Tuberculose

Résumé

La tuberculose est une maladie infectieuse causée par la bactérie Mycobacterium tuberculosis, qui se transmet par voie aérienne, avec des signes cliniques variables. Elle touche le plus souvent les poumons et peut parfois atteindre d’autres organes. Elle arrive en tête des causes de mortalité d'origine infectieuse à l’échelle mondiale, devant le sida. L'Organisation mondiale de la santé (OMS) rapporte à travers son rapport annuel consacré à la tuberculose et sorti en 2015 que de personnes sont mortes de la tuberculose l’année précédente. Parmi les nouveaux cas de tuberculose enregistrés en 2019, 87 % sont survenus dans les trente pays présentant la plus forte charge de la maladie. Deux tiers des cas sont concentrés dans huit pays, avec l’Inde en tête, suivie de l’Indonésie, de la Chine, des Philippines, du Pakistan, du Nigéria, du Bangladesh et de l’Afrique du Sud. Le rapport 2015 de l’OMS rapporte également que la mortalité a baissé de 47 % depuis 1990 grâce en grande partie au d

Source officielle

https://fr.wikipedia.org/wiki/Tuberculose

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Functional characterization of the virulence determinant ESX-1 from Mycobacterium tuberculosis

Paloma Julia Soler Arnedo

Tuberculosis (TB) is a chronic infectious disease that mainly affects the lungs and causes extensive human morbidity and mortality. It results from infection with Mycobacterium tuberculosis, a slow-growing intracellular pathogen that can replicate and survive inside macrophages. M. tuberculosis relies on the specialised ESX-1 secretion system to export virulence factors needed for intracellular spread and pathogenesis. The ESX-1 apparatus is a multi-subunit nanomachine composed of ~20 polypeptides including membrane proteins, ATPases, proteases, chaperones and substrates. Although many of the individual components of this secretion system have been characterised, the overall mechanism underlying ESX-1 secretion is still far from clear. To obtain a more comprehensive picture of the functioning of the ESX-1 apparatus, we have studied various components which were largely unexplored in M. tuberculosis. The structural component EccE1, the ESX-1 specific protein EspL and the transcriptional regulator WhiB6 have been investigated in this thesis using an integrative approach involving genetics, biochemistry, proteomics and microscopy. We have demonstrated that EccE1 is a membrane- and cell-wall associated protein critical for secretion of ESX-1 substrates and M. tuberculosis-mediated cell lysis. Deletion of eccE1 from the chromosome severely compromised secretion of EsxA, EsxB, EspA and EspC but not EspB. Localization studies using a florescent-fusion protein showed that EccE1 localises to the poles of M. tuberculosis in the presence of an active ESX-1 system. Our study also shows that EspL is a cytosolic protein needed for stabilising EspE, EspF and EspH, suggesting that it acts as a specific chaperone of the ESX-1 secretion system. Moreover, EspL was shown to interact with EspD and to be important for the secretion of ESX-1 substrates. Lack of EspL resulted in a growth defect ex vivo, loss of cytotoxicity and reduction of innate cytokine production demonstrating its critical role in M. tuberculosis virulence. Analysis of the transcriptional response revealed that the only gene deregulated in the absence of espL was whiB6, encoding a transcriptional factor that positively controls ESX-1 genes. To explore the role of this regulator in M. tuberculosis virulence, we generated a deletion mutant of whiB6 and discovered that its loss resulted in severe reduction of cytotoxicity ex vivo. Overall this investigation improves our current understanding of the ESX-1 secretion system and of the molecular basis of M. tuberculosis virulence. The increased knowledge of the complex interactions between the pathogen and the human host will hopefully translate into new strategies to control the spread of TB.

EPFL2018

Chargement

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

Chargement

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

Chargement

EPFL2018

Functional characterization of the virulence determinant ESX-1 from Mycobacterium tuberculosis

Paloma Julia Soler Arnedo

EPFL2018

EPFL2019