Loss of glycerol catabolism confers carbon-source-dependent artemisinin resistance in .

In view of the urgent need for new antibiotics to treat human infections caused by multidrug-resistant pathogens, drug repurposing is gaining strength due to the relatively low research costs and shorter clinical trials. Such is the case of artemisinin, an antimalarial drug that has recently been shown to display activity against (Mtb), the causative agent of tuberculosis. To gain insight into how Mtb is affected by artemisinin, we used RNAseq to assess the impact of artemisinin on gene expression profiles, revealing the induction of several efflux pumps and the KstR2 regulon.

An O-methylflavone from Artemisia afra kills non-replicating hypoxic Mycobacterium tuberculosis.

Ethnopharmacological Relevance: African wormwood (Artemisia afra Jacq. ex Willd.) has been used traditionally in southern Africa to treat illnesses causing fever and was recently shown to possess anti-tuberculosis activity.

A methoxylated flavone from kills .

Tuberculosis, caused by (Mtb), is a deadly and debilitating disease globally affecting millions annually. Emerging drug-resistant Mtb strains endanger the efficacy of the current combination therapies employed to treat tuberculosis; therefore, there is an urgent need to develop novel drugs to combat this disease. is used traditionally in southern Africa to treat malaria and recently has shown anti tuberculosis activity.

Azide-Masked Fluorescence Turn-On Probe for Imaging Mycobacteria.

A fluorescence turn-on probe, an azide-masked and trehalose-derivatized carbazole (), was developed to image mycobacteria. The fluorescence turn-on is achieved by photoactivation of the azide, which generates a fluorescent product through an efficient intramolecular C-H insertion reaction. The probe is highly specific for mycobacteria and could image mycobacteria in the presence of other Gram-positive and Gram-negative bacteria.

and Extracts Have Bactericidal Activity against in Physiologically Relevant Carbon Sources and Hypoxia.

(Mtb) is a deadly pathogen and causative agent of human tuberculosis, causing ~1.5 million deaths every year. The increasing drug resistance of this pathogen necessitates novel and improved treatment strategies.

A research program-linked, course-based undergraduate research experience that allows undergraduates to participate in current research on mycobacterial gene regulation.

Undergraduate instructional biology laboratories are typically taught within two paradigms. Some labs focus on protocols and techniques delivered in "cookbook" format with defined experimental outcomes. There is increasing momentum to alternatively employ student-driven, open-ended, and discovery-based strategies, often course-based undergraduate research experiences (CUREs) using crowd-sourcing initiatives.

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis.

Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates.

Regulation of mRNA Stability During Bacterial Stress Responses.

Bacteria have a remarkable ability to sense environmental changes, swiftly regulating their transcriptional and posttranscriptional machinery as a response. Under conditions that cause growth to slow or stop, bacteria typically stabilize their transcriptomes in what has been shown to be a conserved stress response. In recent years, diverse studies have elucidated many of the mechanisms underlying mRNA degradation, yet an understanding of the regulation of mRNA degradation under stress conditions remains elusive.

MamA essentiality in Mycobacterium smegmatis is explained by the presence of an apparent cognate restriction endonuclease.

Objective: Restriction-Modification (R-M) systems are ubiquitous in bacteria and were considered for years as rudimentary immune systems that protect bacterial cells from foreign DNA. Currently, these R-M systems are recognized as important players in global gene expression and other cellular processes such us virulence and evolution of genomes. Here, we report the role of the unique DNA methyltransferase in Mycobacterium smegmatis, which shows a moderate degree of sequence similarity to MamA, a previously characterized methyltransferase that affects gene expression in Mycobacterium tuberculosis and is important for survival under hypoxic conditions.

Artemisia annua and Artemisia afra extracts exhibit strong bactericidal activity against Mycobacterium tuberculosis.

Ethnopharmacological Relevance: Emergence of drug-resistant and multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is a major barrier to tuberculosis (TB) eradication, as it leads to longer treatment regimens and in many cases treatment failure. Thus, there is an urgent need to explore new TB drugs and combinations, in order to shorten TB treatment and improve outcomes. Here, we evaluated the potential of two Asian and African traditional medicinal plants, Artemisia annua, a natural source of artemisinin (AN), and Artemisia afra, as sources of novel antitubercular agents.

mRNA Degradation Rates Are Coupled to Metabolic Status in Mycobacterium smegmatis.

The success of as a human pathogen is due in part to its ability to survive stress conditions, such as hypoxia or nutrient deprivation, by entering nongrowing states. In these low-metabolism states, can tolerate antibiotics and develop genetically encoded antibiotic resistance, making its metabolic adaptation to stress crucial for survival. Numerous bacteria, including , have been shown to reduce their rates of mRNA degradation under growth limitation and stress.

Defining the Transcriptional and Post-transcriptional Landscapes of in Aerobic Growth and Hypoxia.

The ability of to infect, proliferate, and survive during long periods in the human lungs largely depends on the rigorous control of gene expression. Transcriptome-wide analyses are key to understanding gene regulation on a global scale. Here, we combine 5'-end-directed libraries with RNAseq expression libraries to gain insight into the transcriptome organization and post-transcriptional mRNA cleavage landscape in mycobacteria during log phase growth and under hypoxia, a physiologically relevant stress condition.

Transfer RNA Induces IL-12p70 via Synergistic Activation of Pattern Recognition Receptors within a Cell Network.

Upon recognition of a microbial pathogen, the innate and adaptive immune systems are linked to generate a cell-mediated immune response against the foreign invader. The culture filtrate of contains ligands, such as tRNA, that activate the innate immune response and secreted Ags recognized by T cells to drive adaptive immune responses. In this study, bioinformatics analysis of gene-expression profiles derived from human PBMCs treated with distinct microbial ligands identified a mycobacterial tRNA-induced innate immune network resulting in the robust production of IL-12p70, a cytokine required to instruct an adaptive Th1 response for host defense against intracellular bacteria.

Leaderless Transcripts and Small Proteins Are Common Features of the Mycobacterial Translational Landscape.

RNA-seq technologies have provided significant insight into the transcription networks of mycobacteria. However, such studies provide no definitive information on the translational landscape. Here, we use a combination of high-throughput transcriptome and proteome-profiling approaches to more rigorously understand protein expression in two mycobacterial species.

The Psp system of Mycobacterium tuberculosis integrates envelope stress-sensing and envelope-preserving functions.

The bacterial envelope integrates essential stress-sensing and adaptive functions; thus, envelope-preserving functions are important for survival. In Gram-negative bacteria, envelope integrity during stress is maintained by the multi-gene Psp response. Mycobacterium tuberculosis was thought to lack the Psp system since it encodes only pspA and no other psp ortholog.

RNA sequencing for transcript 5'-end mapping in mycobacteria.

Next-generation sequencing technologies facilitate the analysis of multiple important properties of the transcriptome in addition to gene expression levels. Here we describe a method for mapping RNA 5' ends in Mycobacterium tuberculosis, which allows the determination of transcriptional start sites (TSSs), comparative analysis of promoter usage under different conditions, and mapping of endoribonucleolytic processing sites. We describe in detail the procedures for constructing RNA sequencing libraries appropriate for RNA 5' end mapping using an Illumina sequencing platform.

DNA methylation impacts gene expression and ensures hypoxic survival of Mycobacterium tuberculosis.

DNA methylation regulates gene expression in many organisms. In eukaryotes, DNA methylation is associated with gene repression, while it exerts both activating and repressive effects in the Proteobacteria through largely locus-specific mechanisms. Here, we identify a critical DNA methyltransferase in M.

Interaction between the Msh2 and Msh6 nucleotide-binding sites in the Saccharomyces cerevisiae Msh2-Msh6 complex.

Indirect evidence has suggested that the Msh2-Msh6 mispair-binding complex undergoes conformational changes upon binding of ATP and mispairs, resulting in the formation of Msh2-Msh6 sliding clamps and licensing the formation of Msh2-Msh6-Mlh1-Pms1 ternary complexes. Here, we have studied eight mutant Msh2-Msh6 complexes with defective responses to nucleotide binding and/or mispair binding and used them to study the conformational changes required for sliding clamp formation and ternary complex assembly. ATP binding to the Msh6 nucleotide-binding site results in a conformational change that allows binding of ATP to the Msh2 nucleotide-binding site, although ATP binding to the two nucleotide-binding sites appears to be uncoupled in some mutant complexes.

Chimeric Saccharomyces cerevisiae Msh6 protein with an Msh3 mispair-binding domain combines properties of both proteins.

Msh2-Msh3 and Msh2-Msh6 are two partially redundant mispair-recognition complexes that initiate mismatch repair in eukaryotes. Crystal structures of the prokaryotic homolog MutS suggest the mechanism by which Msh6 interacts with mispairs because key mispair-contacting residues are conserved in these two proteins. Because Msh3 lacks these conserved residues, we constructed a series of mutants to investigate the requirements for mispair interaction by Msh3.

The N terminus of Saccharomyces cerevisiae Msh6 is an unstructured tether to PCNA.

The eukaryotic MutS homolog complexes, Msh2-Msh6 and Msh2-Msh3, recognize mismatched bases in DNA during mismatch repair (MMR). The eukaryote-specific N-terminal regions (NTRs) of Msh6 and Msh3 have not been characterized other than by demonstrating that they contain an N-terminal PCNA-interacting motif. Here we have demonstrated genetically that the NTR of Msh6 has an important role in MMR that is partially redundant with PCNA binding.