Unit: Mycobacterial Genetics
Director: Brigitte GICQUEL
Tuberculosis, which is caused by mycobacteria from the Tuberculosis complex, is still a major public health problem: it is responsible for about three million deaths each year. BCG, the only currently available vaccine, is relatively efficient. The use of antibiotics may be compromised by the emergence of multiresistant strains of bacteria. Our unit is involved in the characterisation of Mycobacterium tuberculosis virulence factors and the host immune responses induced by this bacterium and BCG. This work could lead to the identification of targets for new drugs against tuberculosis, the identification of bacterial components that could be included in a new vaccine or even to the isolation of a new attenuated strain that is more efficient than BCG. Our Unit is also studying the possibility of using recombinant strains of BCG to protect against other diseases, such as AIDS. We are identifying genetic markers that are specific to epidemic strains, in particular strains responsible for multidrug resistant outbreaks.
Genetic study of virulence determinants Persons in charge: M. Jackson, O. Neyrolles, B. Gicquel
A temperature-sensitive vector derived from the pAL5000 plasmid of Mycobacterium fortuitum and containing the counterselective marker sacB (Ts/sacB vector) was used to construct allelic exchange and transposon mutants in mycobacterial species of the M. tuberculosis complex. An ordered transposition mutant library of Mycobacterium tuberculosis containing 4000 mutants was constructed by signature-tagged transposon mutagenesis (STM) and directly screened for mutants exhibiting an attenuated virulence in mice. About 4000 mutants have now been screened for their ability to multiply in the lungs of mice during the acute phase of infection. 29 mutants were selected and the corresponding mutations were characterised. 12 different mutations were identified in a 50 kb region of the genome containing genes dedicated to the synthesis and transport of phthiocerol dimycocerosates (DIM). A study on the roles played by these complex lipids in pathogenesis revealed that the presence of DIM in the cell envelope of M. tuberculosis protects this bacterium from the cidal activity of reactive nitrogen intermediates produced by macrophages and modulates the host early immune response to infection. We are now characterising attenuated STM mutants presenting other deficiencies in cell envelope lipids. In a complementary approach, we constructed by allelic replacement mutants of M. tuberculosis deficient in transcriptional regulators, in the less characterized PE/PPE family, and in the synthesis of lipids that share some structural similarities with DIM and that are specifically produced by pathogenic species of mycobacteria. The analysis of these mutants in cellular and animal models allowed us to measure the contribution of these lipids to pathogenesis. We are now focusing on the regulation of the synthesis of these lipids.
The role of these mycobacterial compounds is studied within the integrated project TB-VAC of the European Commission (www.tb-vac.org). Ethical aspects are discussed within the project TBETHICS of the European Commission and within coordinated by Pr. Brigitte Gicquel (www.tbethics.org).
Interactions between M. tuberculosis and phagocytes Persons in charge: O. Neyrolles, B. Gicquel
In order to better understand the mechanisms of macrophage parasitism by M. tuberculosis, new approaches combining cell biology and functional genomics are under development. In particular, libraries of M. tuberculosis mutants have been screened to identify mycobacterial genes involved in intracellular growth, and inhibition of mycobacterial phagosome maturation. Out of about 1,500 mutants, about 30 have been isolated as attenuated in their ability to replicate in macrophages. These clones of interest have been characterized and are now under study at the individual level.
We are also studying early interactions between mycobacteria and human phagocytes. We have recently shown that the lectin DC-SIGN is the major M. tuberculosis receptor on human dendritic cells, and that mycobacterial lipoarabinomannan (LAM) is a key DC-SIGN ligand. We have also detected Dc-SIGN expression on human lung dendritic cells, and were able to detected mycobacteria in DC-SIGN-positive dendritic cells in lymph node biopsies from patients with tuberculosis, thus suggesting that DC-SIGN-mediated interactions between M. tuberculosis and dendritic cells are likely to occur in vivo. We have also identified the molecular determinants of the LAM-DC-SIGN ligation, which explain why DC-SIGN recongizes specifically pathogenic mycobacteria from the "tuberculosis" complex. Altogether, our results provide evidences that pathogenic mycobacteria may have evolved surface motifs in oder to interact with lectins, such as DC-SIGN, on the surface of phagocytes. The functional consequences of DC-SIGN-mycobacteria interactions is now under investigation, both in vitro and in vivo.
Identification of new anti-TB drug targets Persons in charge: M. Jackson, B. Gicquel
Phosphatidylinositol (PI) and metabolically-derived products such as the phosphatidylinositol mannosides (PIM), linear and mature branched lipomannan and lipoarabinomannan are prominent phospholipids/lipoglycans of Mycobacterium spp. believed to play important roles in the structure and physiology of the bacterium as well as during host infection. The characterization of the enzymes involved in their biosynthesis, in addition to providing fundamental knowledge about the synthesis of these molecules, could lead to the identification of attractive drug targets for the development of new anti-TB drugs.
We have identified a cluster of five genes potentially dedicated to the early steps of PIM synthesis. The function of two of these genes, pimA (Rv2610c) and Rv2611c, was examined and we showed that they respectively encode a mannosyltransferase and an acyltransferase involved in the synthesis of di- and tri-acylated phosphatidylinositol mono-mannosides. Moreover, we provided evidence that PimA is essential for mycobacterial growth. Therefore, PimA represents an attractive novel drug target. In collaboration with the Structural Biochemistry Unit of the Institut Pasteur, we undertook to solve the three-dimensional structure of PimA. Knowledge of the 3D-structure of this protein will allow the virtual screening of inhibitors (in silico) and the inhibitory activity of the compounds identified will then be tested using the cell-free assays that we developed for PimA.
In a broader approach to identify other M. tuberculosis enzymes involved in the biosynthesis of PIM, LM and LAM, we undertook to knock-out by homologous recombination several glycosyltransferase genes potentially involved in the synthesis of these molecules. Moreover, we are also screening transposon mutant libraries of M. tuberculosis for mutants presenting a reduced reactivity to a monoclonal anti-LAM antibody. Fifteen mutants have already been isolated and are being characterized.
Molecular Epidemiology of Tuberculosis Person in charge: Brigitte Gicquel
We sought genetic markers specific for highly transmissible M. tuberculosis strains that are responsible for outbreaks, in particular MDR outbreaks. This study was carried out in collaboration with the European Network of Molecular Epidemiology, the Public Health Research Institute and the network of Pasteur Institutes and associated Institutes. mut T- and ogt-specific alleles were found in M. tuberculosis strains of the W-Beijing and Haarlem genotype. These results suggest that these strains, which are well adapted to the host, evolved thanks to the acquisition of mutations in DNA repair enzymes, thus resulting in transient mutator phenotypes. These mutations, which are specific to M. tuberculosis genotypes, may be useful for molecular diagnostic identifying different branches of the W. Beijing or Haarlem family. In addition, molecular methods are being used to type M. tuberculosis strains from different geographical areas. New polymorphic markers specific of major genotypes have been identified. This should allow us to identify the major family by genotyping techniques and to study their transmission.
BCG and new vaccines Person in charge :N. Winter
Mycobactérium bovis BCG is the only prophylactic tool availbale to control TB. Althoug BCG vaccination is widespread, immune mechanisms that lead to -partially effective- vaccination are ill defined. Thus we are interested in understanding such mechanisms. Because BCG is administered intradermally to humans, a murine model of BCG inoculation in the ear dermis has been set up in the laboratory. We have tracked BCG host cells both at injection site and in draining lymph node at early times after injection. Skin mononuclear phagocytes, including Langerhans cells and dermal dendritic cells were barely associated with live BCG suggesting that during vaccination, these cells do not play a major role in bacillus capture and transport to the lymphoid organ. We observed that neutrophils that were rapidly recruited to the vaccination site captured BCG but did not eliminate it. Moreover, neutrophils were detected into skin lymphatic vessels and infiltrated lymph node subcapsular space and sinuses as early as 4 hours post injection where they also represented main BCG host cells. At 12 hpi, neutrophils were detcetd i the T-cell zone of the lymph node, indicating that they where they could play an important role in the establishment of the immune response. Fluorescent-labeled neutrophils injected into left or right ear appeared in the ipsilateral auricular draining lymph node after BCG vaccination. These data show for the first time that neutrophils may leave inflammed tissues to migrate via afferent lymph to the drainant lymph nod. The mechanism is unknown but seems CCR6 and CCR7 independant. Finally, recombinant green fluorescent BCG-egfp and red fluorescent rBCG-dsrfp injected into distinct sites of the same ear were mainly detected into distinct neutrophils in the lymph node capsular space. These results indicate that neutrophils may transport BCG from skin to the draining lymph node, a role reserved to dendritic cells, macrophages and monocytes.
Interactions between DC and M. tuberculosis and BCG are also studied in vitro both in our laboratory and in collaboration with other partners (Prina et al., 2003). We have recently shown the immunodulatory function of DIM molecules on the DC inflammatory response to TB (Rousseau et al., 2004).
BCG also represents an attractive vector for the development of recombinant vaccines against AIDS for example (Méderlé et al., 2003). Integration of expression cassettes into the BCG chromosome allows to obtain stable strains in vivo. Vectors derived from mycobacteriophage Ms6 integrate into different tRNAala genes in M. smegmatis and BCG. This observation allows us to construct multi-recombinant stable BCG strains. New promoters that are highly expressed in dendritic cells are also being evalutated.
Keywords: Mycobacterium, pathogenicity, virulence, tuberculosis, epidemiology, vaccine