Mycobacterial Genetics  


  HEADGicquel Brigitte / bgicquel@pasteur.fr
  MEMBERSBadell-Ocando Edgar, Ph.D. / Boudou Frédéric / Cimino Mena, Ph.D./ Haikine Natacha/ Dos Vultos Tiago, Ph.D./ Favre-Rochex Sandrine / Gillard Marie-Renée / Laurent Marc / Jang Jichan, Ph.D. /Martino Angelo, Ph.D. / Mestre Olga/ Polena Helena/ Rakotosamimanana Niaina/ Sola Christophe, Ph.D. / Tailleux Ludovic, Ph.D. /Murray Alan, Ph.D. / Winter Nathalie, Ph.D. /Charles Patricia/ Dubois Nicolas/Pierre-Audigier Catherine, MD, Ph.D


  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 ofMycobacterium 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 phoP inactivated strain, and 5) responses to BCG vaccination. In addition a group of TB expertise was set up(6).

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. tuberculosisshowed 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 and identifying biomarkers associated with TB infection.

2) The isolation of mutants impaired in intracellular growth by screening representative libraries ofM. tuberculosismutants 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. tuberculosis genomeidentified 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 asM. tuberculosis. A 3R-based phylogenetic tree was constructed. It is a new tool for distinguishing betweenM. tuberculosiscomplex 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. The role of the different in silico identified DNA repair genes is being studied by inactivating them and looking at the resultant phenotypes.

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. tuberculosis phoPknocked out mutant. This new vaccine candidate provided a higher degree of protection against TB than BCG in animal models. The basis of attenuation ofphoPstrains is being studied.

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. 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. A mucosal route of vaccination was investigated using a recombinant BCG expressing a fusion of the 85B and ESAT6 major antigens.

6) A group of expertise was set up with the aim to help laboratories for the implementation and use of molecular tools for the diagnosis, drug susceptibility testing and molecular epidemiology of tuberculosis. Training workshops are organised to exchange expertise between TB laboratories (www.moleculartb.org).

Keywords: Tuberculosis, vaccine, transcriptional profiling , neutrophils, antibiotic resistance, antimutator, DNA repair, virulence, pathogenicity, diagnosis, drug susceptibility testing, molecular epidemiology

Gemyc.jpg

legend: Phylogenetic network based on the total set of 3R genes SNPs. Colors correspond to M. tuberculosisfamily strains.



  Publications

Horizontally acquired genomic islands in the tubercle bacilli. Jang J, Becq J, Gicquel B, Deschavanne P, Neyrolles O. Trends Microbiol. (2008) 16:303-8.

Evolution and diversity of clonal bacteria: the paradigm of Mycobacterium tuberculosis. Dos Vultos T, Mestre O, Rauzier J, Golec M, Rastogi N, Rasolofo V, Tonjum T, Sola C, Matic I, Gicquel B. PLoS ONE. (2008) 3:e1538.

Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages. Tailleux L, Waddell SJ, Pelizzola M, Mortellaro A, Withers M, Tanne A, Castagnoli PR, Gicquel B, Stoker NG, Butcher PD, Foti M, Neyrolles O. PLoS ONE. (2008) 3:e1403.

A point mutation in the two-component regulator PhoP-PhoR accounts for the absence of polyketide-derived acyltrehaloses but not that of phthiocerol dimycocerosates in Mycobacterium tuberculosis H37Ra. Chesne-Seck ML, Barilone N, Boudou F, Gonzalo Asensio J, Kolattukudy PE, Martín C, Cole ST, Gicquel B, Gopaul DN, Jackson M. J Bacteriol. (2008) 190:1329-34.

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



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Activity Reports 2009 - Institut Pasteur
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