Biosynthesis of soluble capsule precursors in <em>Staphylococcus aureus</em>

Abstract

Most invasive pathogens produce polysaccharide capsules, which play an essential role in conferring protection against host immune defense. In the case of <em>Staphylococcus aureus</em>, most clinical isolates are encapsulated, and inhibition of capsule biosynthesis may offer a valuable strategy for novel anti-infective treatment. Despite their importance for pathogenicity, the biochemistry underlying the assembly of staphylococcal capsular polysaccharides is not fully understood. Three nucleotide-activated sugar precursors, synthesized in the cytoplasm of the cell, are required for <em>S. aureus</em> capsule production. In this thesis, the UDP-N-acetyl-D-glucosamine<br /> 4,6-dehydratases CapD and CapE of <em>S. aureus</em> serotype 5, which catalyze the first steps in the synthesis of the soluble capsule precursors UDP-N-acetyl-D-fucosamine (UDP-D-FucNAc) and UDP-N-acetyl-L-fucosamine (UDP-L-FucNAc), respectively, were purified and characterized. CapD is an integral membrane protein and was obtained for the first time in a purified, active form. Using a new, robust and sensitive capillary electrophoresis-based method for detection of the soluble capsule precursors, kinetic studies for CapD and CapE were performed, and a compound library was screened in search for enzyme inhibitors. Several active compounds were identified and characterized, including suramin (IC₅₀ at CapE 1.82 μM) and ampicillin (IC₅₀ at CapD of 40.1 μM). Moreover, the cell wall precursors UDP-<em>N</em>-acetylmuramyl-pentapeptide and lipid II appear to function as inhibitors of CapD enzymatic activity, suggesting an integrated mechanism of regulation for cell envelope biosynthesis pathways in <em>S. aureus.</em> A further aim of the present study was the elucidation of the pathway underlying UDP-D-FucNAc biosynthesis in <em>S. aureus. </em>Using the purified recombinant enzymes CapD and CapN, enzymatic in vitro synthesis of the soluble capsule precursor UDP-D-FucNAc was achieved for the first time. Furthermore, the role of the tyrosine kinase complex CapAB in the regulation of capsular polysaccharide production was investigated. The capsule biosynthetic enzyme CapE was identified as regulatory target of the CapAB kinase complex, and it was shown that tyrosine phosphorylation enhances CapE catalytic activity in vitro. Besides substantial progress in the understanding of capsule biosynthesis and the underlying regulation, the established <em>in vitro</em> systems provide the molecular basis for screening for potential anti-virulence agents.