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Structure-function analysis of the protein secretion system ESX-1 of Mycobacterium tuberculosis

English title Structure-function analysis of the protein secretion system ESX-1 of Mycobacterium tuberculosis
Applicant Cole Stewart
Number 125061
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 01.04.2009 - 31.12.2012
Approved amount 468'000.00
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Keywords (9)

Tuberculosis; Pathogenesis; Crystallography; Virulence factor secretion; Effector proteins; Drug discovery; protein secretion; biochemistry; structural biology.

Lay Summary (French)

Lead
Lay summary
Lead: La tuberculose est un problème de santé publique colossal dont aucun pays n'est épargné. Cette maladie respiratoire réprésente un frein considérable à la croissance économique et ainsi à la stabilité sociale au niveau mondial. Résumé: La tuberculose, maladie humaine majeure, résulte de l'infection par le bacille Mycobacterium tuberculosis. On peut protéger efficacement les enfants contre la tuberculose en les 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 groupe de gènes qui codent pour un système de sécrétion protéique, ESX-1. Malgré le fait que ce système est indispensable pour le pouvoir pathogène de M. tuberculosis on ignore tout de son fonctionnement. But: L'objectif de notre projet multidisciplinaire 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 génétiques, biochimiques et structurales. Parallèlement nous utiliserons la microbiologie moléculaire et cellulaire afin d'établir la fonction de chacune des protéines lors de l'infection. Signification: Les connaissances qui découlent de l'étude d'ESX-1 seront d'une part fort utiles pour mieux comprendre comment la bactérie déclenche la maladie et serviront d'autre part à la mis au point de mesures préventives et thérapeutiques. Par exemple, en bloquant l'activité d'ESX-1 par une drogue on empêchera le bacille tuberculeux de se multiplier.
Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
High-resolution transcriptome and genome-wide dynamics of RNA polymerase and NusA in Mycobacterium tuberculosis
Uplekar Swapna (2012), High-resolution transcriptome and genome-wide dynamics of RNA polymerase and NusA in Mycobacterium tuberculosis, in Nucleic Acids Research, [Epub ahead of print], 1-17.
Streptomycin-Starved Mycobacterium tuberculosis 18b, a Drug Discovery Tool for Latent Tuberculosis
Zhang Ming (2012), Streptomycin-Starved Mycobacterium tuberculosis 18b, a Drug Discovery Tool for Latent Tuberculosis, in Antimicrobial Agents and Chemotherapy, 56(11), 5782-5789.
Virulence Regulator EspR of Mycobacterium tuberculosis Is a Nucleoid-Associated Protein.
Blasco Benjamin (2012), Virulence Regulator EspR of Mycobacterium tuberculosis Is a Nucleoid-Associated Protein., in PLoS Pathog, 8(3), e1002621- e1002631.
EspD is critical for the virulence-mediating ESX-1 secretion system in Mycobacterium tuberculosis
Chen Jeffery M (2011), EspD is critical for the virulence-mediating ESX-1 secretion system in Mycobacterium tuberculosis, in J Bacteriol, 194(4), 884-893.
ESAT-6 Secretion-Independent Impact of ESX-1 Genes espF and espG1 on Virulence of Mycobacterium tuberculosis.
Bottai Daria (2011), ESAT-6 Secretion-Independent Impact of ESX-1 Genes espF and espG1 on Virulence of Mycobacterium tuberculosis., in J Infect Dis , 203, 1155-1164.
Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis
Blasco Benjamin (2011), Atypical DNA recognition mechanism used by the EspR virulence regulator of Mycobacterium tuberculosis, in Mol Microbiol, 82(1), 251-264.
Comparative genomics of Esx genes from clinical isolates of Mycobacterium tuberculosis provides evidence for gene conversion and epitope variation
Uplekar Swapna (2011), Comparative genomics of Esx genes from clinical isolates of Mycobacterium tuberculosis provides evidence for gene conversion and epitope variation, in Infect Immun, 79(10), 4042-4049.
Towards anti-virulence drugs targeting ESX-1 mediated pathogenesis of Mycobacterium tuberculosis.
Chen Jeffery (2010), Towards anti-virulence drugs targeting ESX-1 mediated pathogenesis of Mycobacterium tuberculosis., in Drug Discovery Today, 7(1), e25-e31.
Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis
Sala Claudia (2010), Simple model for testing drugs against nonreplicating Mycobacterium tuberculosis, in Antimicrob Agents Chemother, 54(10), 4150-4158.
High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum
Jungwirth Britta, High-resolution detection of DNA binding sites of the global transcriptional regulator GlxR in Corynebacterium glutamicum, in Microbiology, 158.

Scientific events

Active participation

Title Type of contribution Title of article or contribution Date Place Persons involved
Dublin Academy of Pathogenomics and Infection Biology 12.06.2012 Dublin, Ireland
Infection Biology Seminar - Biozentrum 23.11.2011 Basel, Switzerland
Keynote address Webster Symposium 16.09.2011 Dunedin, New Zealand
Seminar - Malaghan Institute of Medical Research 12.09.2011 Wellington, New Zealand


Associated projects

Number Title Start Funding scheme
164090 Request for an Electron Detection Camera 01.01.2016 R'EQUIP
140778 Structure-function analysis of the protein secretion system ESX-1 of Mycobacterium tuberculosis 01.01.2013 Project funding (Div. I-III)
133797 Biology Needs Ultra High Throughput DNA Sequencing 01.06.2011 R'EQUIP

Abstract

Tuberculosis is arguably the most important infectious disease of humans, afflicting the healthy and immunocompromised alike thus claiming countless millions of lives and causing massive suffering worldwide. Mycobacterium tuberculosis, the airborne pathogen responsible for this disease, is slow-growing and can persist indefinitely in the human body. The principal virulence factor in the infection process and an important mediator of intercellular spread is a dedicated protein secretion system known as ESX-1 (ESAT-6 secretion system 1). Loss of ESX-1 by the live vaccine strains M. bovis BCG and M. microti accounts for their severe attenuation and exemplary safety record. EsxA (ESAT-6) is an immunodominant protein of 95 amino acids, and the prototype of the W-X-G 100 family found in many ESX-proficient Gram positive bacteria, like Staphylococcus aureus, the agent of the nosocomial disease MRSA. A heterodimer forms between EsxA and the related protein EsxB (also known as CFP-10), when two helical hairpins coil around each other in an antiparallel manner to form a four-helix bundle. In the current model, EsxA/B is guided by a cytoplasmic traffic ATPase (Rv3868) to the secretion apparatus, comprising several membrane proteins (Rv3869, Rv3870, Rv3871, Rv3877), then transported across the membrane in an ATP-dependent step. At low pH, following acidification of the phagosome on interferon-gamma-mediated activation of the macrophage, EsxA dissociates from its partner to interact with cholesterol-containing membranes and provoke lysis, which likely allows M. tuberculosis to escape into the cytoplasm or even further. There is growing evidence that other proteins such as EspA (Rv3616c) and EspB (Rv3881) are also secreted via ESX-1 and, by analogy to the type III and IV secretion systems, these may serve as effectors to subvert host cell functions.However, formal evidence is lacking for much of this scheme, which is based largely on genetic and preliminary biochemical findings. Here, we will apply an integrated approach combining molecular and cellular microbiology with structural biology to establish the precise nature and organization of the ESX-1 apparatus and distinguish its components from the substrates. This will be achieved by implementing three specific aims. 1. We will study the regulation of Esx/Esp gene expression in situ by the pH-responsive two component system, PhoP-PhoR, and by EspR, a positive regulator that is also secreted by ESX-1, using ChIP-on-chip (chromatin immunoprecipitation and hybridization to microarrays) and regulon analysis. In parallel, comparative genomics will be applied to the ESX-repertoire of clinical isolates to test the hypothesis that escape variants emerge in response to immune pressure. 2. ESX-1 and selected components will be purified then analysed by X-ray crystallography, cryo-electron microscopy and tomography to determine 3D-structures and build models. Purified proteins will be used in automated screens of chemical libraries to find inhibitors. 3. Acetylation of EsxA modifies its activity so proteomics will be used to study this and other post-translational modifications of ESX-1. Innovative target and cell-based assays will be developed to understand the function of EspA, and partners, and to identify inhibitory ligands.We expect the combined findings of this project to provide deep functional and mechanistic insight into a major determinant of mycobacterial pathogenesis that is also of relevance to other bacterial diseases, including MRSA. In addition, the small molecule inhibitors of ESX-function characterized during the course of this work could serve as lead compounds for the development of new antivirulence drugs of potential use in combination therapy for tuberculosis.
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