Exploration of the role of the penicillin binding protein 2c (Pbp2c) in inducible ß-lactam resistance in Corynebacteriaceae.

Six genes encoding putative high molecular weight penicillin-binding proteins (Pbp) are present in the genome of the ß-lactam-resistant strain Corynebacterium jeikeium K411. In this study, we show that pbp2c, one of these six genes, is present in resistant strains of Corynebacteriaceae but absent from sensitive strains. The molecular study of the pbp2c locus from C. jeikeium and its heterologous expression in Corynebacterium glutamicum allowed us to show that Pbp2c confers high levels of ß-lactam resistance to the host and is under the control of a ß-lactam-induced regulatory system encoded by two adjacent genes, jk0410 and jk0411. The detection of this inducible resistance may require up to 48 h of incubation, particularly in Corynebacterium amycolatum. Finally, the Pbp2c-expressing strains studied were resistant to all the ß-lactam antibiotics tested, including carbapenems, ceftaroline, and ceftobiprole.

Amino-acyl tXNA as inhibitors or amino acid donors in peptide synthesis.

Xenobiotic nucleic acids (XNAs) offer tremendous potential for synthetic biology, biotechnology, and molecular medicine but their ability to mimic nucleic acids still needs to be explored. Here, to study the ability of XNA oligonucleotides to mimic tRNA, we synthesized three L-Ala-tXNAs analogs. These molecules were used in a non-ribosomal peptide synthesis involving a bacterial Fem transferase. We compared the ability of this enzyme to use amino-acyl tXNAs containing 1',5'-anhydrohexitol (HNA), 2'-fluoro ribose (2'F-RNA) and 2'-fluoro arabinose. L-Ala-tXNA containing HNA or 2'F-RNA were substrates of the Fem enzyme. The synthesis of peptidyl-XNA and the resolution of their structures in complex with the enzyme show the impact of the XNA on protein binding. For the first time we describe functional tXNA in an in vitro assay. These results invite to test tXNA also as substitute for tRNA in translation.

Synthesis of RNA-cofactor conjugates and structural exploration of RNA recognition by an m6A RNA methyltransferase.

Chemical synthesis of RNA conjugates has opened new strategies to study enzymatic mechanisms in RNA biology. To gain insights into poorly understood RNA nucleotide methylation processes, we developed a new method to synthesize RNA-conjugates for the study of RNA recognition and methyl-transfer mechanisms of SAM-dependent m6A RNA methyltransferases. These RNA conjugates contain a SAM cofactor analogue connected at the N6-atom of an adenosine within dinucleotides, a trinucleotide or a 13mer RNA. Our chemical route is chemo- and regio-selective and allows flexible modification of the RNA length and sequence. These compounds were used in crystallization assays with RlmJ, a bacterial m6A rRNA methyltransferase. Two crystal structures of RlmJ in complex with RNA-SAM conjugates were solved and revealed the RNA-specific recognition elements used by RlmJ to clamp the RNA substrate in its active site. From these structures, a model of a trinucleotide bound in the RlmJ active site could be built and validated by methyltransferase assays on RlmJ mutants. The methyl transfer by RlmJ could also be deduced. This study therefore shows that RNA-cofactor conjugates are potent molecular tools to explore the active site of RNA modification enzymes.

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.

Traceless Staudinger Ligation for Bioconjugation of RNA.

Staudinger ligation is an attractive bioorthogonal reaction for use in studying biomolecules due to its capacity to form a native amide bond between a tag and a biomolecule. Here, we explore the traceless variant of the Staudinger ligation for 3'-end modification of oligoribonucleotides. The procedure involves (i) synthesis of phosphine-containing reactive groups, affinity purification tags, or photoactivatable benzophenone probe, (ii) synthesis of 2'-azido dinucleotides and 24-nt RNA, and (iii) traceless Staudinger ligation experiments. Each phosphine was characterized by 1 H, 13 C, and 31 P NMR and high-resolution spectrometry and the functionalized nucleotides were characterized by LC/MS. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of phosphines Basic Protocol 2: Synthesis of dinucleotides 4 and 5 Basic Protocol 3: Synthesis of modified RNA 6 Basic Protocol 4: Traceless Staudinger reactions on a dinucleotide Basic Protocol 5: Traceless Staudinger reaction on RNA.

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.

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.

Phosphine-Mediated Bioconjugation of the 3'-End of RNA.

Staudinger ligation is an attractive bio-orthogonal reaction that has been widely used to tag proteins, carbohydrates, and nucleic acids. Here, we explore the traceless variant of the Staudinger ligation for 3'-end modification of oligoribonucleotides. An azido-containing dinucleotide was used to study the ligation. Nine phosphines containing reactive groups, affinity purification tags, or photoswitch probes have been successfully obtained. The corresponding modified dinucleotides were synthesized and characterized by LC/MS. Mechanistic interpretations of the reaction are proposed, in particular, the unprecedented formation of an oxazaphospholane nucleotide derivative, which was favored by the vicinal position of 2'-N3 and 3'-OH functional groups on the terminal ribose has been observed. The post-functionalization of a 24-nt RNA with a photoactivable tag is also reported.

Diazabicyclooctane Functionalization for Inhibition of ß-Lactamases from Enterobacteria.

Second-generation ß-lactamase inhibitors containing a diazabicyclooctane (DBO) scaffold restore the activity of ß-lactams against pathogenic bacteria, including those producing class A, C, and D enzymes that are not susceptible to first-generation inhibitors containing a ß-lactam ring. Here, we report optimization of a synthetic route to access triazole-containing DBOs and biological evaluation of a series of 17 compounds for inhibition of five ß-lactamases representative of enzymes found in pathogenic Gram-negative bacteria. A strong correlation (Spearman coefficient of 0.87; p = 4.7 × 10-21) was observed between the inhibition efficacy of purified ß-lactamases and the potentiation of ß-lactam antibacterial activity, indicating that DBO functionalization did not impair penetration. In comparison to reference DBOs, avibactam and relebactam, our compounds displayed reduced efficacy, likely due to the absence of hydrogen bonding with a conserved asparagine residue at position 132. This was partially compensated for by additional interactions involving certain triazole substituents.

Structural insight into YcbB-mediated beta-lactam resistance in Escherichia coli.

The bacterial cell wall plays a crucial role in viability and is an important drug target. In Escherichia coli, the peptidoglycan crosslinking reaction to form the cell wall is primarily carried out by penicillin-binding proteins that catalyse D,D-transpeptidase activity. However, an alternate crosslinking mechanism involving the L,D-transpeptidase YcbB can lead to bypass of D,D-transpeptidation and beta-lactam resistance. Here, we show that the crystallographic structure of YcbB consists of a conserved L,D-transpeptidase catalytic domain decorated with a subdomain on the dynamic substrate capping loop, peptidoglycan-binding and large scaffolding domains. Meropenem acylation of YcbB gives insight into the mode of inhibition by carbapenems, the singular antibiotic class with significant activity against L,D-transpeptidases. We also report the structure of PBP5-meropenem to compare interactions mediating inhibition. Additionally, we probe the interaction network of this pathway and assay beta-lactam resistance in vivo. Our results provide structural insights into the mechanism of action and the inhibition of L,D-transpeptidation, and into YcbB-mediated antibiotic resistance.

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.

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.

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.

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 tRNA analogues containing a terminal ribose locked in the South conformation to study tRNA-dependent enzymes.

We report here the synthetic route of two constrained dinucleotides and the determination of the sugar puckering by NMR analyses of the starting nucleosides. Enzymatic ligation to microhelix-RNAs provide access to tRNA analogues containing a 3' terminal A76 locked in South conformation. Biological evaluation of our tRNA analogues has been performed using amino-acyl tRNA-dependent transferase FemXWv, which mediates non-ribosomal incorporation of amino acids into the bacterial cell wall. We have shown that our tRNA analogues inhibited the aminoacyl transfer reaction catalyzed by FemXWv with IC50s of 10 and 8 µM. These results indicate that FemXWv displays a moderate preference for tRNAs containing a terminal A76 locked in the South conformation and that a South to North switch in the conformation of the terminal ribose might contribute to the release of the uncharged tRNAAla product of the aminoacyl transfer reaction catalyzed by FemXwv.

Recognition of Peptidoglycan Fragments by the Transpeptidase PBP4 From Staphylococcus aureus.

Peptidoglycan (PG) is an essential component of the cell envelope, maintaining bacterial cell shape and protecting it from bursting due to turgor pressure. The monoderm bacterium Staphylococcus aureus has a highly cross-linked PG, with ~90% of peptide stems participating in DD-cross-links and up to 15 peptide stems connected with each other. These cross-links are formed in transpeptidation reactions catalyzed by penicillin-binding proteins (PBPs) of classes A and B. Most S. aureus strains have three housekeeping PBPs with this function (PBP1, PBP2, and PBP3) but MRSA strains have acquired a third class B PBP, PBP2a, which is encoded by the mecA gene and required for the expression of high-level resistance to ß-lactams. Another housekeeping PBP of S. aureus is PBP4, which belongs to the class C PBPs, and hence would be expected to have PG hydrolase (DD-carboxypeptidase or DD-endopeptidase) activity. However, previous works showed that, unexpectedly, PBP4 has transpeptidase activity that significantly contributes to both the high level of cross-linking in the PG of S. aureus and to the low level of ß-lactam resistance in the absence of PBP2a. To gain insights into this unusual activity of PBP4, we studied by NMR spectroscopy its interaction in vitro with different substrates, including intact peptidoglycan, synthetic peptide stems, muropeptides, and long glycan chains with uncross-linked peptide stems. PBP4 showed no affinity for the complex, intact peptidoglycan or the smallest isolated peptide stems. Transpeptidase activity of PBP4 was verified with the disaccharide peptide subunits (muropeptides) in vitro, producing cyclic dimer and multimer products; these assays also showed a designed PBP4(S75C) nucleophile mutant to be inactive. Using this inactive but structurally highly similar variant, liquid-state NMR identified two interaction surfaces in close proximity to the central nucleophile position that can accommodate the potential donor and acceptor stems for the transpeptidation reaction. A PBP4:muropeptide model structure was built from these experimental restraints, which provides new mechanistic insights into mecA independent resistance to ß-lactams in S. aureus.

Electrophilic RNA for Peptidyl-RNA Synthesis and Site-Specific Cross-Linking with tRNA-Binding Enzymes.

RNA functionalization is challenging due to the instability of RNA and the limited range of available enzymatic reactions. We developed a strategy based on solid phase synthesis and post-functionalization to introduce an electrophilic site at the 3' end of tRNA analogues. The squarate diester used as an electrophile enabled sequential amidation and provided asymmetric squaramides with high selectivity. The squaramate-RNAs specifically reacted with the lysine of UDP-MurNAc-pentapeptide, a peptidoglycan precursor used by the aminoacyl-transferase FemXWv for synthesis of the bacterial cell wall. The peptidyl-RNA obtained with squaramate-RNA and unprotected UDP-MurNAc-pentapeptide efficiently inhibited FemXWv . The squaramate unit also promoted specific cross-linking of RNA to the catalytic Lys of FemXWv but not to related transferases recognizing different aminoacyl-tRNAs. Thus, squaramate-RNAs provide specificity for cross-linking with defined groups in complex biomolecules due to its unique reactivity.

Routes of Synthesis of Carbapenems for Optimizing Both the Inactivation of L,D-Transpeptidase LdtMt1 of Mycobacterium tuberculosis and the Stability toward Hydrolysis by ß-Lactamase BlaC.

Combinations of ß-lactams of the carbapenem class, such as meropenem, with clavulanate, a ß-lactamase inhibitor, are being evaluated for the treatment of drug-resistant tuberculosis. However, carbapenems approved for human use have never been optimized for inactivation of the unusual ß-lactam targets of Mycobacterium tuberculosis or for escaping to hydrolysis by broad-spectrum ß-lactamase BlaC. Here, we report three routes of synthesis for modification of the two side chains carried by the ß-lactam and the five-membered rings of the carbapenem core. In particular, we show that the azide-alkyne Huisgen cycloaddition reaction catalyzed by copper(I) is fully compatible with the highly unstable ß-lactam ring of carbapenems and that the triazole ring generated by this reaction is well tolerated for inactivation of the L,D-transpeptidase LdtMt1 target. Several of our new carbapenems are superior to meropenem both with respect to the efficiency of in vitro inactivation of LdtMt1 and reduced hydrolysis by BlaC.

Synthesis of 3'-fluoro-tRNA analogues for exploring non-ribosomal peptide synthesis in bacteria.

Aminoacyl-tRNAs (aa-tRNAs) participate in a vast repertoire of metabolic pathways, including the synthesis of the peptidoglycan network in the cell walls of bacterial pathogens. Synthesis of aminoacyl-tRNA analogues is critical for further understanding the mechanisms of these reactions. Here we report the semi-synthesis of 3'-fluoro analogues of Ala-tRNA(Ala) . The presence of fluorine in the 3'-position blocks Ala at the 2'-position by preventing spontaneous migration of the residue between positions 2' and 3'. NMR analyses showed that substitution of the 3'-hydroxy group by fluorine in the ribo configuration favours the S-type conformation of the furanose ring of terminal adenosine A76. In contrast, the N-type conformation is favoured by the presence of fluorine in the xylo configuration. Thus, introduction of fluorine in the ribo and xylo configurations affects the conformation of the furanose ring in reciprocal ways. These compounds should provide insight into substrate recognition by Fem transferases and the Ala-tRNA synthetases.

Synthesis of 3'-triazoyl-dinucleotides as precursors of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues.

We report here the synthesis of stable Phe-tRNA(Phe) and Leu-tRNA(Leu) analogues containing a 1,2,3-triazole ring instead of the ribose-amino acid ester bond. The 1,2,3-triazole ring is generated by dipolar cycloaddition of alkyne Phe and Leu analogues to 3'-azido-3'-deoxyadenosine via the Cu(I)-catalysed Huisgen, Meldal, Sharpless 1,3-cycloaddition. The corresponding triazoyl pdCpA dinucleotides, obtained by classical phosphoramidite chemistry, were enzymatically ligated to 22-nt or 74-nt RNA generating stable Phe-tRNA(Phe) analogues containing the acceptor stem or full tRNA moieties, respectively. These molecules represent useful tools to study the contribution of the RNA and amino acid moieties in stabilization of aminoacyl-tRNA/protein complexes.