Demonstration of UDP-glucose dehydrogenase activity in cell extracts of Escherichia coli expressing the pneumococcal cap3A gene required for the synthesis of type 3 capsular polysaccharide

Arrecubieta, Carlos; Garcı́a, Ernesto; López, Rubens

doi:10.1128/jb.178.10.2971-2974.1996

Cited by 32 publications

(23 citation statements)

References 21 publications

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“…We have named this gene ugd. The homologues in the CPS1, CPS2, and CPS3 of S. pneumoniae gene clusters have a high level of similarity (76.3, 90.6, and 61.6% identity, respectively) to the CPS8 gene, and those of CPS1 and CPS3 have been shown by experiment to encode UDP-glucose-6-dehydrogenases (5,53)…”

Section: Bacterialmentioning

confidence: 99%

Molecular Characterization ofStreptococcus pneumoniaeType 4, 6B, 8, and 18C Capsular Polysaccharide Gene Clusters

2001

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Capsular polysaccharide (CPS) is a major virulence factor in Streptococcus pneumoniae. CPS gene clusters of S. pneumoniae types 4, 6B, 8, and 18C were sequenced and compared with those of CPS types 1, 2, 14, 19F, 19A, 23F, and 33F. All have the same four genes at the 5 end, encoding proteins thought to be involved in regulation and export. Sequences of these genes can be divided into two classes, and evidence of recombination between them was observed. Next is the gene encoding the transferase for the first step in the synthesis of CPS. The predicted amino acid sequences of these first sugar transferases have multiple transmembrane segments, a feature lacking in other transferases. Sugar pathway genes are located at the 3 end of the gene cluster. Comparison of the four dTDP-L-rhamnose pathway genes (rml genes) of CPS types 1, 2, 6B, 18C, 19F, 19A, and 23F shows that they have the same gene order and are highly conserved. There is a gradient in the nature of the variation of rml genes, the average pairwise difference for those close to the central region being higher than that for those close to the end of the gene cluster and, again, recombination sites can be observed in these genes. This is similar to the situation we observed for rml genes of O-antigen gene clusters of Salmonella enterica. Our data indicate that the conserved first four genes at the 5 ends and the relatively conserved rml genes at the 3 ends of the CPS gene clusters were sites for recombination events involved in forming new forms of CPS. We have also identified wzx and wzy genes for all sequenced CPS gene clusters by use of motifs.Streptococcus pneumoniae is an important human pathogen causing invasive diseases such as pneumonia, bacteremia, and meningitis. Control of pneumococcal diseases is being complicated by the increasing prevalence of antibiotic-resistant strains and the suboptimal clinical efficacy of existing vaccines. The capsular polysaccharide (CPS) of S. pneumoniae is essential for virulence because it protects S. pneumoniae from the nonspecific defence system of the host organism. All fresh isolates from patients with pneumococcal infection are encapsulated, and spontaneous nonencapsulated derivatives of such strains are almost completely avirulent (7).Ninety different CPS types have been identified so far by immunological and chemical techniques (31). Each has a structurally distinct CPS, composed of repeating oligosaccharide units joined by glycosidic linkages. CPS production requires a complex pathway, including synthesis of the activated monosaccharide nucleotide precursors, sequential transfer of each sugar to a lipid carrier to form repeating CPS units, and subsequent polymerization, export, and attachment to the cell surface. The exception to this is type 3 CPS, which has a very simple CPS structure and appears to be synthesized by a processive transferase (6). Classic genetic studies carried out by Austrian et al. (8) demonstrated that the genes encoding CPS biosynthesis are closely linked on the S. pneumoniae chromoso...

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Section: Bacterialmentioning

confidence: 99%

Molecular Characterization ofStreptococcus pneumoniaeType 4, 6B, 8, and 18C Capsular Polysaccharide Gene Clusters

2001

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“…This enzyme has an NAD-binding domain spanning residues 2 to 29 and an active site spanning residues 251 to 263 (12). The cps3S gene encodes a processive ␤-glycosyltransferase that catalyzes the formation of glycosidic linkages to polymerize UDP-Glc and UDP-GlcA, thereby generating the cellobiuronic acid capsule (4,5). The last two genes in the operon, cps3U and cps3M, have genomic homologues, pgm (26) and galU (18), respectively.…”

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Characterization of In Vitro Biofilm-Associated Pneumococcal Phase Variants of a Clinically Relevant Serotype 3 Clone

2007

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An increasing proportion of children with acute otitis media due to Streptococcus pneumoniae have serotype 3 infections since licensure of the seven-valent pneumococcal conjugate vaccine. These serotype 3 strains are genetically related by molecular subtyping. During otitis media with effusion and recurrent otitis media, biofilms commonly develop. Pneumococcal in vitro biofilms are comprised of phase variants that differ in colony morphology. By using a representative strain of the mucoid serotype 3 clone, rough phase variants with a diverse array of mutations were detected in biofilms formed in vitro. Most phase variants had mutations in the cps3D gene, the first gene of the capsular operon. Eleven had single nucleotide polymorphisms (SNPs) in the cps3D gene, one had an SNP in the ؊10 promoter, and three had large deletions in the cps3D gene. Reversion to the mucoid phenotype was associated with reversion of the mutation in the cps3D gene. Unlike the phase variants detected in the nasopharynx, which have at least 20% of the parental amount of capsule, the in vitro biofilm-associated phase variants had <12% of the parental amount of capsule, as determined by capsule enzyme-linked immunosorbent assays. Using real-time reverse transcription-PCR, we determined that capsule expression in the phase variants was likely regulated at multiple levels. These in vitro phase variation data, which underscore the plasticity of the pneumococcus, need to be confirmed with in vivo analyses of the middle ear mucosa during otitis media.

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“…Group A and C streptococci are mammalian pathogens that use UDPGDH in the synthesis of a capsule composed of hyaluronic acid (a polysaccharide consisting of alternating glucuronic acid and N-acetylglucosamine residues) (9,10). Many of the known strains of Streptococcus pneumoniae also use UDP-glucuronic acid in the construction of their polysaccharide capsule (11), and it has recently been shown that UDPGDH is required for capsule production in S. pneumoniae type 3 (12). The encapsulated Escherichia coli K5 is also known to use the enzyme for a similar purpose (2,13).…”

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Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

Campbell

Sala

Rijn

et al. 1997

Journal of Biological Chemistry

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UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD ؉ -dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDPglucuronate is released last. UDP-xylose was found to be a competitive inhibitor (K i , 2.7 M) of the enzyme. The enzyme is irreversibly inactivated by uridine 5 -diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (k i /K i ) was found to be 2 ؋ 10 3 mM ؊1 min ؊1 . Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5 -diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.The enzyme UDP-glucose dehydrogenase (UDPGDH) 1 catalyzes the NAD ϩ -dependent oxidation of UDP-glucose to UDPglucuronic acid (Fig. 1). It belongs to a small group of dehydrogenases that are able to carry out the 2-fold oxidation of an alcohol to an acid without the release of an aldehyde intermediate (1). Much of the work to date has focused on the properties and mechanism of the beef liver enzyme, and relatively little is known about the enzyme purified from bacterial sources (2-4). In many strains of bacteria that act as human pathogens, UDPGDH provides the UDP-glucuronic acid required for the construction of an antiphagocytic capsular polysaccharide. It is well established that the formation of the capsule is required for virulence (5, 6), and it is thought that the capsule enables the bacteria to evade the host's immune system (7,8). Group A and C streptococci are mammalian pathogens that use UDPGDH in the synthesis of a capsule composed of hyaluronic acid (a polysaccharide consisting of alternating glucuronic acid and N-acetylglucosamine residues) (9, 10). Many of the known strains of Streptococcus pneumoniae also use UDP-glucuronic acid in the construction of their polysaccharide capsule (11), and it has recently been shown that UDPGDH is required for capsule production in S. pneumoniae type 3 (12). The encapsulated Escherichia coli K5 is also known to use the enzyme for a similar purpose (2, 13). The hasB gene that encodes for UDPGDH in group A streptococci (Streptococcus pyogenes) has been cloned and overexpressed in Escherichia coli (14). Its gene product shares 57% sequence identity and 74% sequence similarity with the S. pneumoniae enzyme (15, 16). These properties make th...

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Demonstration of UDP-glucose dehydrogenase activity in cell extracts of Escherichia coli expressing the pneumococcal cap3A gene required for the synthesis of type 3 capsular polysaccharide

Cited by 32 publications

References 21 publications

Molecular Characterization ofStreptococcus pneumoniaeType 4, 6B, 8, and 18C Capsular Polysaccharide Gene Clusters

Molecular Characterization ofStreptococcus pneumoniaeType 4, 6B, 8, and 18C Capsular Polysaccharide Gene Clusters

Characterization of In Vitro Biofilm-Associated Pneumococcal Phase Variants of a Clinically Relevant Serotype 3 Clone

Properties and Kinetic Analysis of UDP-glucose Dehydrogenase from Group A Streptococci

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