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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 140778
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 01.01.2013 - 31.12.2015
Approved amount 627'000.00
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Keywords (7)

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

Lay Summary (French)

Lead
La tuberculose est un problème de santé publique colossal dont aucun pays n'est épargné. Cette maladie respiratoire représente un frein considérable à la croissance économique et ainsi à la stabilité sociale au niveau mondial.
Lay summary
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 disséminer.
Direct link to Lay Summary Last update: 19.12.2012

Responsible applicant and co-applicants

Employees

Publications

Publication
Lansoprazole is an antituberculous prodrug targeting cytochrome bc1.
Rybniker Jan, Vocat Anthony, Sala Claudia, Busso Philippe, Pojer Florence, Benjak Andrej, Cole Stewart T (2015), Lansoprazole is an antituberculous prodrug targeting cytochrome bc1., in Nature communications, 6, 7659-7659.
Mycobacterium tuberculosis Differentially Activates cGAS- and Inflammasome-Dependent Intracellular Immune Responses through ESX-1.
Wassermann Ruth, Gulen Muhammet F, Sala Claudia, Perin Sonia Garcia, Lou Ye, Rybniker Jan, Schmid-Burgk Jonathan L, Schmidt Tobias, Hornung Veit, Cole Stewart T, Ablasser Andrea (2015), Mycobacterium tuberculosis Differentially Activates cGAS- and Inflammasome-Dependent Intracellular Immune Responses through ESX-1., in Cell host & microbe, 17(6), 799-810.
Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis.
Korotkova Natalia, Piton Jérémie, Wagner Jonathan M, Boy-Röttger Stefanie, Japaridze Aleksandre, Evans Timothy J, Cole Stewart T, Pojer Florence, Korotkov Konstantin V (2015), Structure of EspB, a secreted substrate of the ESX-1 secretion system of Mycobacterium tuberculosis., in Journal of structural biology, 191(2), 236-44.
Anticytolytic screen identifies inhibitors of mycobacterial virulence protein secretion.
Rybniker Jan, Chen Jeffrey M, Sala Claudia, Hartkoorn Ruben C, Vocat Anthony, Benjak Andrej, Boy-Röttger Stefanie, Zhang Ming, Székely Rita, Greff Zoltán, Orfi László, Szabadkai István, Pató János, Kéri György, Cole Stewart T (2014), Anticytolytic screen identifies inhibitors of mycobacterial virulence protein secretion., in Cell host & microbe, 16(4), 538-48.
EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis.
Zhang Ming, Chen Jeffrey M, Sala Claudia, Rybniker Jan, Dhar Neeraj, Cole Stewart T (2014), EspI regulates the ESX-1 secretion system in response to ATP levels in Mycobacterium tuberculosis., in Molecular microbiology, 93(5), 1057-65.
In vitro and in vivo activities of three oxazolidinones against nonreplicating Mycobacterium tuberculosis.
Zhang Ming, Sala Claudia, Dhar Neeraj, Vocat Anthony, Sambandamurthy Vasan K, Sharma Sreevalli, Marriner Gwendolyn, Balasubramanian V, Cole Stewart T (2014), In vitro and in vivo activities of three oxazolidinones against nonreplicating Mycobacterium tuberculosis., in Antimicrobial agents and chemotherapy, 58(6), 3217-23.
The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors.
Townsend Philip D, Jungwirth Britta, Pojer Florence, Bußmann Michael, Money Victoria A, Cole Stewart T, Pühler Alfred, Tauch Andreas, Bott Michael, Cann Martin J, Pohl Ehmke (2014), The crystal structures of apo and cAMP-bound GlxR from Corynebacterium glutamicum reveal structural and dynamic changes upon cAMP binding in CRP/FNR family transcription factors., in PloS one, 9(12), 113265-113265.
The cysteine desulfurase IscS of Mycobacterium tuberculosis is involved in iron-sulfur cluster biogenesis and oxidative stress defence.
Rybniker Jan, Pojer Florence, Marienhagen Jan, Kolly Gaëlle S, Chen Jeffrey M, van Gumpel Edeltraud, Hartmann Pia, Cole Stewart T (2014), The cysteine desulfurase IscS of Mycobacterium tuberculosis is involved in iron-sulfur cluster biogenesis and oxidative stress defence., in The Biochemical journal, 459(3), 467-78.
The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis.
Solans Luis, Gonzalo-Asensio Jesús, Sala Claudia, Benjak Andrej, Uplekar Swapna, Rougemont Jacques, Guilhot Christophe, Malaga Wladimir, Martín Carlos, Cole Stewart T (2014), The PhoP-dependent ncRNA Mcr7 modulates the TAT secretion system in Mycobacterium tuberculosis., in PLoS pathogens, 10(5), 1004183-1004183.
The phosphatidyl-myo-inositol mannosyltransferase PimA is essential for Mycobacterium tuberculosis growth in vitro and in vivo.
Boldrin Francesca, Ventura Marcello, Degiacomi Giulia, Ravishankar Sudha, Sala Claudia, Svetlikova Zuzana, Ambady Anisha, Dhar Neeraj, Kordulakova Jana, Zhang Ming, Serafini Agnese, Vishwas K G, Vishwas V G, Kolly Gaëlle S, Kumar Naveen, Palù Giorgio, Guerin Marcelo E, Mikusova Katarina, Cole Stewart T, Manganelli Riccardo (2014), The phosphatidyl-myo-inositol mannosyltransferase PimA is essential for Mycobacterium tuberculosis growth in vitro and in vivo., in Journal of bacteriology, 196(19), 3441-51.
Who will develop new antibacterial agents?
Cole Stewart T (2014), Who will develop new antibacterial agents?, in Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 369(1645), 20130430-20130430.
Mycobacterium tuberculosis EspB binds phospholipids and mediates EsxA-independent virulence.
Chen Jeffrey M, Zhang Ming, Rybniker Jan, Boy-Röttger Stefanie, Dhar Neeraj, Pojer Florence, Cole Stewart T (2013), Mycobacterium tuberculosis EspB binds phospholipids and mediates EsxA-independent virulence., in Molecular microbiology, 89(6), 1154-66.
Phenotypic profiling of Mycobacterium tuberculosis EspA point mutants reveals that blockage of ESAT-6 and CFP-10 secretion in vitro does not always correlate with attenuation of virulence.
Chen Jeffrey M, Zhang Ming, Rybniker Jan, Basterra Laetitia, Dhar Neeraj, Tischler Anna D, Pojer Florence, Cole Stewart T (2013), Phenotypic profiling of Mycobacterium tuberculosis EspA point mutants reveals that blockage of ESAT-6 and CFP-10 secretion in vitro does not always correlate with attenuation of virulence., in Journal of bacteriology, 195(24), 5421-30.

Awards

Title Year
Jan Rybniker was joint winner of the Swiss-TB award in 2015 2015

Associated projects

Number Title Start Funding scheme
133797 Biology Needs Ultra High Throughput DNA Sequencing 01.06.2011 R'EQUIP
125061 Structure-function analysis of the protein secretion system ESX-1 of Mycobacterium tuberculosis 01.04.2009 Project funding (Div. I-III)
162641 Integrated investigation of the ESX-1 protein secretion system of Mycobacterium tuberculosis 01.01.2016 Project funding (Div. I-III)

Abstract

The course of human evolution and economic development has been profoundly influenced by tuberculosis (TB), which still claims nearly 2 millions lives annually and causes massive 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 TB virulence determinant is a dedicated protein secretion system known as ESX-1 (ESAT-6 secretion system 1) that mediates both cell uptake and intercellular spread. There are five ESX systems in M. tb and, because these share a common genetic and biochemical organisation, their core constituents are referred to as Esx Conserved Components or Ecc proteins. These are membrane-bound proteins or ATPases that probably constitute and energize a transmembrane apparatus to secrete the cognate substrates. In the case of ESX-1 the substrates include the heterodimeric ESAT-6 proteins, EsxA and EsxB, and a variety of ESX-1 Secretion-associated Proteins, or Esps. It is far from clear which proteins comprise the apparatus nor what the effector proteins do.To clarify this situation and explore the druggability of ESX-1 we have employed an integrated approach combining studies of gene expression, biochemistry, biophysics and structural biology. Since 2009, we have demonstrated that EspR is a nucleoid-associated protein (NAP) that uses an atypical DNA-recognition mechanism to control gene expression. Most of the genes under EspR control are involved in cell envelope biogenesis and among them are espACD. These genes contribute to, and are essential for, ESX-1 function although the EspD protein seemingly does not require ESX-1 for secretion. EspD does, however, stabilize EspA, EspB and EspC so may function as a chaperone.We have expressed and purified most of the Ecc and Esp proteins to homogeneity in an attempt to glean functional insight from their 3D-structures and have elucidated the membrane topology of MycP1, EccB1, EccE1, EccCa1, EccCb1, and EccD1. Using X-ray crystallography high resolution structures of EspB, EspR, and EspR variants were obtained. EspR is a homodimer with an N-terminal DNA-binding domain and a C-terminal dimerization domain. A five-helix bundle comprises the N-terminal domain of EspB and this is highly similar in size and structure to the PE28-PPE41 heterodimer secreted by the ESX-5 system. As the EsxA-EsxB heterodimer is a four helix bundle it appears that ESX-substrates are likely to be all helical in nature. Examination of EspB by electron microscopy (EM), cryo-EM and other techniques revealed a heptameric donut-shaped protein that could form a channel with a lumen of 30 Å. This is sufficient to allow the passage of EsxA-EsxB.In the next phase, we will complete the EspB structure using SAXS (small angle X-ray scattering), integrate the EM data and build an atomic model. Crystal trials and EM examination of other Esp and Ecc proteins will continue together with studies of their topology and subcellular localization. We will attempt to purify the secretion apparatus from the membrane for characterization by mass spectrometry and EM. The key discovery that EspR is a NAP, involved in stabilizing long-range structures in the genome, highlights the importance to understand its interactions with other NAPs and regulators. This will be achieved by means of ChIP-Seq and chromosome conformation capture experiments. Since loss of EspR severely attenuates M. tb this opens up new avenues for drug discovery. These will be pursued by both rational approaches involving peptide inhibitor design and novel whole cell reporter assays to screen compound libraries. Inhibitors of Ecc ATPase activity will also be identified and pursued.We are confident that our combined findings will provide deep functional and mechanistic insight into a major determinant of mycobacterial pathogenesis and generate a conformational regulatory map of the M. tb chromosome. Furthermore, the small molecule inhibitors of ESX-function discovered 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 TB.
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