Unit: Mycobacterial Genetics
Director: GICQUEL Brigitte
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 (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 2000 mutants were screened for their ability to multiply in the lungs of mice during the acute phase of infection. Sixteen mutants were selected and the corresponding mutations were characterised. Several mutations were identified in a 50 kb region of the genome containing genes dedicated to the synthesis and transport of phtiocerol 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. It is probably the combination of these two effects that favours the multiplication of DIM-producing strains of M. tuberculosis during the acute phase of infection in mice. We are now characterising other attenuated STM mutants presenting deficiencies in other cell envelope lipids. In a complementary approach, we constructed by allelic replacement mutants of M. tuberculosis deficient 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. New screening methods of the transposon mutant libraries are being developed. They should allow the isolation of new attenuated strains of M. tuberculosis with mutations in genes that are important at later stages of the infection in mice or for growth inside macrophages in in vitro systems.
The protective efficacy of the M. tuberculosis attenuated strains isolated in the laboratory is being studied as part of the EEC "TB vaccine cluster" coordinated by Pr. Brigitte Gicquel (http://www.pasteur.fr/EC_TBvaccine/).
Interactions between M. tuberculosis and phagocytes (O. Neyrolles, M. Jackson, 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 are beeing screened to identify mycobacterial genes involved in inhibition of mycobacterial phagosome maturation. 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 detect 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 recognizes specifically pathogenic mycobacteria from the "tuberculosis" complex. Altogether, our results provide evidences that pathogenic mycobacteria may have evolved surface motifs in order to interact with lectins, such as DC-SIGN, on the surface of phagocytes.
Identification of new anti-TB drug targets (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. We examined the function of two of these genes, pimA (Rv2610c) and Rv2611c, and showed that they encode a mannosyltransferase and an acyltransferase, respectively, involved in the synthesis of di- and tri-acylated phosphatidylinositol mono-mannosides. Cell-free assays were developed for both enzymes. In order to determine if PimA is an essential enzyme of mycobacteria, we constructed a pimA conditional mutant of Mycobacterium smegmatis and found that the expression of pimA is essential for growth. Rv2611c is not an essential enzyme in M. smegmatis, due to the existence of redundant acyltransferase activities, but a deficiency in this enzyme severely affects the growth of M. smegmatis. Therefore, the synthesis of phosphatidylinositol mono-mannosides and derived higher PIM/LM and LAM appear to be essential for mycobacterial growth.
PimA, and perhaps also Rv2611c, are therefore attractive drug targets for the development of new anti-TB drugs. Moreover, the cell-free assays that we developed might prove useful for the high-throughput screening of inhibitors of these enzymes.
Molecular Epidemiology of Tuberculosis (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 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 family. In addition, molecular methods are being used to type M. tuberculosis strains from different geographical areas. New markers specific of major genotypes are currently identified. This should allow us to identify the major genotypes and to study their transmission.
BCG and new vaccines (N. Winter)
M. bovis BCG is the only prophylactic tool available 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. Thanks to recombinant BCG strains expressing reporter genes such as ova or egfp, early immune events that are established in the first hours and days after vaccination are being studied. Combination of flow cytometry , immunohistochemistry and immunological tests allows us to define which cells are involved in establishing the immune response to BCG-borne antigens in the draining lymph node. Respective roles of dermal, epidermal dendritic cells but also of other inflammatory cells such as monocytes and polynuclear neutrophils are being analysed.
Interactions between DC and M. tuberculosis and BCG are also studied in vitro both in our laboratory and in collaboration with other partners. We recently showed the immunodulatory function of DIM molecules on the DC inflammatory response to TB (Rousseau et al., Cell. Microbiol. in press).
BCG also represents an attractive vector for the development of recombinant vaccines against AIDS for example. 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.
As soon as 4 hours post BCG (rBCG-egfp, green) vaccination in the mouse ear dermis, the drainig lymph node is filled with Ly6G positive cells (red) composed mainly of polymorphonuclear leukocytes. BCG bacilli often colocalise with those cells. Influence of those early events on the induction of the T-cell response to the vaccine is under study.
Keywords: Mycobacterium, pathogenicity, virulence, tuberculosis, epidemiology, vaccine