Mycobacterium | EPFL Graph Search

Mycobacterium is a genus of over 190 species in the phylum Actinomycetota, assigned its own family, Mycobacteriaceae. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis (M. tuberculosis) and leprosy (M. leprae) in humans. The Greek prefix myco- means 'fungus', alluding to this genus' mold-like colony surfaces. Since this genus has cell walls with a waxy lipid-rich outer layer that contains high concentrations of mycolic acid, acid-fast staining is used to emphasize their resistance to acids, compared to other cell types. Mycobacterial species are generally aerobic, non-motile, and capable of growing with minimal nutrients. The genus is divided based on each species' pigment production and growth rate. While most Mycobacterium species are non-pathogenic, the genus' characteristic complex cell wall contributes to evasion from host defenses. Microbiology Morphology Mycobacteria are aerobic

More Medicines for Tuberculosis: Fuelling Drug Discovery against Mycobacterium tuberculosis

Shi-Yan Caroline Foo

Tuberculosis (TB), whose etiological agent is Mycobacterium tuberculosis (M. tuberculosis), has plagued humanity since antiquity. Even with chemotherapy available today, TB is the leading cause of death due to an infectious disease. Modern day factors, such as the HIV epidemic and the emergence of drug-resistant TB strains, have redefined the complexities and challenges of tackling the TB pandemic. Current treatments for TB are lengthy, and poor adherence to such prolonged treatment further exacerbates the issue of drug resistance. Moreover, therapies for drug-resistant TB have low cure rates. There is therefore a pressing need for improved therapies that are short and efficacious against all TB strains, which can be achieved through new antimicrobials that are more potent and have novel mechanisms of action, in addition to being affordable, orally bioavailable, and without drug-drug interactions. Such new anti-TB drugs need to be discovered and developed through a long, risky, and costly process, in which attrition rates are high. While there are promising compounds currently being developed, including the benzothiazinones (BTZs), it is necessary to further populate and enhance the Global TB drug pipeline to ensure the availability of new drugs. This thesis aims to address this need through the discovery work of two new, highly promising families, the AX and PB compounds, and of BTZs. The piperazine-based AX analogs are easily synthesised and demonstrate potent activity against M. tuberculosis in vitro and in vivo. Their target, identified in this work, is the QcrB subunit of the cytochrome bc1-aa3 complex, a terminal oxidase of the mycobacterial respiratory chain. Notably, AX compounds are bactericidal in the absence of the alternate terminal oxidase, cytochrome bd. As this family interacts differently in the same binding site of QcrB as Q203, a drug candidate in clinical trials, AX compounds could potentially serve as a backup series for QcrB inhibitors. The PB family, derived from the natural product lapachol, is also easily synthesised and shows substantially improved activity against M. tuberculosis in vitro compared to the parent compound. PB analogs also demonstrated activity against the non-replicating bacillus and in infected macrophages. The mechanism of action of PB compounds relies on the F420 cofactor, although not on the F420-dependent nitroreductase Ddn, therefore this family has a novel mechanism of action which is highly specific to M. tuberculosis. To support the clinical development of PBTZ169, the mechanism of resistance to BTZs was further elucidated in this thesis. Five mutations at cysteine 387 of the target enzyme, DprE1, were identified as mediating resistance to BTZs, which would serve as resistance markers for clinical screening. The impact of these mutations on M. tuberculosis and on DprE1 was further characterised, revealing a fitness cost imposed on the bacillus intracellularly and reduced catalytic efficiency of the enzyme. This thesis additionally contributed to the characterisation of a PBTZ169 backup series and the design of an optimal regimen with other TB drugs. The compounds presented herein merit further optimisation so their full antibiotic potential may be realised for TB, and possibly for other mycobacterial diseases as well. Altogether, this thesis has contributed to the fuelling of drug discovery against M. tuberculosis, and a step towards more medicines for TB.

EPFL2018

Innovative Tools and in vivo Methods for Tuberculosis Drug Discovery and Development

Raphael Christopher Sommer

Worldwide, tuberculosis (TB) is the leading cause of death due to a single infectious agent, claiming 1.6 million human lives and causing >10 million new cases in 2017 alone, mostly in low-income regions. The intracellular pathogen Mycobacterium tuberculosis is the etiological agent and generally infects the lungs. Although treatments exist, curing TB requires long and often expensive chemotherapy, frequently accompanied by undesirable side-effects. In the absence of an effective vaccine and with the increasing emergence of multidrug resistance, new and more powerful antibiotics are urgently needed to control the global TB crisis. In this respect, benzothiazinones (BTZs), a class of new anti-TB compounds, have recently been discovered. Macozinone (PBTZ169), that has reached phase I and II clinical trials in Switzerland and Russia, respectively, is undoubtedly the most promising drug of this category. We derivatized PBTZ169 into a fluorescent chemical probe that efficiently, covalently and selectively labels the enzyme DprE1, thereby localizing the target of BTZs to the periplasm at the poles of actinobacteria. We further discuss the potential applications of such probes for in vivo imaging, mycobacterial detection and designing new diagnostic tools. In general, the search for novel anti-mycobacterial drugs is an extremely long process, delaying the progression of new compounds down the pipeline and to the clinic. Assessing drug efficacy in vivo typically requires extensive resources and is time-consuming. This process can be shortened by applying alternative and innovative methods devised in this thesis, namely the implementation of in vivo imaging procedures and use of the zebrafish embryo model of mycobacterial infection for drug discovery and development. In vivo imaging enables bacterial loads in animal models to be assessed in real time, by means of fluorescence or bioluminescence imaging. We constructed, optimized and characterized genetically engineered near-infrared fluorescent reporters of the pathogens Mycobacterium marinum and M. tuberculosis that allow direct visualization of bacteria in infected zebrafish and mice, respectively. Furthermore, we show that the fluorescence level accurately reflects the bacterial load, as determined by colony forming unit enumeration, thus enabling the efficacy of antibiotic treatment to be assessed in live animals. Alternatively, we applied autobioluminescent M. tuberculosis to evaluate drug efficacy in mice and demonstrated that our system enables the therapeutic response to be followed in real time. Longitudinal imaging also reduces the number animals required thus providing a substantial gain of time and resources. Finally, the zebrafish embryo is an inexpensive and faster alternative model that has been applied to investigate mycobacterial pathogenesis. We implemented this model of M. marinum infection and successfully applied it to assess the activity of several drugs in vivo. Altogether, the innovative methods described in this work are advantageous for TB drug discovery and development, especially for the in vivo evaluation of new anti-mycobacterial candidates, and provide more ethically acceptable approaches and biomedical applications.

EPFL2019

Genomics and transcriptomics of the Mycobacterium tuberculosis complex

Swapna Uplekar

The goal of eliminating tuberculosis (TB) by 2050 depends on the development of improved TB diagnostics, drugs and vaccines. Advances in these areas require a deep understanding of the disease and its causative agent, Mycobacterium tuberculosis (M. tb). Mycobacterial species that cause TB in humans and other mammalian hosts are grouped within the M. tb complex. Development of powerful technologies such as next-generation sequencing and microarrays opened up new avenues for comparative and functional genomics of the M. tb complex. Due to the large and increasingly complex datasets generated from these technologies, the bottleneck in biological investigation has shifted from data generation to analysis. The objectives of this thesis were to establish and employ strategies for the analysis, integration, and interpretation of high-throughput sequencing and microarray datasets using a range of bioinformatics and statistical tools. In the area of comparative genomics, we assessed the genetic diversity in the M. tb complex using various methods, such as SNP (single nucleotide polymorphism) genotyping, automated Sanger sequencing and next-generation sequencing. In a study comparing the genomes of the virulent M. bovis and M. bovis BCG vaccine strains, we identified a set of SNPs that were common to all BCG strains, and could provide novel insights on the molecular basis of BCG attenuation. In another study, we surveyed the genetic variation in the highly immunodominant esx gene family among clinical isolates of M. tb and identified sequence polymorphisms in known T- cell epitopes on Esx proteins that could affect their immunogenicity. We exploited the power of next-generation sequencing to detect sequence variation among M. tb strains that could result in phenotypic differences. By comparing the genomes of drug-resistant mutants with the sensitive wild-type strain we were able to identify the target of the anti-TB drug, pyridomycin. Using a similar approach we identified a mutation that makes M. tb strains incapable of producing PDIMs (phthiocerol dimycocerosates), which are cell wall associated lipids involved in M. tb virulence. In the area of functional genomics, we mapped genome-wide binding sites for transcription factors using chromatin immunoprecipitation followed by hybridization to microarrays (ChIP-on-chip) or sequencing (ChIP-seq), and performed transcription profiling by means of high-throughput cDNA sequencing (RNA-seq). We carried out a comprehensive study to characterize the whole transcriptome of M. tb in exponential and stationary phases of growth, and understand the genome-wide dynamics of two key components of the transcription machinery, namely, RNA polymerase and NusA. By systematic integration of the ChIP-seq and RNA-seq data, we identified a set of transcription units (TU) in the M. tb genome, and mapped their putative promoters. Analysis of RNAP and NusA binding across the promoter and body of TUs and their correlation with transcription uncovered new functional aspects of the transcriptional complex in M. tb. We also exploited the ChIP-on-chip and ChIP-seq technologies to define the regulon of the M. tb sigma factor F, and gain a better understanding of the regulatory role of the nucleoid associated protein, EspR. Altogether, this thesis has improved our knowledge of the evolution, physiology and virulence of the M. tb complex. In addition, we have established next generation sequencing as a powerful tool for comparative and functional studies, with potential applications in the clinical setting.

EPFL2012