Engineered Mycobacteriophage TM4::GeNL Rapidly Determines Bedaquiline, Pretomanid, Linezolid, Rifampicin, and Clofazimine Sensitivity in Mycobacterium tuberculosis Clinical Isolates.

Background: Drug-resistant tuberculosis is a growing public health threat, and early characterization of the resistance phenotype is essential for guiding treatment and mitigating the high mortality associated with the disease. However, the slow growth rate of Mycobacterium tuberculosis, the causative agent of tuberculosis, necessitates several weeks for conventional culture-dependent drug susceptibility testing (DST). In addition, there are no widely available molecular diagnostic assays for evaluating resistance to newer tuberculosis drugs or drugs with complex resistance mechanisms.

Propionate prevents loss of the PDIM virulence lipid in Mycobacterium tuberculosis.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine.

A dual-targeting succinate dehydrogenase and FF-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M.

The PDIM paradox of : new solutions to a persistent problem.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of . culturing rapidly selects for spontaneous mutations that cause PDIM loss leading to virulence attenuation and increased cell wall permeability. We discovered that PDIM loss is due to a metabolic deficiency of methylmalonyl-CoA that impedes the growth of PDIM-producing bacilli.

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments.

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy.

Are all antibiotic persisters created equal?

Antibiotic persisters are a sub-population of bacteria able to survive in the presence of bactericidal antibiotic despite the lack of heritable drug resistance mechanisms. This phenomenon exists across many bacterial species and is observed for many different antibiotics. Though these bacteria are often described as "multidrug persisters" very few experiments have been carried out to determine the homogeneity of a persister population to different drugs.

Capturing Structural Variants of Herpes Simplex Virus Genome in Full Length by Oxford Nanopore Sequencing.

Genome sequencing and assembly of viral genomes within the family, particularly herpes simplex virus (HSV), have been challenging due to the large size (~154 Kb), high GC content (68%), and nucleotide variations arising during replication. Oxford Nanopore Technology (ONT) has been successful in obtaining read lengths ranging from 100 Kb up to 2.3 Mb.

An amiloride derivative is active against the FF-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis.

Combining CRISPRi and metabolomics for functional annotation of compound libraries.

Molecular profiling of small molecules offers invaluable insights into the function of compounds and allows for hypothesis generation about small-molecule direct targets and secondary effects. However, current profiling methods are limited in either the number of measurable parameters or throughput. Here we developed a multiplexed, unbiased framework that, by linking genetic to drug-induced changes in nearly a thousand metabolites, allows for high-throughput functional annotation of compound libraries in Escherichia coli.

HS Functions as a Sink to Modulate Central Metabolism, Bioenergetics, and Drug Susceptibility.

HS is a potent gasotransmitter in eukaryotes and bacteria. Host-derived HS has been shown to profoundly alter () energy metabolism and growth. However, compelling evidence for endogenous production of HS and its role in physiology is lacking.

Bioenergetic Inhibitors: Antibiotic Efficacy and Mechanisms of Action in .

Development of novel anti-tuberculosis combination regimens that increase efficacy and reduce treatment timelines will improve patient compliance, limit side-effects, reduce costs, and enhance cure rates. Such advancements would significantly improve the global TB burden and reduce drug resistance acquisition. Bioenergetics has received considerable attention in recent years as a fertile area for anti-tuberculosis drug discovery.

Dual inhibition of the terminal oxidases eradicates antibiotic-tolerant Mycobacterium tuberculosis.

The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F F ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa terminal oxidase.

Nanoluciferase Reporter Mycobacteriophage for Sensitive and Rapid Detection of Mycobacterium tuberculosis Drug Susceptibility.

Phenotypic testing for drug susceptibility of is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent strains.

Derailing the aspartate pathway of Mycobacterium tuberculosis to eradicate persistent infection.

A major constraint for developing new anti-tuberculosis drugs is the limited number of validated targets that allow eradication of persistent infections. Here, we uncover a vulnerable component of Mycobacterium tuberculosis (Mtb) persistence metabolism, the aspartate pathway. Rapid death of threonine and homoserine auxotrophs points to a distinct susceptibility of Mtb to inhibition of this pathway.

Genome-wide mutational biases fuel transcriptional diversity in the Mycobacterium tuberculosis complex.

The Mycobacterium tuberculosis complex (MTBC) members display different host-specificities and virulence phenotypes. Here, we have performed a comprehensive RNAseq and methylome analysis of the main clades of the MTBC and discovered unique transcriptional profiles. The majority of genes differentially expressed between the clades encode proteins involved in host interaction and metabolic functions.

Inhibitors of energy metabolism interfere with antibiotic-induced death in mycobacteria.

Energy metabolism has recently gained interest as a target space for antibiotic drug development in mycobacteria. Of particular importance is bedaquiline (Sirturo), which kills mycobacteria by inhibiting the FF ATP synthase. Other components of the electron transport chain such as the NADH dehydrogenases (NDH-2 and NdhA) and the terminal respiratory oxidase : are also susceptible to chemical inhibition.

Synthesis and degradation of FtsZ quantitatively predict the first cell division in starved bacteria.

In natural environments, microbes are typically non-dividing and gauge when nutrients permit division. Current models are phenomenological and specific to nutrient-rich, exponentially growing cells, thus cannot predict the first division under limiting nutrient availability. To assess this regime, we supplied starving with glucose pulses at increasing frequencies.

Arginine-deprivation-induced oxidative damage sterilizes .

Reactive oxygen species (ROS)-mediated oxidative stress and DNA damage have recently been recognized as contributing to the efficacy of most bactericidal antibiotics, irrespective of their primary macromolecular targets. Inhibitors of targets involved in both combating oxidative stress as well as being required for in vivo survival may exhibit powerful synergistic action. This study demonstrates that the de novo arginine biosynthetic pathway in () is up-regulated in the early response to the oxidative stress-elevating agent isoniazid or vitamin C.

The Capacity of To Survive Iron Starvation Might Enable It To Persist in Iron-Deprived Microenvironments of Human Granulomas.

This study was conducted to investigate the role of iron deprivation in the persistence of We present evidence of iron restriction in human necrotic granulomas and demonstrate that under iron starvation persists, refractive to antibiotics and capable of restarting replication when iron is made available. Transcriptomics and metabolomic analyses indicated that the persistence of under iron starvation is dependent on strict control of endogenous Fe utilization and is associated with upregulation of pathogenicity and intrinsic antibiotic resistance determinants. mutants compromised in their ability to survive Fe starvation were identified.

Exploiting the synthetic lethality between terminal respiratory oxidases to kill and clear host infection.

The recent discovery of small molecules targeting the cytochrome : in triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome : consists of a menaquinone:cytochrome reductase ( ) and a cytochrome -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome reductase.

Oxidative Phosphorylation as a Target Space for Tuberculosis: Success, Caution, and Future Directions.

The emergence and spread of drug-resistant pathogens, and our inability to develop new antimicrobials to combat resistance, have inspired scientists to seek out new targets for drug development. The complex is a group of obligately aerobic bacteria that have specialized for inhabiting a wide range of intracellular and extracellular environments. Two fundamental features in this adaptation are the flexible utilization of energy sources and continued metabolism in the absence of growth.

in the Face of Host-Imposed Nutrient Limitation.

Coevolution of pathogens and host has led to many metabolic strategies employed by intracellular pathogens to deal with the immune response and the scarcity of food during infection. Simply put, bacterial pathogens are just looking for food. As a consequence, the host has developed strategies to limit nutrients for the bacterium by containment of the intruder in a pathogen-containing vacuole and/or by actively depleting nutrients from the intracellular space, a process called nutritional immunity.