title: Analysis of the Staphylococcus aureus capsule biosynthesis pathway in vitro: characterization of the UDP-GlcNAc C6 dehydratases CapD and CapE and identification of enzyme inhibitors.
abstract: Polysaccharide capsules significantly contribute to virulence of invasive pathogens, and inhibition of capsule biosynthesis may offer a valuable strategy for novel anti-infective treatment. We purified and characterized the enzymes CapD and CapE of the Staphylococcus aureus serotype 5 biosynthesis cluster, which catalyze the first steps in the synthesis of the soluble capsule precursors UDP-D-FucNAc and UDP-L-FucNAc, respectively. CapD is an integral membrane protein and was obtained for the first time in a purified, active form. A capillary electrophoresis (CE)-based method applying micellar electrokinetic chromatography (MEKC) coupled with UV detection at 260 nm was developed for functional characterization of the enzymes using a fused-silica capillary, electrokinetic injection, and dynamic coating with polybrene at pH 12.4. The limits of detection for the CapD and CapE products UDP-2-acetamido-2,6-dideoxy-α-D-xylo-hex-4-ulose and UDP-2-acetamido-2,6-dideoxy-β-L-arabino-hex-4-ulose, respectively, were below 1 μM. Using this new, robust and sensitive method we performed kinetic studies for CapD and CapE and screened a compound library 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 40.1 μM). Furthermore, the cell wall precursors UDP-D-MurNAc-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 S. aureus. Corroborating the in vitro findings, staphylococcal cells grown in the presence of subinhibitory concentrations of ampicillin displayed drastically reduced CP production. Our studies contribute to a profound understanding of the capsule biosynthesis in pathogenic bacteria. This approach may lead to the identification of novel anti-virulence and antibiotic drugs.
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, Bonn 53121, Germany.
authors.forename: Wenjin
authors.initials: W
authors.lastname: Li
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology-Pharmaceutical Microbiology Section, University of Bonn, Bonn 53105, Germany.
authors.forename: Hannah
authors.initials: H
authors.lastname: Ulm
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology-Pharmaceutical Microbiology Section, University of Bonn, Bonn 53105, Germany.
authors.forename: Marvin
authors.initials: M
authors.lastname: Rausch
authors.affiliation: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA; Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Li
authors.affiliation: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Katie
authors.initials: K
authors.lastname: O'Riordan
authors.affiliation: Channing Laboratory, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Jean C
authors.initials: JC
authors.lastname: Lee
authors.affiliation: Institute of Medical Microbiology, Immunology and Parasitology-Pharmaceutical Microbiology Section, University of Bonn, Bonn 53105, Germany; German Centre for Infection Research (DZIF), Partner site Bonn-Cologne, Bonn, Germany. Electronic address: tanja@microbiology-bonn.de.
authors.forename: Tanja
authors.initials: T
authors.lastname: Schneider
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry I, University of Bonn, An der Immenburg 4, Bonn 53121, Germany. Electronic address: christa.mueller@uni-bonn.de.
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
mesh_terms: D000667:Ampicillin / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000378:metabolism; D016667:Bacterial Capsules / Q000378:metabolism*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D004791:Enzyme Inhibitors / Q000302:isolation & purification*; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D007700:Kinetics; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D013498:Suramin / Q000378:metabolism
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D013498:Suramin; D000667:Ampicillin; D006836:Hydro-Lyases; C441972:wbpM protein, Pseudomonas aeruginosa
keywords: Antibiotics; Capillary electrophoresis; Capsule; Cell wall; Enzyme inhibitors; Enzyme kinetics; Staphylococcus aureus