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Transcriptional regulation and cellular localization of mycobacterial cell cycle proteins during dormancy

Final Report Summary - MYCOMANCY (Transcriptional regulation and cellular localization of mycobacterial cell cycle proteins during dormancy)

The aim of the MYCOMANCY project was to study the relation between the cell cycle and dormancy development in Mycobacterium tuberculosis (Mtb). Our final goal is to increase the knowledge on dormancy development and to identify potential candidates for the development of antitubercular drugs specifically active in dormant bacilli. Mtb is a facultative intracellular pathogen able to infect the human lung for decades without showing any symptom. Following immunity suppression caused by age, diseases, or malnutrition, tubercle bacilli can reactivate to give overt disease. About one third of the human population is estimated to be affected by latent tuberculosis representing a huge reservoir for the infection. The lack of drugs efficiently active on dormant bacteria is a major problem in fighting this important disease.

As a result of the project, they have identified at least one gene which could be a good drug candidate to hit bacteria during latent infection and a powerful candidate for the development of an antitubercular vaccine. Regarding the possible use of the sigma factor mutant as an attenuated vaccine, a patenting procedure has been activated as well as contacts with possible investors.

During the course of this project we produced and purified 14 different antibody against the Mtb cell division proteins. Eight of them were shown to be of a suitable for immunolocalisation and the respective proteins were already localised in actively growing Mtb. We are now analysing their cytolocalisation during dormancy and in mutants as whiA and whiB2 that are affected for the cell growth or shape. In summary, a set of useful tools for studying cell cycle in Mtb has been developed. These will represent an invaluable tool to characterise the cell cycle proteins in this powerful pathogen and their behaviour in dormant bacteria helping to understand the physiology of latency. We expect that the availability of these tools will boost the studies in this important still neglected field.

We developed an assay to screen an already available M. smegmatis library of mutants obtained by transposon mutagenesis for strains unable to survive to anaerobic condition. In a first screening, we analysed 10 000 mutants, identifying 278 affected clones. These were retested individually: 21 clones resulted to be really affected by hypoxia. Among them, 8 clones were completely unable to grow, so named severely affected and 13 clones were moderately affected by this stressful condition. The site of transposon insertion was identified for most of them to know the gene affected by the insertion. We are actually complementing the mutants with the wt genes of M. smegmatis and of Mtb, when an ortholog exists.

In summary, the project developed a new, simple assay to evaluate the ability of mycobacteria to resist to hypoxia and identified a set of genes involved in mycobacterial resistance in hypoxic environment. Further studies on the proteins encoded by these genes will increase our knowledge of the mechanism used by mycobacteria to survive in low oxygen environment and represent good candidate for the development of drugs active against dormant bacteria.