title: Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets.
abstract: Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Paul A
authors.initials: PA
authors.lastname: Mann
authors.affiliation: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.|German Centre for Infection Research (DZIF), partner site Bonn-Cologne, Bonn, Germany.
authors.forename: Anna
authors.initials: A
authors.lastname: Müller
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Kerstin A
authors.initials: KA
authors.lastname: Wolff
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Thierry
authors.initials: T
authors.lastname: Fischmann
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Laboratory of Bacterial Cell Biology, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Patricia
authors.initials: P
authors.lastname: Reed
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Yan
authors.initials: Y
authors.lastname: Hou
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry, University of Bonn, Bonn, Germany.
authors.forename: Wenjin
authors.initials: W
authors.lastname: Li
authors.affiliation: PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical Chemistry, University of Bonn, Bonn, Germany.
authors.forename: Christa E
authors.initials: CE
authors.lastname: Müller
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Jianying
authors.initials: J
authors.lastname: Xiao
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Murgolo
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Xinwei
authors.initials: X
authors.lastname: Sher
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Todd
authors.initials: T
authors.lastname: Mayhood
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Payal R
authors.initials: PR
authors.lastname: Sheth
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Asra
authors.initials: A
authors.lastname: Mirza
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Marc
authors.initials: M
authors.lastname: Labroli
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Li
authors.initials: L
authors.lastname: Xiao
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Mark
authors.initials: M
authors.lastname: McCoy
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Gill
authors.affiliation: Laboratory of Bacterial Cell Biology, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal.
authors.forename: Mariana G
authors.initials: MG
authors.lastname: Pinho
authors.affiliation: Institute for Pharmaceutical Microbiology, University of Bonn, Bonn, Germany.|German Centre for Infection Research (DZIF), partner site Bonn-Cologne, Bonn, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Schneider
authors.affiliation: Merck Research Laboratories, Kenilworth New Jersey, United States of America.
authors.forename: Terry
authors.initials: T
authors.lastname: Roemer
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development; D002473:Cell Wall / Q000378:metabolism; D018360:Crystallography, X-Ray; D004195:Disease Models, Animal; D055624:Methicillin-Resistant Staphylococcus aureus; D051379:Mice; D008826:Microbial Sensitivity Tests; D008856:Microscopy, Fluorescence; D019906:Nuclear Magnetic Resonance, Biomolecular; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*; D013203:Staphylococcal Infections / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism*; D013212:Staphylococcus epidermidis / Q000378:metabolism*; D013682:Teichoic Acids / Q000096:biosynthesis*
chemical_list: D001426:Bacterial Proteins; D013682:Teichoic Acids; D019998:Racemases and Epimerases