Integration of biophysical and biological approaches to validate fragment-like compounds targeting l,d-transpeptidases from Mycobacterium tuberculosis.

Tuberculosis is one of the major causes of death worldwide; more than a million people die every year because of this infection. The constant emergency of Mycobacterium tuberculosis resistant strains against the most used treatments also contributes to the burden caused by this disease. Consequently, the development of new alternative therapies against this disease is constantly required. In recent years, only a few molecules have reached the market as new antituberculosis agents. The mycobacterial cell wall biosynthesis is for a longstanding considered an important target for drug development. Particularly, in M. tuberculosis, the peptidoglycan cross-links are predominantly formed by nonclassical bridges between the third residues of adjacent tetrapeptides. The responsible enzymes for these reactions are ld-transpeptidases (Ldts), for which M. tuberculosis has five paralogues. Although these enzymes are distinct from the penicillin-binding proteins (PBPs), they can also be inactivated by ß-lactam antibiotics, but since M. tuberculosis has a chromosomal ß-lactamase, most of the antibiotics of these classes can be degraded. Thus, to identify alternative scaffolds for the development of new antimicrobials against tuberculosis, we have integrated several fragment-based drug discovery techniques. Based on that, we identified and validated a number of small molecules that could be the starting point in the synthesis of more potent inhibitors against at least two Ldts from M. tuberculosis, LdtMt2 and LdtMt3. Eight identified molecules inhibited the Ldts activity in at least 20%, and three of them have antimycobacterial activity. The cell ultrastructural analysis suggested that one of the best compounds induced severe effects on the septum and cell wall morphologies, which corroborates our target-based approach to identifying new Ldts hits.

Characterization of Pseudomonas aeruginosa l,d-Transpeptidases and Evaluation of Their Role in Peptidoglycan Adaptation to Biofilm Growth.

Peptidoglycan is an essential component of the bacterial cell envelope that sustains the turgor pressure of the cytoplasm, determines cell shape, and acts as a scaffold for the anchoring of envelope polymers such as lipoproteins. The final cross-linking step of peptidoglycan polymerization is performed by classical d,d-transpeptidases belonging to the penicillin-binding protein (PBP) family and by l,d-transpeptidases (LDTs), which are dispensable for growth in most bacterial species and whose physiological functions remain elusive. In this study, we investigated the contribution of LDTs to cell envelope synthesis in Pseudomonas aeruginosa grown in planktonic and biofilm conditions. We first assigned a function to each of the three P. aeruginosa LDTs by gene inactivation in P. aeruginosa, heterospecific gene expression in Escherichia coli, and, for one of them, direct determination of its enzymatic activity. We found that the three P. aeruginosa LDTs catalyze peptidoglycan cross-linking (LdtPae1), the anchoring of lipoprotein OprI to the peptidoglycan (LdtPae2), and the hydrolysis of the resulting peptidoglycan-OprI amide bond (LdtPae3). Construction of a phylogram revealed that LDTs performing each of these three functions in various species cannot be assigned to distinct evolutionary lineages, in contrast to what has been observed with PBPs. We showed that biofilm, but not planktonic bacteria, displayed an increase proportion of peptidoglycan cross-links formed by LdtPae1 and a greater extent of OprI anchoring to peptidoglycan, which is controlled by LdtPae2 and LdtPae3. Consistently, deletion of each of the ldt genes impaired biofilm formation and potentiated the bactericidal activity of EDTA. These results indicate that LDTs contribute to the stabilization of the bacterial cell envelope and to the adaptation of peptidoglycan metabolism to growth in biofilm. IMPORTANCE Active-site cysteine LDTs form a functionally heterologous family of enzymes that contribute to the biogenesis of the bacterial cell envelope through formation of peptidoglycan cross-links and through the dynamic anchoring of lipoproteins to peptidoglycan. Here, we report the role of three P. aeruginosa LDTs that had not been previously characterized. We show that these enzymes contribute to resistance to the bactericidal activity of EDTA and to the adaptation of cell envelope polymers to conditions that prevail in biofilms. These results indicate that LDTs should be considered putative targets in the development of drug-EDTA associations for the control of biofilm-related infections.

Peptidoglycan Remodeling by an L,D-Transpeptidase, LdtD during Cold Shock in Escherichia coli.

Peptidoglycan (PG) is a unique and essential component of the bacterial cell envelope. It is made up of several linear glycan polymers cross-linked through covalently attached stem peptides making it a fortified mesh-like sacculus around the bacterial cytosolic membrane. In most bacteria, including Escherichia coli, the stem peptide is made up of l-alanine (l-Ala1), d-glutamate (d-Glu2), meso-diaminopimelic acid (mDAP3), d-alanine (d-Ala4), and d-Ala5 with cross-links occurring either between d-ala4 and mDAP3 or between two mDAP3 residues. Of these, the cross-links of the 4-3 (d-Ala4-mDAP3) type are the most predominant and are formed by penicillin-binding D,D-transpeptidases, whereas the formation of less frequent 3-3 linkages (mDAP3-mDAP3) is catalyzed by L,D-transpeptidases. In this study, we found that the frequency of the 3-3 cross-linkages increased upon cold shock in exponentially growing E. coli and that the increase was mediated by an L,D-transpeptidase, LdtD. We found that a cold-inducible RNA helicase DeaD enhanced the cellular LdtD level by facilitating its translation resulting in an increased abundance of 3-3 cross-linkages during cold shock. However, DeaD was also required for optimal expression of LdtD during growth at ambient temperature. Overall, our study finds that E. coli undergoes PG remodeling during cold shock by altering the frequency of 3-3 cross-linkages, implying a role for these modifications in conferring fitness and survival advantage to bacteria growing in diverse environmental conditions. IMPORTANCE Most bacteria are surrounded by a protective exoskeleton called peptidoglycan (PG), an extensively cross-linked mesh-like macromolecule. In bacteria, such as Escherichia coli, the cross-links in the PG are of two types: a major fraction is of 4-3 type whereas a minor fraction is of 3-3 type. Here, we showed that E. coli exposed to cold shock had elevated levels of 3-3 cross-links due to the upregulation of an enzyme, LdtD, that catalyzed their formation. We showed that a cold-inducible RNA helicase DeaD enhanced the cellular LdtD level by facilitating its translation, resulting in increased 3-3 cross-links during cold shock. Our results suggest that PG remodeling contributes to the survival and fitness of bacteria growing in conditions of cold stress.

Overexpression of lpxT Gene in Escherichia coli Inhibits Cell Division and Causes Envelope Defects without Changing the Overall Phosphorylation Level of Lipid A.

LpxT is an inner membrane protein that transfers a phosphate group from the essential lipid undecaprenyl pyrophosphate (C-55PP) to the lipid A moiety of lipopolysaccharide, generating a lipid A tris-phosphorylated species. The protein is encoded by the non-essential lpxT gene, which is conserved in distantly related Gram-negative bacteria. In this work, we investigated the phenotypic effect of lpxT ectopic expression from a plasmid in Escherichia coli. We found that lpxT induction inhibited cell division and led to the formation of elongated cells, mostly with absent or altered septa. Moreover, the cells became sensitive to detergents and to hypo-osmotic shock, indicating that they had cell envelope defects. These effects were not due to lipid A hyperphosphorylation or C-55PP sequestering, but most likely to defective lipopolysaccharide transport. Indeed, lpxT overexpression in mutants lacking the L,D-transpeptidase LdtD and LdtE, which protect cells with outer membrane defects from osmotic lysis, caused cell envelope defects. Moreover, we found that pyrophosphorylated lipid A was also produced in a lpxT deletion mutant, indicating that LpxT is not the only protein able to perform such lipid A modification in E. coli.

The catalytic role of water in the binding site of l,d-transpeptidase 2 within acylation mechanism: A QM/MM (ONIOM) modelling.

Mycobacterium tuberculosis is the causative agent of Tuberculosis. Formation of 3 → 3 crosslinks in the peptidoglycan layer of M. tuberculosis is catalyzed by l,d-transpeptidases. These enzymes can confer resistance against classical ß-lactams that inhibit enzymes that generate 4 → 3 peptidoglycan crosslinks. The focus of this study is to investigate the catalytic role of water molecules in the acylation mechanism of the ß-lactam ring within two models; 4- and 6-membered ring systems using two-layered our Own N-layer integrated Molecular Mechanics ONIOM (B3LYP/6-311++G(2d,2p): AMBER) model. The obtained thermochemical parameters revealed that the 6-membered ring model best describes the inhibition mechanism of acylation which indicates the role of water in the preference of 6-membered ring reaction pathway. This finding is in accordance with experimental data for the rate-limiting step of cysteine protease with the same class of inhibitor and binding affinity for both inhibitors. As expected, the ΔG# results also reveal that the 6-membered ring reaction pathway is the most favourable. The electrostatic potential (ESP) and the natural bond orbital analysis (NBO) showed stronger interactions in 6-membered ring transition state (TS-6) mechanism involving water in the active site of the enzyme. This study could be helpful in the development of novel antibiotics against l,d-transpeptidase.

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.

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.

Self-resistance mechanisms of actinomycetes producing lipid II-targeting antibiotics.

Glycopeptides and several lantibiotics are lipid II-targeting antibiotics produced by actinomycetes. To protect themselves from their own product, antibiotic producers developed self-resistance mechanisms. Inspection of different producer strains revealed that their resistance is not only based on a single determinant but on the synergistic action of different factors. Glycopeptide producers possess different ways to synthesize a modified peptidoglycan to prevent the binding of the glycopeptide antibiotic. One possible modification is the synthesis of peptidoglycan precursors terminating with a D-alanyl-D-lactate (D-Ala-D-Lac) rather than with a D-alanyl-D-alanine (D-Ala-D-Ala) resulting in a 1000-fold decreased binding affinity of the glycopeptide to its target. The reprogramming of the peptidoglycan precursor biosynthesis is based on the action of VanHAX or paralogous enzymes as it was shown for Amycolatopsis balhimycina. A second peptidoglycan modification resulting in glycopeptide resistance was investigated in the glycopeptide A40926 producer Nonomuraea ATCC 39727. Nonomuraea eliminates the glycopeptide target by synthesizing a peptidoglycan with 3-3 cross-linked peptide stems. The carboxypeptidase VanYn provides tetrapeptides which serve as substrates for the L,D-transpeptidase catalyzing the formation of 3-3 cross-links. The occurrence of 3-3 cross-linked dimers is also an important feature of the lantibiotic NAI-107 producer Microbispora ATCC PTA-5024. Moreover, the D-Ala in the fourth position in the acceptor peptide of muropeptides is exchanged to glycine or serine in Microbispora, a side reaction of the L,D-transpeptidase. Together with the lipoprotein MlbQ, the ABC transporter MlbYZ and the transmembrane protein MlbJ it might contribute to the self-resistance in Microbispora ATCC PTA-5024.

Structures of free and inhibited forms of the L,D-transpeptidase LdtMt1 from Mycobacterium tuberculosis.

The modelling of peptidoglycan is responsible for key cellular processes in Mycobacterium tuberculosis such as cell growth, division and resuscitation from dormancy. The structure of M. tuberculosis peptidoglycan is atypical since it contains a majority of 3,3 cross-links synthesized by L,D-transpeptidases that replace the 4,3 cross-links formed by the D,D-transpeptidase activity of classical penicillin-binding proteins. Carbapenems inactivate these L,D-transpeptidases and in combination with clavulanic acid are bactericidal against extensively drug-resistant M. tuberculosis. Here, crystal structures of the L,D-transpeptidase LdtMt1 from M. tuberculosis in a ligand-free form and in complex with the carbapenem imipenem are reported. Elucidation of the structural features of LdtMt1 unveils analogies and differences between the two key transpeptidases of M. tuberculosis: LdtMt1 and LdtMt2. In addition, the structure of imipenem-inactivated LdtMt1 provides a detailed structural view of the interactions between a carbapenem drug and LdtMt1. By providing the key interactions in the binding of carbapenem to LdtMt1, this work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antitubercular agents against drug-resistant M. tuberculosis.

Structural basis for the inhibition of Mycobacterium tuberculosis L,D-transpeptidase by meropenem, a drug effective against extensively drug-resistant strains.

Difficulty in the treatment of tuberculosis and growing drug resistance in Mycobacterium tuberculosis (Mtb) are a global health issue. Carbapenems inactivate L,D-transpeptidases; meropenem, when administered with clavulanate, showed in vivo activity against extensively drug-resistant Mtb strains. LdtMt2 (Rv2518c), one of two functional L,D-transpeptidases in Mtb, is predominantly expressed over LdtMt1 (Rv0116c). Here, the crystal structure of N-terminally truncated LdtMt2 (residues Leu131-Ala408) is reported in both ligand-free and meropenem-bound forms. The structure of meropenem-inhibited LdtMt2 provides a detailed structural view of the interactions between a carbapenem drug and Mtb L,D-transpeptidase. The structures revealed that the catalytic L,D-transpeptidase domain of LdtMt2 is preceded by a bacterial immunogloblin-like Big_5 domain and is followed by an extended C-terminal tail that interacts with both domains. Furthermore, it is shown using mass analyses that meropenem acts as a suicide inhibitor of LdtMt2. Upon acylation of the catalytic Cys354 by meropenem, the `active-site lid' undergoes a large conformational change to partially cover the active site so that the bound meropenem is accessible to the bulk solvent via three narrow paths. This work will facilitate structure-guided discovery of L,D-transpeptidase inhibitors as novel antituberculosis drugs against drug-resistant Mtb.