Tuberculosis remains one of the most important infectious diseases, causing about 3 million deaths every year, the developing world accounting for the majority of cases. This disease is intimately linked to the pathogenesis of the causative agent, Mycobacterium. tuberculosis. This bacterium can lie quiescent in unidentified sites in the human host for years without producing overt disease and then revive to cause lesions and, in many cases, progressive tuberculosis. Bacterial genes that govern the latency state of the infection of M. tuberculosis will be identified and it will be established how they affect the cell cycle division. The project will seek to clarify the mechanism by which this bacteria shifts down to a dormant state and reverts to active growth again, and to describe morphologically and functionally the persistent M. tuberculosis cell. Association of IS6110 transpositions to dormancy will be assessed.
The aim of this project is to clarify the mechanisms by which tuberculosis shift down to dormant state and revert to active growth again. The specific objectives are:
1) to determine the influence of stress conditions (in vivo and in
vitro) on the expression of the genes involved in active duplication,
dormant state and resuscitation
2) to determine the expression of cell division genes as a function of the bacterial cell cycle
3) to determine the role of the genes previously identified by studying knockout mutant strains
4) to investigate the mechanisms involved that convert M. tuberculosis from an active growth to latency and vice versa
5) to determine the possible association of IS6110 transpositions and the evolutionary development of M. tuberculosis (dormancy and progressive disease).
By analysing the nucleotide sequence of the M. tuberculosis genome and by PCR amplification, the project will isolate some genes that are clearly homologues to those involved in replication, chromosome segregation and cell division. Unknown genes shift during dormant state and resuscitation will be identified from mycobacteria cultures using two different approaches. Firstly, flow cytometry will be used to sort cells from a library of M. bovis BCG Tngfp. M. bovis BCG strains emitting fluorescence during dormancy or resuscitation, according to the Wayne model, will be isolated and the sequence interrupted by Tngfp will be used to identify the homologue genes in M. tuberculosis. Secondly, DNA micro or macro–array-based gene response will be used to identify genes selectively expressed during dormancy or resuscitation. Fundamental information, such as transcriptional organisation and promoters’ activity, will be provided. The role of the genes previously identified on the ability of adaptation of this microorganism to environmental changes and eventual development of dormancy will be studied by analysing the gene statement under different conditions. This analysis includes the determination of the genetic expression in mycobacteria cultures and infected macrophages under stress conditions, as well as in infected mice during progressive disease and latent infected cell type. Knockout mutants will be constructed to determine the cellular function of several of these genes. The phenotype of the mutant strains will be analysed as well as their effect on the global genetic expression of the microorganism during progressive diseases, dormancy or reactivation. This last approach will allow identification of putative cellular targets of the latency or resuscitation factors studied. Finally, in order to define the process that led to the latency of M. tuberculosis, a morphological and functional characterisation of the cells during the shift down to the dormant state will be done. The putative role on IS6110 transcriptional events on the control of cell cycle and dormancy in M. tuberculosis will be assessed by analysing strains and samples from latent tuberculosis infections.
The genetic expression of genes involved in active duplication, dormant state and resuscitation under different environmental conditions and in synchronised cultures. The role of those genes along the bacterial cell cycle.
Possible impact of IS6110 insertions on the adaptation M. tuberculosis during the dormant state or reactivation thereafter; Description of the dormant state in M. tuberculosis: main effects and morphological changes that take place during the shift down to dormant state and the eventual regress to active growth.
Maria J. Garcia
Universidad Autonoma de Madrid
Departamento de Medicina Preventiva / Facultad de Medicina
Arzobispo Morcillo, s/n
Tel: +34 913 975440
Fax: +34 913 975353