Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

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.publishe

Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase

<div><p>To combat the threat of antibiotic-resistant Gram-negative bacteria, novel agents that circumvent established resistance mechanisms are urgently needed. Our approach was to focus first on identifying bioactive small molecules followed by chemical lead prioritization and target identification. Within this annotated library of bioactives, we identified a small molecule with activity against efflux-deficient <i>Escherichia coli</i> and other sensitized Gram-negatives. Further studies suggested that this compound inhibited DNA replication and selection for resistance identified mutations in a subunit of <i>E</i>. <i>coli</i> DNA gyrase, a type II topoisomerase. Our initial compound demonstrated weak inhibition of DNA gyrase activity while optimized compounds demonstrated significantly improved inhibition of <i>E</i>. <i>coli</i> and <i>Pseudomonas aeruginosa</i> DNA gyrase and caused cleaved complex stabilization, a hallmark of certain bactericidal DNA gyrase inhibitors. Amino acid substitutions conferring resistance to this new class of DNA gyrase inhibitors reside exclusively in the TOPRIM domain of GyrB and are not associated with resistance to the fluoroquinolones, suggesting a novel binding site for a gyrase inhibitor.</p></div

Mapping of the MRL-770/423/1082 resistance mutations onto a model of <i>E</i>. <i>coli</i> DNA gyrase (GyrBA fusion dimer) suggests a novel inhibitor interaction domain.

<p>Amino acids in the GyrB domain (light blue) where MRL-770/MRL-423 resistant primary mutations reside are rendered in stick form. The GyrA domains of monomers 1 and 2 are colored in light green and green respectively, S83 and N87 of GyrA monomer 1 are shown in stick form. The two ciprofloxacin molecules are displayed in CPK and carbon atoms colored in yellow. Nicked DNA is shown in orange and Mn⁺² ions are in purple.</p

Antibacterial small molecules targeting the conserved TOPRIM domain of DNA gyrase - Fig 3

<p><b>Dose-dependent inhibition of <i>E</i>. <i>coli</i> (A) and <i>P</i>. <i>aeruginosa</i> (B) DNA gyrase by MRL-423 and MRL-1082 respectively</b>. (C) Dose-dependent stabilization of cleavage complex formation in <i>E</i>. <i>coli</i> DNA gyrase by MRL-423. Relaxed, closed circular substrate (rel.), linear (lin.), and super-coiled (sc.) DNA species are indicated to the left of each gel image.</p

Chemical structures of novel DNA gyrase inhibitors and reference compounds.

<p>Chemical structures of novel DNA gyrase inhibitors and reference compounds.</p

Antibacterial susceptibility of <i>E</i>. <i>coli gyrB</i> mutants.

<p>Antibacterial susceptibility of <i>E</i>. <i>coli gyrB</i> mutants.</p

Inhibition of macromolecule synthesis in <i>E</i>. <i>coli</i>.

<p>(A) Dose-dependent, selective inhibition of DNA synthesis by MRL-770 in <i>E</i>. <i>coli</i> JL553. (B) Dose-dependent, selective inhibition of DNA synthesis by ciprofloxacin. (C) Schematic representation of the <i>E</i>. <i>coli</i> GyrB mutations conferring resistance to MRL-770 series compounds. For each mutant listed in the first column a black-filled cell identifies the mutation site and amino acid substitution (top row). GYR106-108 mutants were isolated following reselection for higher-level resistance to MRL-423. Red cells designate second site amino acid location and substitution acquired in the reselected mutants.</p

Mapping of MnaA LOF mutations into the MnaA crystal structure reveal key residues for substrate binding site stability and charge.

<p>(A) Overall MRSA COL MnaA crystal structure. The molecular surface is shown in grey. The protein is represented as a cartoon. In all figures one monomer is consistently colored in orange and the other in cyan, and the bound UDP molecules are shown as sticks, methyl groups colored in light blue. Nitrogen, oxygen and phosphor atoms are in blue, red or orange, respectively. (B,C,D,E) Comparison with the <i>M</i>. <i>jannaschii</i> structure in “opened” form (PDB 3NEQ) or “closed” form (PDB 3NES). Both structures are represented as ribbons, one monomer at a time, and UDP as sticks. (B) and (C) compares the opened form, in grey, with each monomer, while the superposition is with the closed form, in (D) and (E), drawn in purple. The RMS deviation in Cα positions are 1.6Å for 262 atoms, 1.6Å for 256 atoms, 1.5Å for 321 atoms, and 1.3Å for 336 atoms, for the superpositions in cartoon (B), (C), (D) and (E), respectively. (F) Mapping MRSE LOF mutants. Eight mutation sites are mapped onto the X-ray crystal structure of UDP bound MRSA COL MnaA. The allosteric site ligand UDP-GlcNAc was taken from the structure of UDP-GlcNAc bound <i>B</i>. <i>anthracis</i> 2-epimerase (PDB ID 3BEO). UDP and UDP-GlcNAc are displayed as thin lines with the carbon atoms colored in light blue. One monomer of MnaA dimer is colored in cyan and the other in white. The mutation sites are highlighted in stick. The carbon atoms of the wild-type residues are colored in yellow; those of the mutant residues are in green. (G) Mapping MRSA <i>mnaA</i> LOF mutants. LOF mutations isolated in MRSA COL MnaA are highlighted. All coloring as in (C), but for simplicity, only the original sequence is shown.</p

Genetic inactivation of <i>mnaA</i> in methicillin-resistant <i>Staphylococci</i> restores β-lactam susceptibility.

<p>Genetic inactivation of <i>mnaA</i> in methicillin-resistant <i>Staphylococci</i> restores β-lactam susceptibility.</p