Project no.LSHP-CT-037566
Project acronym: MYCOMANCY
Project title: Transcriptional regulation and cellular localization of mycobacterial cell cycle proteins during dormancy
Activity report
Project execution
The aim of this 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.
This project stands on three main pillars: i) the construction and characterization of several mutants lacking putative cell cycle regulators; ii) the development of antibody against the main cell division proteins of Mtb in order to allow their cytolocalization; iii) the identification of M. smegmatis genes required for survival in anoxic conditions.
i) Construction and characterization of mutants lacking putative cell cycle regulators Using different techniques, we obtained deletion mutants for whiB5 and for the two SoJ orthologs Rv3213c and Rc1708. Conditional mutants, however, were obtained for Rv3213c, whiA, whiB1, and whiB2. We also obtained Mtb strains overproducing Rv3213c, whiA, whiB1, and whiB5. The mutant strains were first analyzed for their growth, cell shape, and colony morphology. Both the whiA and the whiB2 mutant were impaired for growth with the latter affected also in the cell shape. Moreover, overexpression of whiA and whiB5 was shown to be toxic.
When the mutants were analyzed in condition mimicking dormancy (as nutrient starvation, hypoxia, and NO exposure) only the whiB5 mutant showed relevant phenotypes. Interestingly, this mutant also showed a major attenuation during both active and persistent infection in mice, suggesting that it could be a very important gene related to virulence and dormancy development. Since WhiB5 is a putative transcriptional regulator, this mutant was also subjected to proteomic analysis and RNA profiling in order to identify its regulon.

The transcription of the genes object of the study was characterized during active and latent infection in mice. Interestingly, during active infection, all of them was expressed, but their expression level peaked at different times after infection; however, during latent infection, the expression of all of them was minimal, to fast increase after reactivation.
During the first year of the project we also analyzed virulence of a sigma factor mutant which was previously obtained. Interestingly, we found that it was able to stimulate a strong and peculiar immune response suggesting that it could represent a powerful attenuated vaccine. Recently, we performed challenge experiments strongly supporting this hypothesis.
In summary we 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.
ii) Development of antibody against the main cell division proteins of Mtb and their cytolocalization
The study of Mtb cell cycle during active growth and dormancy will not be possible until proper tools as clean antibody against the main division proteins will not be developed. During the course of this project we produced and purifies 14 different antibody against the Mtb cell division proteins. Eight of them were shown to be of a suitable for immunolocalization and the respective proteins were already localized in actively growing Mtb. We are now analysing their cytolocalization 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 characterize 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.
iii) Identification of M. smegmatis genes required for survival in anoxic conditions
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 analyzed 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, we 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
Dissemination and use
Members of the project presented data obtained in this project to participated to 12 International and 2 national conferences where they could meet with other international expert of the fields of mycobacterial physiology, latency and cell cycle. Thirteen publication related to the project (11 in peer review international journal, and 2 in a book edited by ASM). At least other 4 publication related to the project are actually in preparation and will be soon submitted for publication. A patent for the use of the sigma factor mutant as a vaccine has been filed and possible investors has been contacted.
Co-ordinator
Riccardo Manganelli
Dipartimento di Istologia, Microbiologia e Biotecnologie Mediche
Università di Padova
35121 Padova, Italy
Tel. +39 0490272366
Fax +39 0498272355
e-mail riccardo.manganelli@unipd.it
Partners
Jean-Marc Reyrat
Inserm U570
Université Descartes Paris V, Site Necker,
156 rue de Vaugirard
75730 PARIS cedex 15

jmreyrat@necker.fr
Leiria Salazar
IVIC
Km 11 Carretera Panamericana
Altos de Pipe
Estdo Miranda, Venezuela
or: PO Box: Apartado 21827 Caracas 1020A, Venezuela
Tel.: +58 212 5041715
Fax: +58 212 5041444
e-mail: lsalazar@ivic.ve.
Miguel Vicente
Centro Nacional de Biotecnología
C/ Darwin 3
CSIC Campus de Cantoblanco
E-28049 Madrid, Spain
Tel: +34 91 585 46 99
Fax: +34 91 585 45 06
e-mail: mvicente@cnb.uam.es
institutional web: http://www.cnb.uam.es/
group web: http://www.cnb.uam.es/~mvicente/
Rogelio Hernandez Pando
Experimental Pathology Section
Department of Pathology
National Institute of Medical Sciences and Nutrition "Salvador Zubiran"
Street- Vasco de Quiroga, no-12,
CP-14000, Tlalpan
Mexico city, Mexico
Tel: +52 55 54 85 34 91
Fax:+52 55 56 55 10 76
email: rhdezpando@hotmail.com