Structural and Molecular Dynamics of Mycobacterium tuberculosis Malic Enzyme, a Potential Anti-TB Drug Target.

Tuberculosis (TB) is the most lethal bacterial infectious disease worldwide. It is notoriously difficult to treat, requiring a cocktail of antibiotics administered over many months. The dense, waxy outer membrane of the TB-causing agent, Mycobacterium tuberculosis (Mtb), acts as a formidable barrier against uptake of antibiotics. Subsequently, enzymes involved in maintaining the integrity of the Mtb cell wall are promising drug targets. Recently, we demonstrated that Mtb lacking malic enzyme (MEZ) has altered cell wall lipid composition and attenuated uptake by macrophages. These results suggest that MEZ contributes to lipid biosynthesis by providing reductants in the form of NAD(P)H. Here, we present the X-ray crystal structure of MEZ to 3.6 Å. We use biochemical assays to demonstrate MEZ is dimeric in solution and to evaluate the effects of pH and allosteric regulators on its kinetics and thermal stability. To assess the interactions between MEZ and its substrate malate and cofactors, Mn2+ and NAD(P)+, we ran a series of molecular dynamics (MD) simulations. First, the MD analysis corroborates our empirical observations that MEZ is unusually flexible, which persists even with the addition of substrate and cofactors. Second, the MD simulations reveal that dimeric MEZ subunits alternate between open and closed states, and that MEZ can stably bind its NAD(P)+ cofactor in multiple conformations, including an inactive, compact NAD+ form. Together the structure of MEZ and insights from its dynamics can be harnessed to inform the design of MEZ inhibitors that target Mtb and not human malic enzyme homologues.

Synthetic Antimicrobial Peptide Tuning Permits Membrane Disruption and Interpeptide Synergy.

The ribosomally produced antimicrobial peptides of bacteria (bacteriocins) represent an unexplored source of membrane-active antibiotics. We designed a library of linear peptides from a circular bacteriocin and show that pore-formation dynamics in bacterial membranes are tunable via selective amino acid substitution. We observed antibacterial interpeptide synergy indicating that fundamentally altering interactions with the membrane enables synergy. Our findings suggest an approach for engineering pore-formation through rational peptide design and increasing the utility of novel antimicrobial peptides by exploiting synergy.

Mechanical Genomic Studies Reveal the Role of d-Alanine Metabolism in Pseudomonas aeruginosa Cell Stiffness.

The stiffness of bacteria prevents cells from bursting due to the large osmotic pressure across the cell wall. Many successful antibiotic chemotherapies target elements that alter mechanical properties of bacteria, and yet a global view of the biochemistry underlying the regulation of bacterial cell stiffness is still emerging. This connection is particularly interesting in opportunistic human pathogens such as Pseudomonas aeruginosa that have a large (80%) proportion of genes of unknown function and low susceptibility to different families of antibiotics, including beta-lactams, aminoglycosides, and quinolones. We used a high-throughput technique to study a library of 5,790 loss-of-function mutants covering ~80% of the nonessential genes and correlated P. aeruginosa individual genes with cell stiffness. We identified 42 genes coding for proteins with diverse functions that, when deleted individually, decreased cell stiffness by >20%. This approach enabled us to construct a "mechanical genome" for P. aeruginosa d-Alanine dehydrogenase (DadA) is an enzyme that converts d-Ala to pyruvate that was included among the hits; when DadA was deleted, cell stiffness decreased by 18% (using multiple assays to measure mechanics). An increase in the concentration of d-Ala in cells downregulated the expression of genes in peptidoglycan (PG) biosynthesis, including the peptidoglycan-cross-linking transpeptidase genes ponA and dacC Consistent with this observation, ultraperformance liquid chromatography-mass spectrometry analysis of murein from P. aeruginosa cells revealed that dadA deletion mutants contained PG with reduced cross-linking and altered composition compared to wild-type cells.IMPORTANCE The mechanical properties of bacteria are important for protecting cells against physical stress. The cell wall is the best-characterized cellular element contributing to bacterial cell mechanics; however, the biochemistry underlying its regulation and assembly is still not completely understood. Using a unique high-throughput biophysical assay, we identified genes coding proteins that modulate cell stiffness in the opportunistic human pathogen Pseudomonas aeruginosa This approach enabled us to discover proteins with roles in a diverse range of biochemical pathways that influence the stiffness of P. aeruginosa cells. We demonstrate that d-Ala-a component of the peptidoglycan-is tightly regulated in cells and that its accumulation reduces expression of machinery that cross-links this material and decreases cell stiffness. This research demonstrates that there is much to learn about mechanical regulation in bacteria, and these studies revealed new nonessential P. aeruginosa targets that may enhance antibacterial chemotherapies or lead to new approaches.

Novel inhibitors of the rRNA ErmC' methyltransferase to block resistance to macrolides, lincosamides, streptogramine B antibiotics.

In erythromycin-resistant bacteria, the N6 position of A2058 in 23S rRNA is mono- or dimethylated by Erm family methyltransferases. This modification results in cross-resistance to macrolides, lincosamides and streptogramin B. Most inhibitors of Erm methyltransferases developed up-to-date target the cofactor-binding pocket, resulting in a lack of selectivity whereas inhibitors that bind the substrate-binding pocket demonstrate low in vitro activity. In this study, a molecular docking approach followed by biochemical screening was applied to search for inhibitors targeting both cofactor- and substrate-binding pockets of ErmC' methyltransferase. Based on the results of the molecular docking-based virtual screening of the clean-leads subset of the ZINC database, 29 compounds were chosen for experimental verification. Among them inhibitor 28 (ZINC code 32747906), with an IC50 of 100 µM, decreased the minimal inhibitory concentration of erythromycin in the Escherichia coli strain overexpressing ErmC'. Docking analysis of 28 to the ErmC' structure and the competitive ligand binding assay revealed a non-competitive model of inhibition. Inhibitor 28 served as a template for similarity-based virtual screening, which resulted in the identification of two derivatives 3s (ZINC code 62022572) and 4s (ZINC code 49032257) with an IC50 of 116 µM and 110 µM, respectively. Our results provide a basis for the development of inhibitors against the Erm-family of enzymes.