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Integrated investigation of the ESX-1 protein secretion system of Mycobacterium tuberculosis

English title Integrated investigation of the ESX-1 protein secretion system of Mycobacterium tuberculosis
Applicant Cole Stewart
Number 162641
Funding scheme Project funding (Div. I-III)
Research institution Global Health Institute EPFL SV-DO
Institution of higher education EPF Lausanne - EPFL
Main discipline Molecular Biology
Start/End 01.01.2016 - 31.12.2018
Approved amount 834'000.00
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Keywords (7)

Structural biology; Virulence factor secretion; Pathogenesis; Effector proteins; Drug discovery; Inhibitor screens ; Tuberculosis

Lay Summary (French)

Lead
La tuberculose, maladie humaine majeure, résulte de l'infection par le bacille Mycobacterium tuberculosis. On peut protéger efficacement les enfants contre la tuberculose en immunisant avec le vaccin vivant, le Bacille de Calmette et Guérin (BCG), cousin proche atténué du bacille tuberculeux. Le BCG est inoffensif chez l'homme en raison de la perte d'un bloc de gènes qui codent pour une partie du système de sécrétion protéique, ESX-1. Malgré le fait que ce système est indispensable pour le pouvoir pathogène de M. tuberculosis on ignore presque tout de sa régulation génétique et de son fonctionnement.
Lay summary

Contenu et objectifs du travail de recherche

Le but de notre projet est de déterminer le rôle de chaque composant d'ESX-1 et d'identifier et de caractériser les protéines qu'il sécrète en tirant profit d'approches génétique, biochimique et structurale.  Nous étudierons le contrôle de l’expression génétique et mettrons en œuvre des cribles afin d’identifier des inhibiteurs chimiques du système.  Par ce biais on pourrait atténuer M. tuberculosis lors de l’infection et mettre au point des médicaments anti-virulence.

 

Contexte scientifique et social du projet de recherche

En 2014 il y avait 9 millions nouveaux cas de tuberculose ainsi que 1,5 millions de morts. Les connaissances qui découlent de nos études seront d'une part fort utiles pour mieux comprendre comment le bacille tuberculeux déclenche la maladie et serviront d'autre part à la mis au point de mesures préventive et thérapeutique.

Direct link to Lay Summary Last update: 19.10.2015

Responsible applicant and co-applicants

Employees

Publications

Publication
Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation.
Odermatt Nina T, Sala Claudia, Benjak Andrej, Kolly Gaëlle S, Vocat Anthony, Lupien Andréanne, Cole Stewart T (2017), Rv3852 (H-NS) of Mycobacterium tuberculosis Is Not Involved in Nucleoid Compaction and Virulence Regulation., in Journal of bacteriology, 199(16), 129-17.
Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase.
Ahmed Wareed, Sala Claudia, Hegde Shubhada R, Jha Rajiv Kumar, Cole Stewart T, Nagaraja Valakunja (2017), Transcription facilitated genome-wide recruitment of topoisomerase I and DNA gyrase., in PLoS genetics, 13(5), 1006754-1006754.
EspC forms a filamentous structure in the cell envelope of Mycobacterium tuberculosis and impacts ESX-1 secretion.
Lou Ye, Rybniker Jan, Sala Claudia, Cole Stewart T (2017), EspC forms a filamentous structure in the cell envelope of Mycobacterium tuberculosis and impacts ESX-1 secretion., in Molecular microbiology, 103(1), 26-38.
Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b.
Benjak Andrej, Uplekar Swapna, Zhang Ming, Piton Jérémie, Cole Stewart T, Sala Claudia (2016), Genomic and transcriptomic analysis of the streptomycin-dependent Mycobacterium tuberculosis strain 18b., in BMC genomics, 17, 190-190.

Collaboration

Group / person Country
Types of collaboration
EPFL Switzerland (Europe)
- in-depth/constructive exchanges on approaches, methods or results
- Publication
- Research Infrastructure

Associated projects

Number Title Start Funding scheme
133797 Biology Needs Ultra High Throughput DNA Sequencing 01.06.2011 R'EQUIP
140778 Structure-function analysis of the protein secretion system ESX-1 of Mycobacterium tuberculosis 01.01.2013 Project funding (Div. I-III)

Abstract

The course of human evolution and global economic development have been profoundly influenced by tuberculosis (TB), which still claims over 1.5 million lives annually and causes extensive morbidity and loss of wealth. New drugs are required to treat this airborne disease that results from infection with Mycobacterium tuberculosis (M. tb), a slow-growing intracellular pathogen, which can persist indefinitely in the human body. The principal virulence determinant of M. tb is a dedicated protein secretion system known as ESX-1 (ESAT-6 secretion system 1) that is required for cell uptake, cell exit and intercellular spread. The ESX-1 system is composed of ~20 polypeptides referred to as Ecc (Esx Conserved Components) or Esp (ESX Secretion-associated protein) proteins. The main ESX-1 substrate is a heterodimer of the ESAT-6 proteins, EsxA and EsxB (EsxA/B) but it remains unclear exactly which proteins comprise the apparatus nor what the effector proteins do. Attenuation of the live TB vaccine, BCG (Bacille de Calmette et Guérin) resulted from loss of ESX-1.To investigate the function, composition and druggability of ESX-1, we will employ an integrated approach combining studies of gene expression, biochemistry, biophysics, structural biology and screening of chemical libraries. We have demonstrated that EspR, a nucleoid-associated protein (NAP), controls expression of genes involved in cell envelope biogenesis notably the espACD operon, which is essential for ESX-1 function and EsxA/B secretion. The contribution of other NAPs and regulators to ESX-1 expression and nucleoid structure is being explored by means of ChIP-Seq, RNA-Seq, proteomics and chromosome conformation capture experiments. EspA, EspC and EspD have been extensively studied thus revealing that EspD stabilizes EspA and EspC, and that EspC self-assembles during secretion. Inactivation of these three proteins blocks EsxA/B secretion and reduces virulence but does not cause full attenuation as in BCG. This is likely due to the secretion of EspB, a protein that binds bioactive phospholipids. Our structural analysis revealed that the N-terminal domain of EspB consists of a five-helix bundle, which is highly similar in size and structure to the PE28-PPE41 heterodimer secreted by the ESX-5 system. Examination of EspB by electron microscopy (EM), cryo-EM and other techniques revealed a heptameric donut that could form a channel. Other data indicate that the C-terminal domain of EspC mediates oligomerization and self-assembly in a similar manner to the needle protein of type 3 secretion systems. We are currently further investigating these aspects using structural, functional and immunological approaches.An innovative cell-based screen discovered several compounds that protected host fibroblasts and macrophages from ESX-1-mediated cytotoxicity following infection with M. tb. Two series are being pursued: the BTP series appears to inhibit MprB, the histidine kinase that controls espACD expression; the mechanism of action of the BBH series remains to be elucidated. Importantly, we showed that BTP or BBH treatment not only protects host cells from M. tb-mediated cytotoxicity but also modulates the innate immune response towards protection. Pursuing this line will help chemically dissect ESX-1 function and may give rise to antivirulence drugs. We are confident that our integrated investigation will generate regulatory, functional and mechanistic understanding of a major determinant of mycobacterial pathogenesis. Furthermore, the ESX-1 inhibitors being investigated could serve as leads for new antivirulence drugs for use in combination therapy for TB.
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