Characterization of cpsF and its product CMP‐N‐acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli

Haft, Rachel F.; Wessels, Michael R.; Mebane, Mary Fisk; Conaty, Neil; Rubens, Craig E.

doi:10.1046/j.1365-2958.1996.395931.x

Cited by 43 publications

(32 citation statements)

References 42 publications

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“…The genes neu III B, neu III C, neu III D, and neu III A encode the final four proteins in the locus. Neu III D (formerly called CpsE) and Neu III A have been previously described (8,15). These four genes are most similar to the neuABCD genes from the gram-negative organism E. coli K1 (6) and the siaCAB genes of N. meningitidis serogroup B (11), which are responsible for the synthesis and activation of sialic acid for capsule production in these species.…”

Section: Resultsmentioning

confidence: 99%

“…These four genes are most similar to the neuABCD genes from the gram-negative organism E. coli K1 (6) and the siaCAB genes of N. meningitidis serogroup B (11), which are responsible for the synthesis and activation of sialic acid for capsule production in these species. We previously demonstrated that neu III A encodes a CMP-NeuNAc synthetase, catalyzing the activation of sialic acid with CMP (15). Homologues of Neu III B are NeuNAc synthetases (2).…”

Section: Resultsmentioning

confidence: 99%

“…The asialo mutants had a common transposon insertion site in a gene approximately 9 kb downstream of cps III E, which was designated cpsF (now referred to as neu III A). The neu III A gene was shown to encode a CMP-N-acetylneuraminic acid synthetase (15,46) which complemented an Escherichia coli K1 neuA mutant, restoring synthesis of the polysialic acid capsule (15). A gene homologous to E. coli K1 neuD (formerly designated cpsE and now called neu III D) was found adjacent to the 5Ј end of neu III A (8).…”

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The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon

et al. 2000

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Streptococcus agalactiae is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes, cps III FGHIJKL, neu III B, and neu III C. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (cpsIII) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their cpsIII loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated cpsIII sequences were compared to those of the S. agalactiae cpsIa locus, and the primary difference between the operons was found to reside in cps III H, the putative CPS polymerase gene. Expression of cps III H in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that cps Ia H and cps III H encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.Group B streptococci (GBS) (Streptococcus agalactiae) are the leading cause of serious bacterial infections (bacteremia, pneumonia, and meningitis) in newborns, causing two to three cases per 1,000 live births (47). An indispensable GBS virulence determinant is the production of a type-specific capsular polysaccharide (CPS), which prevents the deposition of host complement factor C3b and inhibits opsonophagocytosis (45). Nine distinct capsular serotypes, Ia, Ib, and II to VIII, have been identified (54), and their chemical compositions and structures have been determined (16-19, 51, 55, 56, 58). Type Ia, Ib, II to V, and VII CPS consist of the monosaccharides glucose, galactose, N-acetylglucosamine, and Nacetylneuraminic acid. Serotypes VI and VIII lack N-acetylglucosamine, and type VIII contains rhamnose (19). Although serotypes Ia, III, and V are currently the most common isolates from the United States associated with early-onset disease (within 1 week of birth), comprising 82% of isolates (27), type III GBS are the most prevalent isolates associated with neonatal disease (5).We previously identified a region of the GBS chromo...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon

et al. 2000

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“…The Neu III C gene of GBS, however, complements ⌬neuC. It has also been shown that the Neu III A gene (previously designated cpsF) of GBS can complement a mutation in the E. coli K1 neuA gene, which encodes the CMP-N-acetylneuraminic acid synthetase (13). Indeed, a plasmid containing the four GBS genes involved in sialic acid synthesis, pDC128, successfully complemented strains with mutations in neuD, neuB, neuC, or neuA, the E. coli K1 region This result indicates that the sialic acid biosynthetic pathways for incorporation into capsular polysaccharides are probably identical in these two pathogens.…”

Section: Discussionmentioning

confidence: 99%

The NeuC Protein ofEscherichia coliK1 Is a UDPN-Acetylglucosamine 2-Epimerase

et al. 2004

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The K1 capsule is an essential virulence determinant of Escherichia coli strains that cause meningitis in neonates. Biosynthesis and transport of the capsule, an ␣-2,8-linked polymer of sialic acid, are encoded by the 17-kb kps gene cluster. We deleted neuC, a K1 gene implicated in sialic acid synthesis, from the chromosome of EV36, a K-12-K1 hybrid, by allelic exchange. Exogenously added sialic acid restored capsule expression to the deletion strain (⌬neuC), confirming that NeuC is necessary for sialic acid synthesis. The deduced amino acid sequence of NeuC showed similarities to those of UDP-N-acetylglucosamine (GlcNAc) 2-epimerases from both prokaryotes and eukaryotes. The NeuC homologue from serotype III Streptococcus agalactiae complements ⌬neuC. We cloned the neuC gene into an intein expression vector to facilitate purification. We demonstrated by paper chromatography that the purified neuC gene product catalyzed the formation of

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“…GBS type III strain COH1, initially isolated from a newborn infant with sepsis, and S. pneumoniae serotype-2 strain D39 have been described previously (13)(14)(15)(16). All strains were grown on blood agar plates (REMEL).…”

Section: Generation Of Ethanol-inactivated Gbs and S Pneumoniaementioning

confidence: 99%

c-Jun Kinase Is a Critical Signaling Molecule in a Neonatal Model of Group B Streptococcal Sepsis

Kenzel¹,

Mancuso

Malley

et al. 2006

The Journal of Immunology

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Group B streptococcus (GBS) is the major cause of sepsis in newborn infants. In vitro, inactivated GBS stimulates macrophages to produce inflammatory proteins via the TLR adapter protein MyD88. Furthermore, inflammatory cytokine release in response to GBS greatly exceeds that following stimulation with pneumococci. In this study, we attempted to unravel signaling events that are involved in GBS-, but not Streptococcus pneumoniae-stimulated phagocytes to identify molecular targets for adjunctive sepsis therapy. We found that inactivated GBS and S. pneumoniae differed in the activation of the MAPK JNK, but not IκB kinase. Furthermore, JNK was essential for the transcriptional activation of inflammatory cytokine genes in response to GBS. Inhibition of JNK by the anthrapyrazolone SP600125 abrogated GBS-induced cytokine formation via an AP-1- and NF-κB-dependent mechanism without impairing antibacterial properties such as phagocytosis of GBS and the formation of intracellular oxidative species. In contrast, inhibition of the MAPK p38 impaired both antibacterial processes. In a neonatal mouse model of GBS sepsis SP600125 inhibited the inflammatory response and improved survival. In conclusion, JNK plays a major role in the inflammatory, but not in the direct antibacterial response to inactivated GBS, and may thus serve as a rational target for an adjunctive GBS sepsis therapy.

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Characterization of cpsF and its product CMP‐N‐acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli

Cited by 43 publications

References 42 publications

The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon

The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon

The NeuC Protein ofEscherichia coliK1 Is a UDPN-Acetylglucosamine 2-Epimerase

c-Jun Kinase Is a Critical Signaling Molecule in a Neonatal Model of Group B Streptococcal Sepsis

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