The characterization of WclY opens the door for the development of antibacterial approaches.
Asunto(s)
Proteínas de Escherichia coli/genética , Glucosiltransferasas/genética , Biología Computacional , Glucosiltransferasas/metabolismoRESUMEN
A series of compounds was designed and synthesized having two imidazolium rings separated by a polymethylene spacer and having alkyl substituents on each of the imidazolium rings. The compounds were assayed for their effects on the activity of galactosyltransferase WbwC, and also on the growth of Gram-negative and Gram-positive bacteria, as well as human cells. The inhibition observed on enzyme activities and cell growth was dependent on the total number of carbons in the spacer and the alkyl substituents on the imidazolium rings. These readily synthesized, achiral compounds have potential as antimicrobial and antiseptic agents.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Escherichia coli/antagonistas & inhibidores , Galactosiltransferasas/antagonistas & inhibidores , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Imidazoles/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Proteínas de Escherichia coli/metabolismo , Galactosiltransferasas/metabolismo , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Grampositivas/crecimiento & desarrollo , Humanos , Imidazoles/síntesis química , Imidazoles/química , Estructura Molecular , Sales (Química)/síntesis química , Sales (Química)/química , Sales (Química)/farmacología , Relación Estructura-ActividadRESUMEN
The biosynthesis of O antigenic polysaccharides in enteric bacteria from nucleoside diphosphate sugars (donor substrates) is catalyzed by the corresponding glycosyltransferases and proceeds through the intermediate formation of undecaprenyl diphosphate sugars (acceptor substrates). To study this process, a chemical synthesis of the compounds having the natural structure or their modified analogs is necessary. The phosphoroimidazolidate method is a universal method for synthesis of lipid diphosphate disaccharides containing 2-acetamido-2-deoxyglycosyl residue at the reducing end of the disaccharide moiety and 11-phenoxyundecyl residue as lipid fragment of the molecule. We report here protocols to synthesize the disaccharides P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-α-D-rhamnopyranosyl-α-D-glucopyranosyl) diphosphate [D-Rha(α1-3)-D-GlcNAcα-PP-PhU, Compound 1] and P1-(11-phenoxyundecyl)-P2-(2-acetamido-2-deoxy-3-O-ß-D-galactopyranosyl-α-D-galactopyranosyl) diphosphate [D-Gal(ß1-3)-D-GalNAcα-PP-PhU, Compound 6]. We describe the procedures for identification and structure estimation of compounds by TLC, NMR, and MS. We also include the biochemical testing of Compound 6 with α2,3-sialyltransferase WbwA from Escherichia coli O104.