Role of endopeptidases in peptidoglycan synthesis mediated by alternative cross-linking enzymes in Escherichia coli.

Bacteria resist to the turgor pressure of the cytoplasm through a net-like macromolecule, the peptidoglycan, made of glycan strands connected via peptides cross-linked by penicillin-binding proteins (PBPs). We recently reported the emergence of ß-lactam resistance resulting from a bypass of PBPs by the YcbB L,D-transpeptidase (LdtD), which form chemically distinct 3→3 cross-links compared to 4→3 formed by PBPs. Here we show that peptidoglycan expansion requires controlled hydrolysis of cross-links and identify among eight endopeptidase paralogues the minimum enzyme complements essential for bacterial growth with 4→3 (MepM) and 3→3 (MepM and MepK) cross-links. Purified Mep endopeptidases unexpectedly displayed a 4→3 and 3→3 dual specificity implying recognition of a common motif in the two cross-link types. Uncoupling of the polymerization of glycan chains from the 4→3 cross-linking reaction was found to facilitate the bypass of PBPs by YcbB. These results illustrate the plasticity of the peptidoglycan polymerization machinery in response to the selective pressure of ß-lactams.

Monitoring Drug-Protein Interactions in the Bacterial Periplasm by Solution Nuclear Magnetic Resonance Spectroscopy.

The rapid spread of antimicrobial resistance across bacterial pathogens poses a serious risk to the efficacy and sustainability of available treatments. This puts pressure on research concerning the development of new drugs. Here, we present an in-cell NMR-based research strategy to monitor the activity of the enzymes located in the periplasmic space delineated by the inner and outer membranes of Gram-negative bacteria. We demonstrate its unprecedented analytical power in monitoring in situ and in real time (i) the hydrolysis of ß-lactams by ß-lactamases, (ii) the interaction of drugs belonging to the ß-lactam family with their essential targets, and (iii) the binding of inhibitors to these enzymes. We show that in-cell NMR provides a powerful analytical tool for investigating new drugs targeting the molecular components of the bacterial periplasm.

In vitro and intracellular activity of vaborbactam combined with ß-lactams against Mycobacterium abscessus.

OBJECTIVES: Mycobacterium abscessus has emerged as an opportunistic pathogen responsible for lung infections, especially in cystic fibrosis patients. In spite of the production of the broad-spectrum ß-lactamase BlaMab, the carbapenem imipenem is recommended in the initial phase of the treatment of pulmonary infections. Here, we determine whether the addition of vaborbactam, a second-generation ß-lactamase inhibitor belonging to the boronate family, improves the activity of ß-lactams against M. abscessus. METHODS: The activity of ß-lactams, alone or in combination with vaborbactam, was evaluated against M. abscessus CIP104536 by determining MICs, time-killing and intramacrophage activity. Kinetic parameters for the inhibition of BlaMab by vaborbactam were determined by spectrophotometry. RESULTS: The combination of vaborbactam (8 mg/L) with ß-lactams decreased more than 8 times the MIC of amoxicillin (from >1024 to 128 mg/L) and 2 times the MICs of meropenem (from 16 to 8 mg/L) and imipenem (from 4 to 2 mg/L). The reduction of the MICs was less than that obtained with avibactam at 4 mg/L for amoxicillin (from >1024 to 16 mg/L, more than 64 times less) and for meropenem (from 16 to 4 mg/L, 4 times less). In vitro and intracellularly, M. abscessus was not killed by the meropenem/vaborbactam combination, in spite of significant in vitro inhibition of BlaMab by vaborbactam. CONCLUSIONS: Inhibition of BlaMab by vaborbactam decreases the MIC of ß-lactams, including that of meropenem. As meropenem/vaborbactam is clinically available, this combination offers an alternative therapeutic option that should be evaluated for the treatment of pulmonary infections due to M. abscessus.

Penicillin-Binding Proteins and Alternative Dual-Beta-Lactam Combinations for Serious Enterococcus faecalis Infections with Elevated Penicillin MICs.

Ampicillin-ceftriaxone has become a first-line therapy for Enterococcus faecalis endocarditis. We characterized the penicillin-binding protein (PBP) profiles of various E. faecalis strains and tested for synergy to better inform beta-lactam options for the treatment of E. faecalis infections. We assessed the affinity of PBP2B from elevated-MIC strain E. faecalis LS4828 compared to type strain JH2-2 using the fluorescent beta-lactam Bocillin FL. We also characterized pbp4 and pbpA structures and PBP4 and PBP2B expression and used deletion and complementation studies to assess the impact of PBP2B on the levels of resistance. We tested penicillin-susceptible and -resistant E. faecalis isolates against ceftriaxone or ceftaroline combinations with other beta-lactams in 24-h time-kill studies. Two penicillin-susceptible strains (JH2-2 and L2052) had identical pbp sequences and similar PBP expression levels. One reduced-penicillin-susceptibility strain (L2068) had pbp sequences identical to those of the susceptible strains but expressed more PBP4. The second decreased-penicillin-susceptibility strain (LS4828) had amino acid substitutions in both PBP4 and PBP2B and expressed increased quantities of both proteins. PBP2B did not appear to contribute significantly to the elevated beta-lactam MICs. No synergy was demonstrable against the strains with both mutated PBPs and increased expression (L2068 and LS4828). Meropenem plus ceftriaxone or ertapenem plus ceftriaxone demonstrated the most consistent synergistic activity. PBP2B of strain LS4828 does not contribute significantly to reduced penicillin susceptibility. Neither the MIC nor the level of PBP expression correlated directly with the identified synergistic combinations when tested at static subinhibitory concentrations.

Partition of tRNAGly isoacceptors between protein and cell-wall peptidoglycan synthesis in Staphylococcus aureus.

The sequence of tRNAs is submitted to evolutionary constraints imposed by their multiple interactions with aminoacyl-tRNA synthetases, translation elongation factor Tu in complex with GTP (EF-Tu•GTP), and the ribosome, each being essential for accurate and effective decoding of messenger RNAs. In Staphylococcus aureus, an additional constraint is imposed by the participation of tRNAGly isoacceptors in the addition of a pentaglycine side chain to cell-wall peptidoglycan precursors by transferases FmhB, FemA and FemB. Three tRNAGly isoacceptors poorly interacting with EF-Tu•GTP and the ribosome were previously identified. Here, we show that these 'non-proteogenic' tRNAs are preferentially recognized by FmhB based on kinetic analyses and on synthesis of stable aminoacyl-tRNA analogues acting as inhibitors. Synthesis of chimeric tRNAs and of helices mimicking the tRNA acceptor arms revealed that this discrimination involves identity determinants exclusively present in the D and T stems and loops of non-proteogenic tRNAs, which belong to an evolutionary lineage only present in the staphylococci. EF-Tu•GTP competitively inhibited FmhB by sequestration of 'proteogenic' aminoacyl-tRNAs in vitro. Together, these results indicate that competition for the Gly-tRNAGly pool is restricted by both limited recognition of non-proteogenic tRNAs by EF-Tu•GTP and limited recognition of proteogenic tRNAs by FmhB.

Modulation of the Specificity of Carbapenems and Diazabicyclooctanes for Selective Activity against Mycobacterium tuberculosis.

Treatment of multidrug-resistant tuberculosis with combinations of carbapenems and ß-lactamase inhibitors carries risks for dysbiosis and for the development of resistances in the intestinal microbiota. Using Escherichia coli producing carbapenemase KPC-2 as a model, we show that carbapenems can be modified to obtain drugs that are inactive against E. coli but retain antitubercular activity. Furthermore, functionalization of the diazabicyclooctanes scaffold provided drugs that did not effectively inactivate KPC-2 but retained activity against Mycobacterium tuberculosis targets.

Synthesis of Carbapenems Containing Peptidoglycan Mimetics and Inhibition of the Cross-Linking Activity of a Transpeptidase of l,d Specificity.

The carbapenem class of ß-lactams has been optimized against Gram-negative bacteria producing extended-spectrum ß-lactamases by introducing substituents at position C2. Carbapenems are currently investigated for the treatment of tuberculosis as these drugs are potent covalent inhibitors of l,d-transpeptidases involved in mycobacterial cell wall assembly. The optimization of carbapenems for inactivation of these unusual targets is sought herein by exploiting the nucleophilicity of the C8 hydroxyl group to introduce chemical diversity. As ß-lactams are structure analogs of peptidoglycan precursors, the substituents were chosen to increase similarity between the drug and the substrate. Fourteen peptido-carbapenems were efficiently synthesized. They were more effective than the reference drug, meropenem, owing to the positive impact of a phenethylthio substituent introduced at position C2 but the peptidomimetics added at position C8 did not further improve the activity. Thus, position C8 can be modified to modulate the pharmacokinetic properties of highly efficient carbapenems.

Click and Release Chemistry for Activity-Based Purification of ß-Lactam Targets.

ß-Lactams, the cornerstone of antibiotherapy, inhibit multiple and partially redundant targets referred to as transpeptidases or penicillin-binding proteins. These enzymes catalyze the essential cross-linking step of the polymerization of cell wall peptidoglycan. The understanding of the mechanisms of action of ß-lactams and of resistance to these drugs requires the development of reliable methods to characterize their targets. Here, we describe an activity-based purification method of ß-lactam targets based on click and release chemistry. We synthesized alkyne-carbapenems with suitable properties with respect to the kinetics of acylation of a model target, the Ldtfm L,D-transpeptidase, the stability of the resulting acylenzyme, and the reactivity of the alkyne for the cycloaddition of an azido probe containing a biotin moiety for affinity purification and a bioorthogonal cleavable linker. The probe provided access to the fluorescent target in a single click and release step.

Copper inhibits peptidoglycan LD-transpeptidases suppressing ß-lactam resistance due to bypass of penicillin-binding proteins.

The peptidoglycan (PG) layer stabilizes the bacterial cell envelope to maintain the integrity and shape of the cell. Penicillin-binding proteins (PBPs) synthesize essential 4-3 cross-links in PG and are inhibited by ß-lactam antibiotics. Some clinical isolates and laboratory strains of Enterococcus faecium and Escherichia coli achieve high-level ß-lactam resistance by utilizing ß-lactam-insensitive LD-transpeptidases (LDTs) to produce exclusively 3-3 cross-links in PG, bypassing the PBPs. In E. coli, other LDTs covalently attach the lipoprotein Lpp to PG to stabilize the envelope and maintain the permeability barrier function of the outermembrane. Here we show that subminimal inhibitory concentration of copper chloride sensitizes E. coli cells to sodium dodecyl sulfate and impair survival upon LPS transport stress, indicating reduced cell envelope robustness. Cells grown in the presence of copper chloride lacked 3-3 cross-links in PG and displayed reduced covalent attachment of Braun's lipoprotein and reduced incorporation of a fluorescent d-amino acid, suggesting inhibition of LDTs. Copper dramatically decreased the minimal inhibitory concentration of ampicillin in E. coli and E. faecium strains with a resistance mechanism relying on LDTs and inhibited purified LDTs at submillimolar concentrations. Hence, our work reveals how copper affects bacterial cell envelope stability and counteracts LDT-mediated ß-lactam resistance.

Ceftazidime-Avibactam Resistance Mediated by the N346Y Substitution in Various AmpC ß-Lactamases.

Chromosomal and plasmid-borne AmpC cephalosporinases are a major resistance mechanism to ß-lactams in Enterobacteriaceae and Pseudomonas aeruginosa The new ß-lactamase inhibitor avibactam effectively inhibits class C enzymes and can fully restore ceftazidime susceptibility. The conserved amino acid residue Asn346 of AmpC cephalosporinases directly interacts with the avibactam sulfonate. Disruption of this interaction caused by the N346Y amino acid substitution in Citrobacter freundii AmpC was previously shown to confer resistance to the ceftazidime-avibactam combination (CAZ-AVI). The aim of this study was to phenotypically and biochemically characterize the consequences of the N346Y substitution in various AmpC backgrounds. Introduction of N346Y into Enterobacter cloacae AmpC (AmpCcloacae), plasmid-mediated DHA-1, and P. aeruginosa PDC-5 led to 270-, 12,000-, and 79-fold decreases in the inhibitory efficacy (k2/Ki ) of avibactam, respectively. The kinetic parameters of AmpCcloacae and DHA-1 for ceftazidime hydrolysis were moderately affected by the substitution. Accordingly, AmpCcloacae and DHA-1 harboring N346Y conferred CAZ-AVI resistance (MIC of ceftazidime of 16 µg/ml in the presence of 4 µg/ml of avibactam). In contrast, production of PDC-5 N346Y was associated with a lower MIC (4 µg/ml) since this ß-lactamase retained a higher inactivation efficacy by avibactam in comparison to AmpCcloacae N346Y. For FOX-3, the I346Y substitution did not reduce the inactivation efficacy of avibactam and the substitution was highly deleterious for ß-lactam hydrolysis, including ceftazidime, preventing CAZ-AVI resistance. Since AmpCcloacae and DHA-1 display substantial sequence diversity, our results suggest that loss of hydrogen interaction between Asn346 and avibactam could be a common mechanism of acquisition of CAZ-AVI resistance.

Inhibition Activity of Avibactam against Nocardia farcinica ß-Lactamase FARIFM10152.

Nocardia farcinica, one of the most frequent pathogenic species responsible for nocardiosis, is characterized by frequent brain involvement and resistance to ß-lactams mediated by a class A ß-lactamase. Kinetic parameters for hydrolysis of various ß-lactams by FARIFM10152 from strain IFM 10152 were determined by spectrophotometry revealing a high catalytic activity (kcat/Km ) for amoxicillin, aztreonam, and nitrocefin. For cephems, kcat/Km was lower but remained greater than 104 M-1 s-1 A low catalytic activity was observed for meropenem, imipenem, and ceftazidime hydrolysis. FARIFM10152 inhibition by avibactam and clavulanate was compared using nitrocefin as a reporter substrate. FARIFM10152 was efficaciously inhibited by avibactam with a carbamoylation rate constant (k2/Ki ) of (1.7 ± 0.3) × 104 M-1 s-1 The 50% effective concentrations (EC50s) of avibactam and clavulanate were 0.060 ± 0.007 µM and 0.28 ± 0.06 µM, respectively. Amoxicillin, cefotaxime, imipenem, and meropenem MICs were measured for ten clinical strains in the presence of avibactam and clavulanate. At 4 µg/ml, avibactam and clavulanate restored amoxicillin susceptibility in all but one of the tested strains but had no effect on the MICs of cefotaxime, imipenem, and meropenem. At 0.4 µg/ml, amoxicillin susceptibility (MIC ≤ 8 µg/ml) was restored for 9 out of 10 strains by avibactam but only for 4 out of 10 strains by clavulanate. Together, these results indicate that avibactam was at least as potent as clavulanate, suggesting that the amoxicillin-avibactam combination could be considered as an option for the rescue treatment of N. farcinica infections if clavulanate cannot be used.

Impact of relebactam-mediated inhibition of Mycobacterium abscessus BlaMab ß-lactamase on the in vitro and intracellular efficacy of imipenem.

OBJECTIVES: Imipenem is one of the recommended ß-lactams for the treatment of Mycobacterium abscessus pulmonary infections in spite of the production of BlaMab ß-lactamase. Avibactam, a second-generation ß-lactamase inhibitor, was previously shown to inactivate BlaMab, but its partner drug, ceftazidime, is devoid of any antibacterial activity against M. abscessus. Here, we investigate whether relebactam, a novel second-generation inhibitor developed in combination with imipenem, improves the activity of this carbapenem against M. abscessus. METHODS: The impact of BlaMab inhibition by relebactam was evaluated by determining MICs, time-kill curves and M. abscessus intracellular proliferation in human macrophages. Kinetic parameters for the inhibition of BlaMab by relebactam were determined by spectrophotometry using nitrocefin as the substrate. The data were compared with those obtained with avibactam. RESULTS: Combination of relebactam (4 mg/L) with ß-lactams led to >128- and 2-fold decreases in the MICs of amoxicillin (from >4096 to 32 mg/L) and imipenem (from 8 to 4 mg/L). In vitro, M. abscessus was not killed by the imipenem/relebactam combination. In contrast, relebactam increased the intracellular activity of imipenem, leading to 88% killing. Relebactam and avibactam similarly potentiated the antibacterial activities of ß-lactams although BlaMab was inactivated 150-fold less effectively by relebactam than by avibactam. CONCLUSIONS: Inhibition of BlaMab by relebactam improves the efficacy of imipenem against M. abscessus in macrophages, indicating that the imipenem/relebactam combination should be clinically considered for the treatment of infections due to M. abscessus.

In Vitro and Intracellular Activity of Imipenem Combined with Tedizolid, Rifabutin, and Avibactam against Mycobacterium abscessus.

Mycobacterium abscessus infections are difficult to treat because of their resistance to many antibiotics. In vitro, tedizolid combined with imipenem displayed a moderate synergistic effect (fractional inhibitory concentration index, 0.41) but no bactericidal activity. Intracellularly, tedizolid 2 µg/ml (half of the MIC), corresponding to the peak serum concentration, increased the efficacy of imipenem at 8 and 32 µg/ml. Addition of avibactam and rifabutin, alone or in combination, improved the activity of the imipenem-tedizolid combination.

Negative Impact of Carbapenem Methylation on the Reactivity of ß-Lactams for Cysteine Acylation as Revealed by Quantum Calculations and Kinetic Analyses.

The Enterococcus faecium l,d-transpeptidase (Ldtfm) mediates resistance to most ß-lactam antibiotics in this bacterium by replacing classical peptidoglycan polymerases. The catalytic Cys of Ldtfm is rapidly acylated by ß-lactams belonging to the carbapenem class but not by penams or cephems. We previously reported quantum calculations and kinetic analyses for Ldtfm and showed that the inactivation profile is not determined by differences in drug binding (KD [equilibrium dissociation constant] values in the 50 to 80 mM range). In this study, we analyzed the reaction of a Cys sulfhydryl with various ß-lactams in the absence of the enzyme environment in order to compare the intrinsic reactivity of drugs belonging to the penam, cephem, and carbapenem classes. For this purpose, we synthesized cyclic Cys-Asn (cCys-Asn) to generate a soluble molecule with a sulfhydryl closely mimicking a cysteine in a polypeptide chain, thereby avoiding free reactive amino and carboxyl groups. Computational studies identified a thermodynamically favored pathway involving a concerted rupture of the ß-lactam amide bond and formation of an amine anion. Energy barriers indicated that the drug reactivity was the highest for nonmethylated carbapenems, intermediate for methylated carbapenems and cephems, and the lowest for penams. Electron-withdrawing groups were key reactivity determinants by enabling delocalization of the negative charge of the amine anion. Acylation rates of cCys-Asn determined by spectrophotometry revealed the same order in the reactivity of ß-lactams. We concluded that the rate of Ldtfm acylation is largely determined by the ß-lactam reactivity with one exception, as the enzyme catalytic pocket fully compensated for the detrimental effect of carbapenem methylation.

Combination of Amino Acid Substitutions Leading to CTX-M-15-Mediated Resistance to the Ceftazidime-Avibactam Combination.

Single amino acid substitutions in the Ω loop of KPC ß-lactamases are known to lead to resistance to the ceftazidime-avibactam combination. Here, we investigate this mechanism of resistance in CTX-M enzymes, which are the most widely spread extended-spectrum ß-lactamases worldwide. Nine single amino acid polymorphisms were identified in the Ω loop of the 172 CTX-M sequences present in the Lahey database of ß-lactamases. The corresponding modifications were introduced in CTX-M-15 by site-directed mutagenesis. None of the nine substitutions was associated with ceftazidime-avibactam resistance in Escherichia coli TOP10. However, two substitutions led to 4-fold (P167S) and 16-fold (L169Q) increases in the MIC of ceftazidime. We determined whether these substitutions favor the in vitro selection of mutants resistant to ceftazidime-avibactam. The selection provided mutants for the L169Q substitution but not for the P167S substitution or for the parental enzyme CTX-M-15. Resistance to the drug combination (MIC of ceftazidime, 16 µg/ml in the presence of 4 µg/ml of avibactam) resulted from the acquisition of the S130G substitution by CTX-M-15 L169Q. Purified CTX-M-15 with the two substitutions, L169Q and S130G, was only partially inhibited by avibactam at concentrations as high as 50,000 µM but retained ceftazidime hydrolysis activity with partially compensatory decreases in kcat and Km These results indicate that emergence of resistance to the ceftazidime-avibactam combination requires more than one mutation in most CTX-M-encoding genes. Acquisition of resistance could be restricted to rare variants harboring predisposing polymorphisms such as Q at position 169 detected in a single naturally occurring CTX-M enzyme (CTX-M-93).

In Vitro and Intracellular Activity of Imipenem Combined with Rifabutin and Avibactam against Mycobacterium abscessus.

Repurposing drugs may be useful as an add-on in the treatment of Mycobacterium abscessus pulmonary infections, which are particularly difficult to cure. M. abscessus naturally produces a ß-lactamase, BlaMAb, which is inhibited by avibactam. The recommended regimens include imipenem, which is hydrolyzed by BlaMAb and used without any ß-lactamase inhibitor. Here, we determine whether the addition of rifabutin improves the activity of imipenem alone or in combination with avibactam against M. abscessus CIP104536. Rifabutin at 16 µg/ml was only bacteriostatic (MIC of 4 µg/ml) and was moderately synergistic in combination with imipenem (fractional inhibitory concentration [FIC] index of 0.38). Addition of rifabutin (16 µg/ml) moderately increased killing by a low (8 µg/ml) but not by a high (32 µg/ml) concentration of imipenem. Addition of avibactam (4 µg/ml) did not further increase killing by the former combination. In infected macrophages, rifabutin (16 µg/ml) increased the activity of imipenem at 8 and 32 µg/ml, achieving 3- and 100-fold reductions in the numbers of intracellular bacteria, respectively. Avibactam (16 µg/ml) improved killing by imipenem at 8 µg/ml. A 5-fold killing was obtained for a triple combination comprising avibactam (16 µg/ml) and therapeutically achievable doses of imipenem (8 µg/ml) and rifabutin (1 µg/ml). These results indicate that the imipenem-rifabutin combination should be further considered for the treatment of M. abscessus pulmonary infections in cystic fibrosis patients and that addition of a ß-lactamase inhibitor might improve its efficacy. Mechanistically, the impact of BlaMAb inhibition by avibactam on antibiotic activity was assessed by comparing CIP104536 and a ß-lactamase-deficient derivative.

Peptidoglycan Cross-Linking Activity of l,d-Transpeptidases from Clostridium difficile and Inactivation of These Enzymes by ß-Lactams.

In most bacteria, the essential targets of ß-lactam antibiotics are the d,d-transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4→3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3→3 cross-links generated by l,d-transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l,d-transpeptidase paralogues LdtCd1, LdtCd2, and LdtCd3, which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. LdtCd2 and LdtCd3 catalyzed the formation of 3→3 cross-links (l,d-transpeptidase activity), the hydrolysis of the C-terminal d-Ala residue of the disaccharide-tetrapeptide substrate (l,d-carboxypeptidase activity), and the exchange of the C-terminal d-Ala for d-Met. LdtCd1 displayed only l,d-carboxypeptidase activity. Mass spectrometry analyses indicated that LdtCd1 and LdtCd2 were acylated by ß-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of LdtCd3 by these ß-lactams was not detected. The acylation efficacy of LdtCd1 and LdtCd2 was higher for the carbapenems (480 to 6,600 M-1 s-1) than for ampicillin and ceftriaxone (3.9 to 82 M-1 s-1). In contrast, the efficacy of the hydrolysis of ß-lactams by LdtCd1 and LdtCd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that LdtCd1 and LdtCd2 are inactivated only by ß-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.

Synthesis of Lipid-Carbohydrate-Peptidyl-RNA Conjugates to Explore the Limits Imposed by the Substrate Specificity of Cell Wall Enzymes on the Acquisition of Drug Resistance.

Conjugation of RNA with multiple partners to obtain mimics of complex biomolecules is limited by the identification of orthogonal reactions. Here, lipid-carbohydrate-peptidyl-RNA conjugates were obtained by post-functionalization reactions, solid-phase synthesis, and enzymatic steps, to generate molecules mimicking the substrates of FmhB, an essential peptidoglycan synthesis enzyme of Staphylococcus aureus. Mimics of Gly-tRNAGly and lipid intermediate II (undecaprenyl-diphospho-disaccharide-pentapeptide) were combined in a single "bi-substrate" inhibitor (IC50 =56 nm). The synthetic route was exploited to generate substrates and inhibitors containing d-lactate residue (d-Lac) instead of d-Ala at the C-terminus of the pentapeptide stem, a modification responsible for vancomycin resistance in the enterococci. The substitution impaired recognition of peptidoglycan precursors by FmhB. The associated fitness cost may account for limited dissemination of vancomycin resistance genes in S. aureus.

Critical Impact of Peptidoglycan Precursor Amidation on the Activity of l,d-Transpeptidases from Enterococcus faecium and Mycobacterium tuberculosis.

The bacterial cell wall peptidoglycan contains unusual l- and d-amino acids assembled as branched peptides. Insight into the biosynthesis of the polymer has been hampered by limited access to substrates and to suitable polymerization assays. Here we report the full synthesis of the peptide stem of peptidoglycan precursors from two pathogenic bacteria, Enterococcus faecium and Mycobacterium tuberculosis, and the development of a sensitive post-derivatization assay for their cross-linking by l,d-transpeptidases. Access to series of stem peptides showed that amidation of free carboxyl groups is essential for optimal enzyme activity, in particular the amidation of diaminopimelate (DAP) residues for the cross-linking activity of the l,d-transpeptidase LdtMt2 from M. tuberculosis. Accordingly, construction of a conditional mutant established the essential role of AsnB indicating that this DAP amidotransferase is an attractive target for the development of anti-mycobacterial drugs.

Synthesis of Avibactam Derivatives and Activity on ß-Lactamases and Peptidoglycan Biosynthesis Enzymes of Mycobacteria.

There is a renewed interest for ß-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus because their ß-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction is reported. The amoxicillin-DBO combinations were active, indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 µg mL-1 for both species). Mechanistically, ß-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, the latter compounds were investigated as inhibitors of l,d-transpeptidases (Ldts), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of Ldts by S-carbamoylation indicating that optimization of DBOs for Ldt inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria.