Mycobacterial Genetics  


  HEADGicquel Brigitte / bgicquel@pasteur.fr
  MEMBERSDr. Badell-Ocando Edgar / Boudou Frédéric / Dr. Dasgupta Arunava / Dauger Pauline / Dos Vultos Tiago/ Favre-Rochex Sandrine / Gillard Marie-Renée / Dr Jackson Mary/Dr. Jang Jichan / Dr Martino Angelo / Mestre Olga
Dr Neyrolles Olivier / Polena Helena/ Rakotosamimanana Niaina/Dr Sola Christophe / Dr Tailleux Ludovic / Tanné Antoine / Dr Winter Nathalie


  Annual Report

Tuberculosis (TB) remains a major public health problem, causing about two million deaths each year. The currently used BCG vaccine is only efficient to protect against the severe forms of TB during infancy, and the efficacy of antibiotic treatments for TB may be reduced by the emergence of multidrug-resistant (MDR) or extremely resistant (XDR) tuberculosis. The understanding of host pathogen interactions during TB infection is important for the design of new therapeutic and preventive initiatives. Interactions of M. tuberculosiswith its host was undertaken using global strategies, 1) transcriptional profiling, 2) the isolation of attenuated mutants, 3) the identification of polymorphism in a series of genes involved in genome plasticity, 4) the study of a vaccine candidate based on a phoPinactivated M. tuberculosis strain, and 5) responses to BCG vaccination.

1) Transcriptional profiling using two types of micro-arrays, one covering the genome of M. tuberculosisand the other covering the human genome has allowed for the first time to investigate gene expression changes in both M. tuberculosisand its human host cells, macrophages and dendritic cells. In addition to common responses, we could identify eukaryotic and microbial transcriptional signatures that are specific to the cell type involved in the infection process. M. tuberculosis showed a marked stress response when inside dendritic cells, which is in accordance with the low permissivity of these specialized phagocytes to the tubercle bacillus and to other pathogens. By contrast, the mycobacterial transcriptome inside macrophages reflects that of replicating bacteria. On the host cell side, differential responses to infection in macrophages and dendritic cells were identified in genes involved in oxidative stress, intracellular vesicle trafficking and phagosome acidification. This study provided a solid framework for individually investigating pathogenicity mechanisms at molecular level.

2) The isolation of mutants impaired in intracellular growth by screening representative libraries of M. tuberculosis mutants allowed the identification of several virulence loci. One of them was shown to be located on a DNA segment horizontally transferred from another bacterial species to the M. tuberculosisancestor. Thanks to a bioinformatics approach, a subsequent analysis of the M. tuberculosisgenome identified several loci where horizontal transfer occurred. Comparison of these loci in different clinical strains showed that horizontal transfers had occurred in ancestral strain and are subjected to variations that are now studied in recent clinical strains.

3) We have analyzed the polymorphism of 56 genes involved in DNA repair, replication and recombination (3R genes) in a representative collection of 92 clinical isolates. We identified essentially single nucleotide polymorphisms. A higher level of polymorphism was observed in 3R genes as compared to other house-keeping genes. Site frequency spectrum comparison of synonymous and non-synonymous variants and Ka/Ks ratio analysis suggest a general negative/purifying selection acting on these sets of genes that may lead to suboptimal 3R system activity. This situation, and the consequent lack of fidelity in genome maintenance, may serve as a starting point for the evolution of antibiotic resistance, fitness for survival and pathogenicity, possibly conferring a selective advantage in certain stressful situations. These findings suggest that 3R genes may play an important role in the evolution of highly clonal bacteria, such as M. tuberculosis. A 3R-based phylogenetic tree was constructed. It is a new tool for distinguishing between M. tuberculosis complex strains. With many more microbial genomes being sequenced, our results open the door to 3R gene-based studies of adaptation and evolution of other, highly clonal bacteria.

4) A two-component system (PhoP/PhoR) which belongs to the ompR/phoBsubfamily of response regulators family and which controls a large number of genes involved in lipid biosynthesis, has been identified, thus leading to the construction of a M. tuberculosisphoP knocked out mutant. This new vaccine candidate provided a higher degree of protection against TB than BCG in animal models.

5) Although BCG is widely used, the immune mechanisms resulting in the establishment of partial protection are poorly understood. In a mouse model of intradermal vaccination, we observed that neutrophils are the principal BCG host cells, rapidly recruited in both the dermis and the lymph nodes upon vaccination Thus, neutrophils, like Dcs, migrate to the lymphoid tissues via the afferent lymphatic system and can transport live micro-organisms. We recently demonstrated using mouse or human cells, that Mycobacterium bovisBCG–infected neutrophils cooperate with dendritic cells to induce specific T-cell responses against BCG antigens.

Keywords: Tuberculosis, vaccine, transcriptional profiling , neutrophils, antibiotic resistance, antimutator, DNA repair, virulence, pathogenicity

Gemyc.jpg

Transcriptional profiling of dendritic cells (DC) and macrophages (MP) infected by Mybobacterium tuberculosis, at different times post infection (p.i.)



  Publications

Neyrolles O, Hernández-Pando R, Pietri-Rouxel F, Fornès P, Tailleux L, Barrios Payán JA, Pivert E, Bordat Y, Aguilar D, Prévost MC, Petit C, Gicquel B. Is adipose tissue a place for Mycobacterium tuberculosispersistence? PloS ONE. 2006 1:e43.

Dos Vultos T, Blázquez J, Rauzier J, Matic I, Gicquel B. Identification of Nudix hydrolase family members with an antimutator role in Mycobacterium tuberculosisand Mycobacterium smegmatis.J Bacteriol. 2006 188:3159-61.

Rosas-Magallanes V, Deschavanne P, Quintana-Murci L, Brosch R, Gicquel B, Neyrolles O. Horizontal transfer of a virulence operon to the ancestor of Mycobacterium tuberculosis. Mol Biol Evol. 2006 23:1129-35.

Tailleux L, Pham-Thi N, Bergeron-Lafaurie A, Herrmann JL, Charles P, Schwartz O, Scheinmann P, Lagrange PH, de Blic J, Tazi A, Gicquel B, Neyrolles O. DC-SIGN induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis. PloS Med. 2005 12:e381.

Abadie V, Badell E, Douillard P, Ensergueix D, Leenen PJ, Tanguy M, Fiette L, Saeland S, Gicquel B, Winter N. Neutrophils rapidly migrate via lymphatics after Mycobacterium bovisBCG intradermal vaccination and shuttle live bacilli to the draining lymph nodes. Blood. 2005 106:1843-50.





Activity Reports 2007 - Institut Pasteur
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