Staphylococcus aureus Cap5O has UDP-ManNAc dehydrogenase activity and is essential for capsule expression

Area of Science:

• Microbiology and Molecular Biology
• Bacterial Pathogenesis
• Glycobiology

Background:

• Staphylococcus aureus serotype 5 capsular polysaccharide (CP5) is a key virulence factor.
• CP5 synthesis involves multiple genes, including cap5A through cap5P.
• Cap5P, a 2-epimerase, converts UDP-N-acetyl glucosamine (UDP-GlcNAc) to UDP-N-acetylmannosamine (UDP-ManNAc).

Purpose of the Study:

• To identify and characterize the enzyme responsible for oxidizing UDP-ManNAc to UDP-N-acetylmannosaminuronic acid (UDP-ManNAcA).
• To investigate the role of the UDP-ManNAc dehydrogenase in CP5 synthesis and bacterial virulence.

Main Methods:

• Cloning and expression of the S. aureus cap5O gene in E. coli.
• Purification of recombinant Cap5O protein and in vitro enzymatic assays using UDP-GlcNAc, Cap5P, NAD+, and a reducing agent.
• Quantification of enzymatic activity by measuring NADH formation and confirmation of UDP-ManNAcA product via gas chromatography-mass spectroscopy.
• Construction of a cap5O deletion mutant in S. aureus and assessment of its virulence in mouse models.

Main Results:

• The S. aureus cap5O gene encodes a UDP-ManNAc dehydrogenase that catalyzes the conversion of UDP-ManNAc to UDP-ManNAcA.
• Purified Cap5O exhibited UDP-ManNAc dehydrogenase activity, confirmed by product analysis.
• A cap5O deletion mutant was CP5 negative and exhibited reduced virulence in mouse models of kidney infection and subcutaneous abscess formation compared to the wild-type strain.

Conclusions:

• The S. aureus cap5O gene is essential for the synthesis of CP5.
• The CP5 capsule produced by S. aureus promotes staphylococcal virulence.
• Cap5O-mediated UDP-ManNAcA production is critical for capsule biosynthesis and subsequent pathogenesis.