title: Staphylococcus aureus Cap5O has UDP-ManNAc dehydrogenase activity and is essential for capsule expression.
abstract: The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a repeating unit composed of (-->4)-3-O-acetyl-beta-D-ManNAcA-(1-->4)-alpha-L-FucNAc (1-->3)-beta-D-FucNAc-(1-->)(n). Sixteen chromosomal genes (cap5A through cap5P) are involved in the synthesis of CP5. We recently demonstrated that Cap5P, a 2-epimerase, catalyzes the conversion of UDP-N-acetyl glucosamine (UDP-GlcNAc) to UDP-N-acetylmannosamine (UDP-ManNAc). In this study, we show that UDP-ManNAc is oxidized to UDP-N-acetylmannosaminuronic acid (UDP-ManNAcA) by a UDP-ManNAc dehydrogenase encoded by S. aureus cap5O. We expressed Cap5O in Escherichia coli and purified the recombinant protein. The UDP-ManNAc dehydrogenase activity of purified Cap5O was assessed by incubating Cap5P and UDP-GlcNAc (to produce UDP-ManNAc), together with Cap5O, NAD(+), and a reducing agent. Enzymatic activity was quantitated indirectly by measuring the increase in absorbance at 340 nm resulting from NADH formation. The product of the reaction was confirmed as UDP-ManNAcA by gas chromatography-mass spectroscopy. A cap5O mutation, created by deletion of 727 bp in the 5' end of the gene, was introduced by allelic replacement into S. aureus Reynolds, rendering it CP5 negative. Mice inoculated intravenously or subcutaneously with the wild-type strain Reynolds had greater numbers of S. aureus recovered from their kidneys (P = 0.019) or their subcutaneous abscesses (P = 0.0018), respectively, than did animals inoculated with the cap5O mutant. The results of this study indicate that S. aureus cap5O is essential for capsule production and that capsule promotes staphylococcal virulence in mouse models of abscess formation.
authors.affiliation: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: M
authors.initials: M
authors.lastname: Portolés
authors.forename: K B
authors.initials: KB
authors.lastname: Kiser
authors.forename: N
authors.initials: N
authors.lastname: Bhasin
authors.forename: K H
authors.initials: KH
authors.lastname: Chan
authors.forename: J C
authors.initials: JC
authors.lastname: Lee
mesh_terms: D000818:Animals; D016667:Bacterial Capsules / Q000502:physiology*; D001426:Bacterial Proteins / Q000302:isolation & purification / Q000502:physiology*; D002237:Carbohydrate Dehydrogenases / Q000502:physiology*; D008297:Male; D051379:Mice; D008813:Mice, Inbred ICR; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000472:pathogenicity; D014774:Virulence
chemical_list: D001426:Bacterial Proteins; C107803:Cap5 protein, Staphylococcus; D002237:Carbohydrate Dehydrogenases; C023215:UDP-N-acetylmuramate dehydrogenase