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  Director : GICQUEL Brigitte (bgicquel@pasteur.fr)


  abstract

 

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 M. 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.



  report

cale

Genetic study of virulence determinants (M. Jackson, O. Neyrolles, B. Gicquel)

We used the temperature-sensitive vector derived from the pAL5000 plasmid of Mycobacterium fortuitum and containing the counterselective marker sacB (Ts/sacB vector) to construct allelic exchange and transposon mutants in mycobacterial species from the M. tuberculosis complex. An M. tuberculosis transposition mutant library containing 7000 mutants was created and screened for mutants carrying insertions in defined genes. This approach led to the isolation of mutants carrying transposon insertions in three different phospholipase C genes (plcA, plcB and plcC). A mutant deficient in a fourth phospholipase C gene (plcD), a triple mutant (plcABC) and a quadruple mutant (plcABCD) were also generated by allelic replacement. The four Plc enzymes were found to be active in M. tuberculosis and to catalyse the hydrolysis of phosphatidylcholine. The analysis of the residual virulence of the plc mutants in the mouse model of infection revealed that phospholipases C are important for the persistence of the tubercle bacillus in vivo.

A second transposon mutant library containing 4000 mutants was constructed by signature-tagged transposon mutagenesis (STM) and directly screened for mutants exhibiting 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 characterised. Four mutations were identified in a 50 kb region of the genome containing 13 genes dedicated to the synthesis and transport of phthiocerol dimycocerosates. The role of these complex lipids in pathogenesis is currently being studied in the laboratory.

The protective efficiency of the M. tuberculosis attenuated strains isolated in the laboratory is being studied as part of the EEC "TB vaccine cluster" coordinated by Prof. Brigitte Gicquel ( http://www.pasteur.fr/EC_TBvaccine/ ).

Interactions between M. tuberculosis and phagocytes (O. Neyrolles, M. Jackson, B. Gicquel)

To further our understanding of the mechanisms of macrophage parasitism by M. tuberculosis, we are developing new approaches combining cell biology and functional genomics. We are studying early interactions between mycobacteria and human phagocytes. We have 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 on human lung dendritic cells and mycobacteria in DC-SIGN-positive dendritic cells in lymph node biopsies from patients with tuberculosis. This suggests that DC-SIGN-mediated interactions between M. tuberculosis and dendritic cells occur in vivo. We have also identified the molecular determinants of the LAM-DC-SIGN ligation, explaining why DC-SIGN specifically recognises pathogenic mycobacteria from the "tuberculosis" complex. Taken together, our results suggest that pathogenic mycobacteria evolved surface motifs to allow them to interact with lectins, such as DC-SIGN, on the surface of phagocytes. (Tailleux et al. (2003)J. Exp. Med. 197, 121-127; Maeda et al. J. Biol. Chem. in press).

Identification of new anti-TB drug targets (M. Jackson, B. Gicquel)

Phosphatidylinositol (PI) and its metabolites, such as the phosphatidylinositol mannosides (PIM), linear and mature branched lipomannans and lipoarabinomannan, are the major phospholipids/lipoglycans of Mycobacterium spp. They are believed to play important roles in the structure and physiology of the bacterium as well as during host infection. The enzymes involved in their biosynthesis may be attractive drug targets for the development of new anti-TB drugs.

We have identified a cluster of five genes potentially involved in the early steps of PIM synthesis. Examination of the function of one of these genes, pimA (Rv2610c), showed that it encodes a mannosyltransferase involved in the synthesis of phosphatidylinositol mono-mannosides. A cell-free assay was developed in which a recombinant PimA enzyme produced in Escherichia coli synthesises phosphatidylinositol mono-mannosides from mannose and phosphatidylinositol. To determine whether PimA is an essential enzyme in mycobacteria, we also constructed a pimA conditional mutant of Mycobacterium smegmatis and found that the expression of pimA is essential for growth. Therefore, the synthesis of phosphatidylinositol mono-mannosides and its derivatives in mycobacteria appears to be dependent on PimA and essential for growth.

PimA is therefore an attractive drug target for the development of new anti-TB drugs. Moreover, the cell-free assay that we developed could be used for the high-throughput screening of PimA inhibitors.

Functional analysis of virulence factors (Jean-Marc Reyrat, Brigitte Gicquel)

Reporter genes (alkaline phosphatase), tagged insertional mutagenesis and computer tools have been used to identify a number of virulence genes.

Erp, which is secreted by members of the tuberculosis complex, was identified by a method in which alkaline phosphatase was used as a reporter. In M. tuberculosis, inactivation of the erp gene by allelic exchange greatly attenuated virulence in vitro, in cultured macrophages and in vivo, in the mouse model of tuberculosis. We recently showed that Erp is ubiquitous in mycobacteria. This secreted protein is composed of three domains. The central domain consists of a tandem repeat of the PGLTS domain. This central domain is the most variable between species: it contains four repeats in M. leprae and 24 repeats in M. xenopi. Analysis of the genomic region surrounding the erp gene showed that most of the genes in this region are cell wall biosynthesis genes. Using gene fusion, we showed that the allelic form of the erp gene influences the colonisation of the lungs but not the spleen of infected mice. Moreover, quantitative histological analysis of the lungs of infected animals showed that the nature of the erp allele strongly affected the number and the size of lung lesions (de Mendonça-Lima et al., Cell. Microbiol., in press).

Another aspect of the project concerns the molecules secreted by mycobacteria. A certain number of virulence factors and antigens are present on the surface of the bacillus or in the extracellular environment. We used the Staphylococcus aureus nuclease gene as a reporter gene to show that his nuclease is secreted into the extracellular environment independently of any potential signal sequence. Cytoplasmic markers showed that this phenomenon is independent of bacterial autolysis. These results suggest the existence of an unidentified secretion pathway that is independent of the general pathway.

Another potential virulence factor, Mramp is being studied in collaboration with A. Hance (Inserm U552, Hopital Bichat). Mramp is an orthologue of the mammalian Nramp gene and is involved in the transport of divalent ions across the bacterial cytoplasmic membrane. We have constructed a knock-out mutant of Mramp in M. tuberculosis and shown that this mutation impairs growth when iron is limited. The growth and survival of the Mramp mutant are not impaired in murine macrophages (derived from bone marrow or a cell line) or in vivo in the mouse model. Furthermore, we have shown that the Mramp phenotype is not affected by the genetic background of the host at the Nramp locus.

BCG and derived vaccines (Nathalie Winter)

Mycobacterium bovis BCG, a live attenuated bacterium, is an attractive vector for the development of recombinant vaccines. To propose candidate vaccines against AIDS, we have obtained several recombinant BCG (rBCG) strains expressing antigens from the Simian Immunodeficiency Virus SIVmac251. We have studied the immune responses induced by these strains in cynomolgus macaques, a relevant animal model for human AIDS. A mixture of three strains —rBCG-SIV3- expressing the nef, gag(p26) and env genes from SIVmac251 has been administered intradermally to macaques. An oral or rectal booster dose was then administered. The rBCG-SIV3 vaccine induced MHCI restricted CD8+ cytotoxic T cell responses against the SIV antigens but undetectable CD4+ lymphoproliferation. After rectal challenge with fully pathogenic SIVmac251, all animals were infected. We observed that the rBCG-SIV3 vaccine induced memory responses against the SIV antigens. Several approaches have been undertaken to improve the immune response to the heterologous antigens carried by rBCG strains. Vectors that are genetically stable in vivo (Méderlé et al., 2002) have been constructed. rBCG strains secreting SIV antigens and new promoters induced in professional antigen presenting cells are currently being studied.

Our laboratory is also interested in determining the role of dendritic cells in the anti-mycobacterial immune response. Dermal dendritic cells and epidermal Langerhans cells are ideally located to initiate the anti-tuberculous immune response after BCG vaccination, which is typically administered in the dermis. In collaboration with several partners (both on and off the Campus), we are studying the behaviour of skin dendritic cells after BCG intradermal immunisation in the mouse ear. Migration to the draining lymph node, antigen presentation capacity and the potential role of lectinic surface receptors in these mechanisms are currently being studied.

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 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 (Rad et al., Molecular Pathogenicity of Tuberculosis, Stockholm June 2002). These mutations, which are specific to M. tuberculosis genotypes, might be useful for molecular diagnostic. In addition, molecular methods are being used to type M. tuberculosis strains from different geographical areas. This should allow us to identify the major genotypes and to study their transmission.

Photo: Trafficking of DC-SIGN (red) and Mycobacterium tuberculosis (green)in a human dendritic cell (DC). Cells and bacteria were incubated 4 h at 4°C (left), 15 min at 37°C (middle) and 3 h at 37°C (right). DC-SIGN is engulfed with the bacillus in the nascent vacuole (middle), then recycled back to the plasma membrane (right).

  1. "A cluster for tuberculosis vaccine development" (N° QLK2-CT-1999-01093). http://www.pasteur.fr/EC_TBvaccine/

  2. "Mucosal Immunization — Cluster Project", (N° QLK2-CT-1999-00228).

  3. "New generation genetic markers and techniques for the epidemiology and control of tuberculosis" (N° QLK2-CT-2000-00630).

  4. "New strategies for treatment and prevention of mycobacterial diseases" (N° QLK2-CT-2000-01761).

  5. "Development of recombinant BCG multivaccine and complementary diagnostics for predominant parasitic and epizootic disease of ruminants in Latin America", (N° ICA4-CT-2000-30032 (INCO-DEV).

  6. "Improved diagnosis, drug resistance detection and control of tuberculosis in Latin America".Projet (N°: ICA4-2000-10054).

  7. "Structural and functional genomics of M. tuberculosis", (N° QLRT-2000-02018).

  8. "TBETHICS", (N° QLK2-CT-2002-30592).

Keywords: Mycobacterium, pathogenicity, virulence, tuberculosis, epidemiology



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  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  SINNO Helena, executive secretary IP,hsinno@pasteur.fr GICQUEL Brigitte, Professor, I.P. ;bgicquel@pasteur.fr

FILLON-JACKSON Mary, CR1 I.P.,mjackson@pasteur.fr

NEYROLLES Olivier, CR CNRS,neyrolle@pasteur.fr

REYRAT Jean-Marc, CR 1, INSERM,jmreyrat@pasteur.fr

WINTER Nathalie, CR 2 IP,nwinter@pasteur.fr

ABADIE Valérie, PhD student,vabadie@pasteur.fr

BLAZQUEZ-GOMEZ Jesus, Postdoc

BOECHAT Néio, M.D., PhD student,boechat@pasteur.fr

GRELLET Sheyla, PhD student

KOCINCOVA Dana, PhD student

MAEDA Norihiro, Postdoc,nmaeda@pasteur.fr

MARTINEZ Valérie, MD, PhD student,vmartinez@pasteur.fr

MAYA MARQUES Catarina, PhD student

MICK Virginie, PhDstudentvmick@pasteur.fr

RECCHI Chiara, PhD student,chiarare@pasteur.fr

ROUSSEAU Cécile, PhD student,rousseau@pasteur.fr

ROSAS MAGALLANES Vania, PhD student,vrosas@pasteur.fr

SONDEN Berit, Postdoc,bsonden@pasteur.fr

AUBERT-PIVERT Elisabeth, engineer I.P,epivert@pasteur.fr

BADELL-OCANDO Edgar, engineer I.P.,ebadell@pasteur.fr

BORDAT Yann, technician I.P.,ybordat@pasteur.fr

CHARLES Patricia, technical agent, I.P.,pcharles@pasteur.fr

ENSERGUEIX Danielle, technician I.P.,densergu@pasteur.fr

GILLARD Marie-Renée, responsible of preparation I.P.

LALLEMAND Catherine, laboratory agent I.P.

RAUZIER Jean, technician I.P.,jrauzier@pasteur.fr


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