Sophie Magnet

Towards a new tuberculosis drug: pyridomycin – nature's isoniazid

Tuberculosis, a global threat to public health, is becoming untreatable due to widespread drug resistance to frontline drugs such as the InhA‐inhibitor isoniazid. Historically, by inhibiting highly vulnerable targets, natural products have been an important source of antibiotics including potent anti‐tuberculosis agents. Here, we describe pyridomycin, a compound produced by Dactylosporangium fulvum with specific cidal activity against mycobacteria. By selecting pyridomycin‐resistant mutants of Mycobacterium tuberculosis, whole‐genome sequencing and genetic validation, we identified the NADH‐dependent enoyl‐ (Acyl‐Carrier‐Protein) reductase InhA as the principal target and demonstrate that pyridomycin inhibits mycolic acid synthesis in M. tuberculosis. Furthermore, biochemical and structural studies show that pyridomycin inhibits InhA directly as a competitive inhibitor of the NADH‐binding site, thereby identifying a new, druggable pocket in InhA. Importantly, the most frequently encountered isoniazid‐resistant clinical isolates remain fully susceptible to pyridomycin, thus opening new avenues for drug development.

Covalent attachment of proteins to peptidoglycan

Bacterial surface proteins are key players in host-symbiont or host-pathogen interactions. How these proteins are targeted and displayed at the cell surface are challenging issues of both fundamental and clinical relevance. While surface proteins of Gram-negative bacteria are assembled in the outer membrane, Gram-positive bacteria predominantly utilize their thick cell wall as a platform to anchor their surface proteins. This surface display involves both covalent and noncovalent interactions with either the peptidoglycan or secondary wall polymers such as teichoic acid or lipoteichoic acid. This review focuses on the role of enzymes that covalently link surface proteins to the peptidoglycan, the well-known sortases in Gram-positive bacteria, and the recently characterized l,d-transpeptidases in Gram-negative bacteria.

Identification of the l,d-Transpeptidases Responsible for Attachment of the Braun Lipoprotein to Escherichia coli Peptidoglycan

The L,D-transpeptidase Ldt(fm) catalyzes peptidoglycan cross-linking in beta-lactam-resistant mutant strains of Enterococcus faecium. Here, we show that in Escherichia coli Ldt(fm) homologues are responsible for the attachment of the Braun lipoprotein to murein, indicating that evolutionarily related domains have been tailored to use muropeptides or proteins as acyl acceptors in the L,D-transpeptidation reaction.

Identification of the l,d-Transpeptidases for Peptidoglycan Cross-Linking in Escherichia coli

Three active-site cysteine L,D-transpeptidases can individually anchor the Braun lipoprotein to the Escherichia coli peptidoglycan. We show here that two additional enzymes of the same family form peptide bonds between the third residues of peptidoglycan stems, generating meso-DAP(3)-->meso-DAP(3) unusual cross-links. This activity partially replaces the D,D-transpeptidase activity of penicillin-binding proteins.

Specificity of L,D-Transpeptidases from Gram-positive Bacteria Producing Different Peptidoglycan Chemotypes

We report here the first direct assessment of the specificity of a class of peptidoglycan cross-linking enzymes, the l,d-transpeptidases, for the highly diverse structure of peptidoglycan precursors of Gram-positive bacteria. The lone functionally characterized member of this new family of active site cysteine peptidases, Ldtfm from Enterococcus faecium, was previously shown to bypass the d,d-transpeptidase activity of the classical penicillin-binding proteins leading to high level cross-resistance to glycopeptide and β-lactam antibiotics. Ldtfm homologues from Bacillus subtilis (LdtBs) and E. faecalis (Ldtfs) were found here to cross-link their cognate disaccharide-peptide subunits containing meso-diaminopimelic acid (mesoDAP3) and l-Lys3-l-Ala-l-Ala at the third position of the stem peptide, respectively, instead of l-Lys3-d-iAsn in E. faecium. Ldtfs differed from Ldtfm and LdtBs by its capacity to hydrolyze the l-Lys3-d-Ala4 bond of tetrapeptide (l,d-carboxypeptidase activity) and pentapeptide (l,d-endopeptidase activity) stems, in addition to the common cross-linking activity. The three enzymes were specific for their cognate acyl acceptors in the cross-linking reaction. In contrast to Ldtfs, which was also specific for its cognate acyl donor, Ldtfm tolerated substitution of l-Lys3-d-iAsn by l-Lys3-l-Ala-l-Ala. Likewise, LdtBs tolerated substitution of mesoDAP3 by l-Lys3-d-iAsn and l-Lys3-l-Ala-l-Ala in the acyl donor. Thus, diversification of the structure of peptidoglycan precursors associated with speciation has led to a parallel evolution of the substrate specificity of the l,d-transpeptidases affecting mainly the recognition of the acyl acceptor. Blocking the assembly of the side chain could therefore be used to combat antibiotic resistance involving l,d-transpeptidases.

Quantitative high-performance liquid chromatography analysis of the pool levels of undecaprenyl phosphate and its derivatives in bacterial membranes

Undecaprenyl phosphate is the essential lipid involved in the transport of hydrophilic motifs across the bacterial membranes during the synthesis of cell wall polymers such as peptidoglycan. A HPLC procedure was developed for the quantification of undecaprenyl phosphate and its two derivatives, undecaprenyl pyrophosphate and undecaprenol. During the exponential growth phase, the pools of undecaprenyl phosphate and undecaprenyl pyrophosphate were ca. 75 and 270 nmol/g of cell dry weight, respectively, in Escherichia coli, and ca. 50 and 150 nmol/g, respectively, in Staphylococcus aureus. Undecaprenol was detected in S. aureus (70 nmol/g), but not in E. coli (<1 nmol/g).

Colicin M hydrolyses branched lipids II from Gram-positive bacteria.

Lipids II found in some Gram-positive bacteria were prepared in radioactive form from l-lysine-containing UDP-MurNAc-pentapeptide. The specific lateral chains of Enterococcus faecalis, Enterococcus faecium and Staphylococcus aureus (di-L-alanine, D-isoasparagine, and pentaglycine, respectively) were introduced by chemical peptide synthesis using the Fmoc chemistry. The branched nucleotides obtained were converted into the corresponding lipids II by enzymatic synthesis using the MraY and MurG enzymes. All of the lipids were hydrolysed by Escherichia coli colicin M at approximately the same rate as the meso-diaminopimelate-containing lipid II found in Gram-negative bacteria, thereby opening the way to the use of this enzyme as a broad spectrum antibacterial agent.

Sigma Factor F Does Not Prevent Rifampin Inhibition of RNA Polymerase or Cause Rifampin Tolerance in Mycobacterium tuberculosis

The tolerance of Mycobacterium tuberculosis to antituberculosis drugs is a major reason for the lengthy therapy needed to treat a tuberculosis infection. Rifampin is a potent inhibitor of RNA polymerase (RNAP) in vivo but has been shown to be less effective against stationary-phase bacteria. Sigma factor F is associated with bacteria entering stationary phase and has been proposed to impact rifampin activity. Here we investigate whether RNAP containing SigF is more resistant to rifampin inhibition in vitro and whether overexpression of sigF renders M. tuberculosis more tolerant to rifampin. Real-time and radiometric in vitro transcription assays revealed that rifampin equally inhibits transcription by RNAP containing sigma factors SigA and SigF, therefore ruling out the hypothesis that SigF may be responsible for increased resistance of the enzyme to rifampin in vitro. In addition, overexpression or deletion of sigF did not alter rifampin susceptibility in axenic cultures of M. tuberculosis, indicating that SigF does not affect rifampin tolerance in vivo.

Discovery of the first inhibitors of bacterial enzyme d-aspartate ligase from Enterococcus faecium (Aslfm)

The D-aspartate ligase of Enterococcus faecium (Aslfm) is an attractive target for the development of narrow-spectrum antibacterial agents that are active against multidrug-resistant E. faecium. Although there is currently little available information regarding the structural characteristics of Aslfm, we exploited the knowledge that this enzyme belongs to the ATP-grasp superfamily to target its ATP binding site. In the first design stage, we synthesized and screened a small library of known ATP-competitive inhibitors of ATP-grasp enzymes. A series of amino-oxazoles derived from bacterial biotin carboxylase inhibitors showed low micromolar activity. The most potent inhibitor compound 12, inhibits Aslfm with a Ki value of 2.9 μM. In the second design stage, a validated ligand-based pharmacophore modeling approach was used, taking the newly available inhibition data of an initial series of compounds into account. Experimental evaluation of the virtual screening hits identified two novel structural types of Aslfm inhibitors with 7-amino-9H-purine (18) and 7-amino-1H-pyrazolo[3,4-d]pyrimidine (30 and 34) scaffolds, and also with Ki values in the low micromolar range. Investigation the inhibitors modes of action confirmed that these compounds are competitive with respect to the ATP molecule. The binding of inhibitors to the target enzyme was also studied using isothermal titration calorimetry (ITC). Compounds 6, 12, 18, 30 and 34 represent the first inhibitors of Aslfm reported to date, and are an important step forward in combating infections due to E. faecium.

The Enterococcus hirae Mur-2 enzyme displays N-acetylglucosaminidase activity

Enterococcus hirae produces two autolytic enzymes named Mur‐1 and Mur‐2, both previously described as N‐acetylmuramidases. We used tandem mass spectrometry to show that Mur‐2 in fact displays N‐acetylglucosaminidase activity. This result reveals that Mur‐2 and its counterparts studied to date, which are members of glycosyl hydrolase family 73 from the CAZy (Carbohydrate‐Active enZyme) database, display the same catalytic activity.