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Published on: September 5, 2013
Identification and molecular cloning of a chondroitin synthase from Pasteurella multocida type F
P L DeAngelis1, A J Padgett-McCue
1Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA.
Abstract:
Pasteurella multocida Type F, the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor. It was reported that the capsule was removed by treating microbes with chondroitin AC lyase. We found by acid hydrolysis that the polysaccharide contained galactosamine and glucuronic acid. We molecularly cloned a Type F polysaccharide synthase and characterized its enzymatic activity. The 965-residue enzyme, called P. multocida chondroitin synthase (pmCS), is 87% identical at the nucleotide and the amino acid level to the hyaluronan synthase, pmHAS, from P. multocida Type A. A recombinant Escherichia coli-derived truncated, soluble version of pmCS (residues 1-704) was shown to catalyze the repetitive addition of sugars from UDP-GalNAc and UDP-GlcUA to chondroitin oligosaccharide acceptors in vitro. Other structurally related sugar nucleotide precursors did not substitute in the elongation reaction. Polymer molecules composed of approximately 10(3) sugar residues were produced, as measured by gel filtration chromatography. The polysaccharide synthesized in vitro was sensitive to the action of chondroitin AC lyase but resistant to the action of hyaluronan lyase. This is the first report identifying a glycosyltransferase that forms a polysaccharide composed of chondroitin disaccharide repeats, [beta(1,4)GlcUA-beta(1,3)GalNAc](n). In analogy to known hyaluronan synthases, a single polypeptide species, pmCS, possesses both transferase activities.
Insights
Pasteurella multocida Type F produces a chondroitin capsule, a virulence factor. Researchers cloned and characterized the chondroitin synthase (pmCS) enzyme responsible for its synthesis.
Area of Science:
- Microbiology
- Glycobiology
- Bacterial Pathogenesis
Background:
- Pasteurella multocida Type F, a fowl cholera pathogen, possesses an extracellular polysaccharide capsule.
- This capsule is considered a potential virulence factor and can be removed by chondroitin AC lyase treatment.
- Previous analysis indicated the polysaccharide contains galactosamine and glucuronic acid.
Purpose of the Study:
- To identify and characterize the enzyme responsible for synthesizing the chondroitin capsule in P. multocida Type F.
- To elucidate the enzymatic mechanism and substrate specificity of the novel polysaccharide synthase.
Main Methods:
- Molecular cloning of the Type F polysaccharide synthase gene.
- Expression and characterization of a truncated, soluble version of the enzyme (pmCS) in recombinant Escherichia coli.
- In vitro enzymatic assays using UDP-sugars and oligosaccharide acceptors.
- Analysis of synthesized polysaccharide structure and enzyme sensitivity to lyases.
Main Results:
- The cloned enzyme, P. multocida chondroitin synthase (pmCS), shares high sequence identity with hyaluronan synthase.
- Recombinant pmCS catalyzes the in vitro synthesis of chondroitin polysaccharide from UDP-GalNAc and UDP-GlcUA.
- The synthesized polysaccharide consists of [beta(1,4)GlcUA-beta(1,3)GalNAc](n) repeats and is sensitive to chondroitin AC lyase.
Conclusions:
- This study identifies the first known glycosyltransferase, pmCS, responsible for synthesizing a chondroitin polysaccharide.
- The findings reveal that a single polypeptide species possesses both transferase activities, similar to hyaluronan synthases.
- Understanding pmCS function provides insights into bacterial capsule synthesis and potential virulence mechanisms.
