Mycobacterium tuberculosis structural biology
Coordinator: Martin Cohen-Gonsaud.

The first M. tuberculosis structure was elucidated at the CBS in 2002. Since then several others structures have been solved in the lab either using NMR or X-ray crystallography with 10 original solution and 5 original X-ray structures solved (10 published to date). Today, in collaboration with other researchers at the CBS numerous projects are on going in the lab. A strong emphasis is placed on the collaboration with microbiologists, immunologists or cell biologists. We thing that structural biology is an important technic to understand the mechanism behind the virulence and persistence of the bacillus. Our current project can be regrouped in three axes detailed below.

M. tuberculosis phospho-regulation
Regulation via Ser/Thr Protein Kinases (STPK) has emerged as an essential mechanism in controlling key pathways in the bacillus (metabolism, cell division, expression regulation). Despite the fact that the STPKs have been extensively studied, the molecular mechanisms of its phosphoregulation were poorly understood. In collaboration with different laboratories we engaged studies to understand the activity modulation of STPKs substrates to unravel new regulation pathways within the bacteria. We solved the solution structure of the unphosphorylated and phosphorylated isoforms of the OdhI protein (also named GarA), a central regulator of the tricarboxylic acid (TCA). We discovered major conformational changes upon phosphorylation of a disordered region of the protein (Barthe 2009). We studied a FHA modular protein called Rv0020c. We solved the structure of its domains by NMR and studied their interaction with STPK demonstrating a fine tuning of the STPK/Rv0020c interaction via the phosphorylation of an unstructured STPK domain (Roumestand 2011). Furthermore, we solved the structure of a transcriptional regulator in solution, and revealed a DNA binding regulation mediated by STPK phosphorylation (Cohen-Gonsaud 2009). Other studies in M.tuberculosis focussed on the STPK regulation of the essential mycolic acid biosynthesis (Veyron-Churley 2012), the S-Adenosylhomocysteine hydrolase activity (Corrales 2013), and the CcpA regulation in S. aureus (Leiba 2012).
One of the keys of Mycobacterium tuberculosis' success as a pathogen is its ability to persist in the host organism in a latent state for years after the first phase of infection. We have been interested for long time in the exit mechanism of latency (Cohen-Gonsaud 2005). The STPK PknB has been proposed to be the receptor for a dormancy exit signal. With proven combined experience in the field, our team and Galiana Mukamolova's group (University of Leicester) initiated a study to isolate and study the potential molecular determinant of resuscitation in M. tuberculosis. We first solved the structure of the external domain of the kinase in solution that revealed an original fold (Barthe 2010) and lead to new hypothesis about the PknB activation.

 

 

Persistence and latency
One of the main projects over the last years has been related to the mechanism that has been unravelled by our collaborator Galiana Mukamolova at the University of Leicester. They demonstrated that three of the main TB antibiotics have the ability to enhance the survival of the bacillus during the stationary phase. Combining molecular modelling, biochemical, genetic, transcriptomic, and animal studies we proposed a complete model to explain the observed phenomenon (Turapov 2014). Another important project has been initiated in collaboration with the group of Pricille Brodin (Institut Pasteur Lille). The ability of the bacillus to arrest phagosome maturation is a major mechanism that allows its survival within host macrophages. To identify mycobacterial genes involved in this process, they developed a high-throughput phenotypic cell-based assay enabling individual sub-cellular analysis of over 11,000 M.tuberculosis mutants. Structural biology studies on essential identified modulator are undergoing at the CBS.

 

Internal Collaborations :

P. Barthe, G. Labesse, J. Gracy, J. Bonnet.

 

External Collaborations:

· G. Mukamolova – University of Leicester – Leicester – UK

· N. Keep – Birkbeck College – London – UK

· V. Molle – DIMNP – Montpellier – France

· François Letourneur – DIMNP – Montpellier – France

· P. Brodin – Institut Pasteur Lille – Lille – France