Summary:

Mycobacterium tuberculosis is a leading threat to human health, killing 40,000 people a week and causing 8 million new cases of disease every year. Current vaccination and chemotherapy strategies are unsatisfactory, making development of novel control strategies an important objective.
Rational design of such strategies requires an understanding of the mechanisms by which M.tuberculosis is able to infect individuals and cause disease.

Central to the success of M. tuberculosis as a pathogen is an ability to replicate inside the normally destructive phagosomal compartments of macrophages. TB-MACS propose to use innovative genetic screens to reveal the genetic and mechanistic basis of this characteristic. Specifically TB-MACS will use a microarray based screening of an M. tuberculosis mutant library in two separate selections to identify mutants that are unable to inhibit phagosome acidification and/or unable to inhibit fusion of the phagosome with hydrolytic lysosomes. Mutants identified in these assays will be isolated and characterised with respect to intracellular trafficking and growth in human macrophages. The in vivo phenotypes of the phagosome mutants will be assessed in murine and guinea pig models of tuberculosis.

TB-MACS brings together three laboratories with complementary skills and facilities in high-throughput genetic screening, flow-cytometry, cell biology, transcriptomics and proteomics - the net result of which is a unique project that is able to provide system-wide answers to important unanswered questions about M.tuberculosis virulence.

TB-MACS will contribute to improving human health by defining bacterial products and processes that are essential to infection, and which will be useful in the rational development of new drugs and vaccines against tuberculosis.

Background:

To form a rational platform on which to determine novel drug targets and vaccine strategies against M. tuberculosis, the processes that allow the bacterium to thrive in vivo need to be identified. Furthermore, the molecular mechanisms behind these processes need to be understood. A key faculty of the bacterium is its ability to grow inside the cells of its host, principally within the macrophage. Multiple factors are involved in intracellular survival including a variety of strategies to avoid and subvert the immune response. Perhaps the most fundamental requirement of intracellular growth is the ability of M. tuberculosis to interfere with the host cell endosomal pathway, preventing acidification of the phagosome compartment in which it resides and stopping its fusion with hydrolytic microbicidal lysosomes .The mechanisms by which M. tuberculosis interferes with the host cell endosomal machinery have yet to be fully understood.

Aim:

The overarching aim of TB-MACS is to understand the genetic and molecular basis by which Mycobacterium tuberculosis is able to manipulate the macrophage phagosome, leading to bacterial survival and replication. Understanding these mechanisms will be important to the future development of novel drug and vaccine strategies. Specifically, TB-MACS will:

1. Screen the genome of M.tuberculosis to identify the genes responsible for inhibiting phagosome acidification;
2. Screen the genome of M.tuberculosis to identify the genes responsible for inhibiting phagosome/lysosome fusion;
3. Characterise the intra-macrophage growth/survival of mutant M.tuberculosis strains which are defective in their ability to manipulate the phagosome (phagosome mutants);
4. Characterise the in vivo survival of phagosome mutants in mice and guinea pigs and;
5. Analyse the proteomes and transcriptomes of phagosome mutants to help understand the mechanisms responsible for phagosome manipulation.

Expected results:

Identification of the genes and molecular mechanisms that allow M.tuberculosis to modulate the macrophage phagosome allowing bacteria to grow and cause disease

Potential applications:

Information derived from the project will help identify novel drug targets for consideration in the development of chemotherapy strategies. Mycobacterial mutants generated during TBMACS may form the basis of novel attenuated vaccine strains.

Coordinator:

Partners:

Nº	Principal Scientific Participants	Official Address	Other Information
2	Olivier Neyrolles	Unit of Mycobacterial Genetics Institute Pasteur 28 rue de Dr Roux, Paris 75015 France	Tel: +33 1 45688840 Fax: +33 1 45688843 E-mail: neyrolle@pasteur.fr Website: www.pasteur.fr
3	Jelle Thole	Edelhertweg 15, PO box 65 ID-Lelystad, Instituut voor Dierhouderij en Diergezondheid bv, Lelystad 8200 AB Netherlands	Tel: +31 320 238508 Fax: +31 320 238961 E-mail: jelle.thole@wur.nl Website: www.asg.wur.nl