Ammunition for the TB Wars: Tuberculosis is a major human disease of global importance resulting from infection with the air-borne pathogen Mycobacterium tuberculosis , which is becoming increasingly resistant to all available drugs. An antituberculosis benzothiazinone compound kills mycobacterium in infected cells and in mice. Makarov et al. (p. 801 ) have identified a sulfur atom and nitro residues important for benzothiazinone's activity and used genetic methods and biochemical analysis to identify its target in blocking arabinogalactan biosynthesis during cell-wall synthesis. The compound affects the same pathway as ethambutol, and thus a benzothiazinone drug has the potential to become an important part of treatment of drug-resistant disease and, possibly, replace the less effective ethambutol in the primary treatment of tuberculosis.

Tuberculosis, a global threat to public health, is becoming untreatable due to widespread drug resistance to frontline drugs such as the InhA‐inhibitor isoniazid. Historically, by inhibiting highly vulnerable targets, natural products have been an important source of antibiotics including potent anti‐tuberculosis agents. Here, we describe pyridomycin, a compound produced by Dactylosporangium fulvum with specific cidal activity against mycobacteria. By selecting pyridomycin‐resistant mutants of Mycobacterium tuberculosis, whole‐genome sequencing and genetic validation, we identified the NADH‐dependent enoyl‐ (Acyl‐Carrier‐Protein) reductase InhA as the principal target and demonstrate that pyridomycin inhibits mycolic acid synthesis in M. tuberculosis. Furthermore, biochemical and structural studies show that pyridomycin inhibits InhA directly as a competitive inhibitor of the NADH‐binding site, thereby identifying a new, druggable pocket in InhA. Importantly, the most frequently encountered isoniazid‐resistant clinical isolates remain fully susceptible to pyridomycin, thus opening new avenues for drug development. →See accompanying article http://dx.doi.org/10.1002/emmm.201201811

Better antibiotics capable of killing multi-drug-resistantMycobacterium tuberculosisare urgently needed. Despite extensive drug discovery efforts, only a few promising candidates are on the horizon and alternative screening protocols are required. Here, by testing a panel of FDA-approved drugs in a host cell-based assay, we show that the blockbuster drug lansoprazole (Prevacid), a gastric proton-pump inhibitor, has intracellular activity againstM. tuberculosis.Ex vivopharmacokinetics and target identification studies reveal that lansoprazole killsM. tuberculosisby targeting its cytochromebc₁complex through intracellular sulfoxide reduction to lansoprazole sulfide. This novel class of cytochromebc₁inhibitors is highly active against drug-resistant clinical isolates and spares the human H⁺K⁺-ATPase thus providing excellent opportunities for targeting the major pathogenM. tuberculosis. Our finding provides proof of concept for hit expansion by metabolic activation, a powerful tool for antibiotic screens.

The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects through proteins like EspR, which controls Esx1 function, via regulation of the espACD operon. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in an M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. One of the best characterized two-component systems in Mycobacterium tuberculosis is represented by the PhoPR pair, with PhoR being the transmembrane sensor kinase and PhoP playing an essential part in controlling expression of virulence-associated genes, such as those encoding the ESX-1 secretion apparatus. Previous studies showed that mutations in phoP resulted in attenuation in the mouse model of infection, thus providing the basis for the development of a novel live attenuated Mycobacterium tuberculosis vaccine carrying a deletion in phoP which is today in clinical trials. To thoroughly investigate the role of PhoP in M. tuberculosis, we undertook a systems biology approach comprising ChIP-seq and RNA-seq technologies. We demonstrated binding of PhoP to at least 35 targets on the M. tuberculosis chromosome and direct impact on expression of 30 genes, while further amplification of the signal is provided by regulators acting downstream. The strongest binding site was located between rv2395 and PE_PGRS41, where transcription of the non-coding RNA (ncRNA) Mcr7 was demonstrated. Expression of Mcr7 was found to be restricted to M. tuberculosis species and totally silenced in a phoP mutant. Genetics and proteomics approaches proved that Mcr7 controls activity of the Twin Arginine (Tat) secretion system, thus modulating secretion of the immunodominant antigen Ag85 complex and the BlaC beta-lactamase.

The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes. A major infection mechanism employed by the causative agent of tuberculosis, Mycobacterium tuberculosis (Mtb), is the ESX-1 secretion system. It has been postulated that the DNA-binding protein EspR controls the virulence of Mtb by specifically regulating expression of the exported EspA protein, which is required for ESX-1 to function. Previous structural studies indicated that EspR forms dimers capable of multimerizing on DNA and forming loop structures, thus bringing together otherwise distant chromosomal regions. Such characteristics are reminiscent of nucleoid-associated proteins (NAPs), the histone equivalent in bacteria. Here we use ChIP-Seq technology to map EspR binding sites on the Mtb chromosome in living bacterial cells. Genome-wide analysis of EspR identified hundreds of binding-sites, with almost equal inter- and intra-genic distribution, and mostly found in proximity to genes associated with cell wall function. We validated a subset of EspR-binding sites experimentally and identified a consensus motif required for optimal binding affinity. Moreover, our study reveals that EspR expression varies with bacterial growth and that intracellular levels are not linked to EspR secretion. These findings corroborate the NAP nature of EspR and its dual roles, architectural and regulatory, that impact the Mtb chromosome and pathogenesis globally rather than the ESX-1 loci specifically.