Staphylococcus aureus Cap50 has UDP-ManNAc dehydrogenase activity and is essential for capsule expression

The Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) has a re peating 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 demonstrat ed that Cap5P, a 2-epimerase, catalyzes the conversion of UDP-N-acetyl gluc osamine (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. T he UDP-ManNAc dehydrogenase activity of purified Cap5O was assessed by incu bating Cap5P and UDP-GlcNAc (to produce UDP-ManNAc), together with Cap5O, N AD(+), and a reducing agent. Enzymatic activity was quantitated indirectly by measuring the increase in absorbance at 340 nm resulting from NADH forma tion. The product of the reaction was confirmed as UDP-ManNAcA by gas chrom atography-mass spectroscopy. A cap5O mutation, created by deletion of 727 b p in the 5' end of the gene, was introduced by allelic replacement into S. aureus Reynolds, rendering it CP5 negative. Mice inoculated intravenously o r subcutaneously with the wild-type strain Reynolds had greater numbers of S. aureus recovered from their kidneys (P = 0.019) or their subcutaneous ab scesses (P = 0.0018), respectively, than did animals inoculated with the ca p5O mutant. The results of this study indicate that S. aureus cap5O is esse ntial for capsule production and that capsule promotes staphylococcal virul ence in mouse models of abscess formation.