A Family of T6SS Antibacterial Effectors Related to l,d-Transpeptidases Targets the Peptidoglycan.

Type VI secretion systems (T6SSs) are nanomachines used by bacteria to inject toxic effectors into competitors. The identity and mechanism of many effectors remain unknown. We characterized a Salmonella T6SS antibacterial effector called Tlde1 that is toxic in target-cell periplasm and is neutralized by its cognate immunity protein (Tldi1). Microscopy analysis reveals that cells expressing Tlde1 stop dividing and lose cell envelope integrity. Bioinformatic analysis uncovers similarities between Tlde1 and the catalytic domain of l,d-transpeptidases. Point mutations on conserved catalytic residues abrogate toxicity. Biochemical assays reveal that Tlde1 displays both l,d-carboxypeptidase activity by cleaving peptidoglycan tetrapeptides between meso-diaminopimelic acid3 and d-alanine4 and l,d-transpeptidase exchange activity by replacing d-alanine4 by a non-canonical d-amino acid. Phylogenetic analysis shows that Tlde1 homologs constitute a family of T6SS-associated effectors broadly distributed among Proteobacteria. This work expands our current knowledge about bacterial effectors used in interbacterial competition and reveals a different mechanism of bacterial antagonism.

Structural Basis for the Interaction and Processing of ß-Lactam Antibiotics by l,d-Transpeptidase 3 (LdtMt3) from Mycobacterium tuberculosis.

Targeting Mycobacterium tuberculosis peptidoglycans with ß-lactam antibiotics represents a strategy to address increasing resistance to antitubercular drugs. ß-Lactams inhibit peptidoglycan synthases such as l,d-transpeptidases, a group of carbapenem-sensitive enzymes that stabilize peptidoglycans through 3 → 3 cross-links. M. tuberculosis encodes five l,d-transpeptidases (LdtMt1-5), of which LdtMt3 is one of the less understood. Herein, we structurally characterized the apo and faropenem-acylated forms of LdtMt3 at 1.3 and 1.8 Å resolution, respectively. These structures revealed a fold and catalytic diad similar to those of other LdtsMt enzymes, supporting its involvement in transpeptidation reactions despite divergences in active site size and charges. The LdtMt3-faropenem structure indicated that faropenem is degraded after Cys-246 acylation, and possibly only a ß-OH-butyrate or an acetyl group (C2H3O) covalently attached to the enzyme remains, an observation that strongly supports the notion that LdtMt3 is inactivated by ß-lactams. Docking simulations with intact ß-lactams predicted key LdtMt3 residues that interact with these antibiotics. We also characterized the heat of acylation involved in the binding and reaction of LdtMt3 for ten ß-lactams belonging to four different classes, and imipenem had the highest inactivation constant. This work provides key insights into the structure, binding mechanisms, and degradation of ß-lactams by LdtMt3, which may be useful for the development of additional ß-lactams with potential antitubercular activity.

Targeting the cell wall of Mycobacterium tuberculosis: a molecular modeling investigation of the interaction of imipenem and meropenem with L,D-transpeptidase 2.

The single crystal X-ray structure of the extracellular portion of the L,D-transpeptidase (ex-LdtMt2 - residues 120-408) enzyme was recently reported. It was observed that imipenem and meropenem inhibit activity of this enzyme, responsible for generating L,D-transpeptide linkages in the peptidoglycan layer of Mycobacterium tuberculosis. Imipenem is more active and isothermal titration calorimetry experiments revealed that meropenem is subjected to an entropy penalty upon binding to the enzyme. Herein, we report a molecular modeling approach to obtain a molecular view of the inhibitor/enzyme interactions. The average binding free energies for nine commercially available inhibitors were calculated using MM/GBSA and Solvation Interaction Energy (SIE) approaches and the calculated energies corresponded well with the available experimentally observed results. The method reproduces the same order of binding energies as experimentally observed for imipenem and meropenem. We have also demonstrated that SIE is a reasonably accurate and cost-effective free energy method, which can be used to predict carbapenem affinities for this enzyme. A theoretical explanation was offered for the experimental entropy penalty observed for meropenem, creating optimism that this computational model can serve as a potential computational model for other researchers in the field.

Simulating the inhibition reaction of Mycobacterium tuberculosis L,D-transpeptidase 2 by carbapenems.

A theoretical free energy study describes the inactivation of a new tuberculosis target, the l,d-transpeptidase 2 enzyme. A new reaction mechanism of two carbapenem inhibitors is proposed and their molecular features are determined using QM/MM and PMF approaches. The theoretical findings with the new proposed mechanism agree in principle with the experimental data.

Catalytic mechanism of L,D-transpeptidase 2 from Mycobacterium tuberculosis described by a computational approach: insights for the design of new antibiotics drugs.

Tuberculosis is perhaps the most persistent human disease caused by an infections bacterium, Mycobacterium tuberculosis. The L,D-transpeptidase enzyme catalyzes the formation of 3 → 3 peptidoglycan cross-links of the Mtb cell wall and facilitates resistance against classical ß-lactams. Herein, the experimentally proposed mechanism for LdtMt2 was studied by performing QM/MM MD simulations. The whole mechanistic process includes two stages: acylation and deacylation. During the acylation step, two steps were observed: the first step is a thiolate/imidazole ion-pair in the zwitterionic form, and the second step is the nucleophilic attack on the carboxyl carbon of the natural substrate accompanied by the breaking of the peptide bond on substrate. In the deacylation step the acyl-enzyme suffers a nucleophilic attack on the carboxyl carbon by the amine group of the second substrate. Our free energy results obtained by PMF analysis reveal that the first step (acylation) is the rate-limiting step in the whole catalytic mechanism in accordance with the experimental proposal. Also, the residues responsible for binding of the substrate and transition state stabilization were identified by energy decomposition methods.

Compensatory evolution of pbp mutations restores the fitness cost imposed by ß-lactam resistance in Streptococcus pneumoniae.

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, ß-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of ß-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of ß-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for ß-lactam resistance evolution in Streptococcus pneumoniae.

All detectable high-molecular-mass penicillin-binding proteins are modified in a high-level beta-lactam-resistant clinical isolate of Streptococcus mitis.

All detectable high-molecular-mass penicillin-binding proteins (HMM PBPs) are altered in a clinical isolate of Streptococcus mitis for which the beta-lactam MICs are increased from those previously reported in our region (cefotaxime MIC, 64 microg/ml). These proteins were hardly detected at concentrations that saturate all PBPs in clinical isolates and showed, after densitometric analysis, 50-fold-lower radiotracer binding. Resistance was related to mosaic structure in all HMM PBP-coding genes, where critical region replacement was complemented not only by substitutions already reported for the closely related Streptococcus pneumoniae but also by other specific replacements that are presumably close to the active-site serine. Mosaic structure was also presumed in a pbp1a-sensitive strain used for comparison, confirming that these structures do not unambiguously imply, by themselves, detectable critical changes in the kinetic properties of these proteins.

Participation of PBP 3 in the acquisition of dicloxacillin resistance in Listeria monocytogenes.

Purified membranes of Listeria monocytogenes ATCC 15313 contain at least five penicillin-binding proteins. In two dicloxacillin-resistant mutants, derived from a sensitive parent strain, a 16-fold increase in the MIC of dicloxacillin was observed. A less-significant increase was detected in the MICs of other beta-lactam drugs. In the mutants, PBP 3 lost its strong affinity for dicloxacillin, but remained fully susceptible to binding of 125I-penicillin X, as compared with the wild-type strain. PBP 2 could not be detected in one of the mutants. No decrease in affinity for the radioactive tracer or dicloxacillin was detected in any other PBP of the resistant mutants.