Tuberculosis (TB), caused by the intracellular pathogen Mycobacterium tuberculosis, remains one of the leading causes of mortality across the world. There is an urgent requirement to build a robust arsenal of effective antimicrobials, targeting novel molecular mechanisms to overcome the challenges posed by the increase of antibiotic resistance in TB. Mycobacterium tuberculosis has a unique cell envelope structure and composition, containing a peptidoglycan layer that is essential for maintaining cellular integrity and for virulence. The enzymes involved in the biosynthesis, degradation, remodelling and recycling of peptidoglycan have resurfaced as attractive targets for anti-infective drug discovery. Here, we review the importance of peptidoglycan, including the structure, function and regulation of key enzymes involved in its metabolism. We also discuss known inhibitors of ATP-dependent Mur ligases, and discuss the potential for the development of pan-enzyme inhibitors targeting multiple Mur ligases.

The undersigned authors wish to note, "The KefFC system of E. coli is maintained in an inactive state by the binding of glutathione (GSH) and is activated by the formation of GSH adducts (GSX), particularly those with bulky substituents. We described two crystal structures with density present in the ligand-binding domain that we interpreted as GSH and GSX. Recently, an independent, experienced crystallographer, who had viewed the structures from our study in a different context, made representations to us that cast doubt on position of the succinimido ring of GSX. We have further reviewed the density maps with the aid of an experienced crystallographer. As a consequence, we believe it is important to draw this altered interpretation of the crystal structures to the attention of readers. In both structures, the density for the backbone of GSH is clear and allows unequivocal assignment of the position of the tripeptide. In PDB coordinate set 3L9X, the density for the succinimido ring is very weak, making interpretation very speculative and the assignment rests on the identity of the ligand added to the crystallization mixture, for which there are two diastereomers in the solutiona possibility that provides some basis for weakening the density. However, in 3L9W there are two anomalies that affect the interpretation of the bound ligand. First, there is no density for the carbon atom attached to the sulfur of GSH and second, there is extra density adjacent to the position of sulfur that could be modelled as a constrained succinimido ring. However, this density could also be water or any other molecule that is trapped in the structure. Thus, while there is good evidence for the peptide, the evidence that it is in the GSH form is uncertain."There are no new data on either the structures or on the gating mechanism. However, we believe that we should be cautious in interpreting the structural data and that the field in general should be made aware of the alternative views of the electron density data. Note that the mutagenesis and spectroscopic data that were presented in the original manuscript are not affected by this alternative interpretation." Tarmo P. The authors note that the following grant should be added to the Acknowledgments: "NIH Grant AG002132." The authors note "The method used for exogenous expression of Ca V 1.2 channels in ref. 32 was incorrectly described as 'viral transduction' in the text. In fact, Yang et al. created transgenic mice with inducible, cardiomyocyte-specific expression of exogenous Ca V 1.2 channels regulated by a tetracycline-inducible promoter. When crossed with a transgenic mouse line expressing doxycycline-regulated reverse transcriptional activator under control of the α-myosin heavy chain protomer, the resulting double transgenic offspring expressed exogenous Ca V 1.2 channels in their cardiac myocytes after treatment with doxycycline. The authors regret the error in describing these methods."www.pnas.org/cgi

The potassium efflux system, Kef, protects bacteria against the detrimental effects of electrophilic compounds via acidification of the cytoplasm. Kef is inhibited by glutathione (GSH) but activated by glutathione-S-conjugates (GS-X) formed in the presence of electrophiles. GSH and GS-X bind to overlapping sites on Kef, which are located in a cytosolic regulatory domain. The central paradox of this activation mechanism is that GSH is abundant in cells (at concentrations of ∼10–20 mM), and thus, activating ligands must possess a high differential over GSH in their affinity for Kef. To investigate the structural requirements for binding of a ligand to Kef, a novel fluorescent reporter ligand, S-{[5-(dimethylamino)naphthalen-1-yl]sulfonylaminopropyl} glutathione (DNGSH), was synthesized. By competition assays using DNGSH, complemented by direct binding assays and thermal shift measurements, we show that the well-characterized Kef activator, N-ethylsuccinimido-S-glutathione, has a 10–20-fold higher affinity for Kef than GSH. In contrast, another native ligand that is a poor activator, S-lactoylglutathione, exhibits a similar Kef affinity to GSH. Synthetic ligands were synthesized to contain either rigid or flexible structures and investigated as ligands for Kef. Compounds with rigid structures and high affinity activated Kef. In contrast, flexible ligands with similar binding affinities did not activate Kef. These data provide insight into the structural requirements for Kef gating, paving the way for the development of a screen for potential therapeutic lead compounds targeting the Kef system.

The rise of multi-drug resistance in bacterial pathogens is one of the grand challenges facing medical science. A major concern is the speed of development of β-lactamase-mediated resistance in Gram-negative species, thus putting at risk the efficacy of the most recently approved antibiotics and inhibitors, including carbapenems and avibactam, respectively. New strategies to overcome resistance are urgently required, which will ultimately be facilitated by a deeper understanding of the mechanisms that regulate the function of β-lactamases such as the Klebsiella Pneumoniae carbapenemases (KPCs). Using enhanced sampling computational methods together with site-directed mutagenesis, we report the identification of two “hydrophobic networks” in the KPC-2 enzyme, the integrity of which has been found to be essential for protein stability and corresponding resistance. Present throughout the structure, these networks are responsible for the structural integrity and allosteric signaling. Disruption of the networks leads to a loss of the KPC-2 mediated resistance phenotype, resulting in restored susceptibility to different classes of β-lactam antibiotics including carbapenems and cephalosporins. The ”hydrophobic networks” were found to be highly conserved among class-A β-lactamases, which implies their suitability for exploitation as a potential target for therapeutic intervention.