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  Lrmyc


  Director : Véronique Vincent (vvincent@pasteur.fr)


  abstract

 

New genetical and immunological tools have been described in the laboratory : linear plasmids able to replicate in fast or slow growing mycobacteria, characterization of M. africanum strains, demonstration that mannosylation of proteins plays a key role in their antigenicity. These specific molecules have been used to develop new tools for the diagnosis of active tuberculosis.



  report

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Mycobacterium National Reference Center (Gilles Marchal, Véronique Vincent, Cristina Gutierrez-Perez, Anne Varnerot, Maria de Moura Goncalves, Jean Sautereau, Michèle Gilbert)

Our laboratory is a National Reference Center and is implied in the surveillance of tuberculosis and other mycobacterial infections. The molecular typing applied on M. tuberculosis allowed to elucidate several cases of hypothesis of transmission between patients at hospital or in the community, and hypotheses of laboratory contamination, especially with multi-drug resistant strains. In collaboration with Institut Pasteur in Lille, we evaluated the epidemiological interest of MIRUs, variable number tandem repeat loci, for the M. tuberculosis typing with a rapid and easy method.

Our CNR contributed to the characterisation of the 58 cases of nosocomial spinal infections, diagnosed in patients who had undergone spinal surgery in a private hospital. Contaminated tap water, used for rinsing surgical devices after disinfection, was identified as the source of the outbreak. In a parisian public hospital, molecular typing allowed to demonstrate water from refrigerated fountains as source of a M. gordonae pseudo-outbreak.

Our laboratory is one of the co-coordinators of the WHO network for the surveillance of antituberculous drug resistance in Europe. We participated to the WHO writing committee for the guidelines for drug susceptibility testing for second-line anti-tuberculous drugs. A large study of multi-drug resistant strains is currently performed in the Institut Pasteur network.

Molecular epidemiology and phylogeny of M. tuberculosis (Cristina Gutierrez-Perez, Véronique Vincent)

The analysis of a spoligotype database, consisting of the genomic profiles based on the polymorphism of the DR region, was performed with the Institut Pasteur Guadeloupe on M. tuberculosis strains isolated all over the world. The analysis showed few major families distributed throughout the world and others specific for certain geographic areas, suggesting persistence of ancestral clones and recent emergence of new families like the Beijing type.

Linear plasmids in mycobacteria (Corinne Le Dantec, Véronique Vincent, Mathieu Picardeau)

Our laboratory described the first linear plasmids in mycobacteria. They are invertron structures with telomeres consisting of terminal inverted repeats with covalently linked proteins. The complete nucleotide sequence of pCLP, a 23-kb plasmid from M. celatum, identified 19 putative open reading frames among which 12 were expressed. PCLP presents loci similar to M. tuberculosis, suggesting horizontal transfer between M. celatum and M. tuberculosis. Moreover, genes similar to genes of bacterial circular plasmids involved in partition and postsegregational mechanisms have been identified. Functional analysis suggests that they probably carry out similar maintenance role in M. celatum.

Mycobacterial biodiversity (Véronique Vincent)

With international collaborations, M. immunogenum, a new species has been described from environmental isolates and clinical isolates from disease-associated nosocomial outbreaks, and new typing methods have been developed for M. marinum and M. ulcerans. In collaboration with finnish teams, strains from the new species M. bohemicum were characterised from human, veterinary and environmental sources.

Immune responses against tuberculosis

The immune protection against tuberculosis can be achieved only by prior vaccination with a living vaccine. The attenuated M. bovis strain bacillus Calmette-Guerin (BCG) is widely administered to protect against tuberculosis. Two major ways of research have been explored during the last years. First to explore the efficacy of the enteral route of immunization in order to decrease the incidence of side effects and furthermore to secure the administration of BCG in developing countries in which risks of AIDS and hepatitis transmission are high. Second to define the major immunodominant molecules recognized by the immune system during a protective immunization.

BCG induces potent immune protection by oral or rectal immunization routes (Micheline Lagranderie, Mohammad Abolhassani, Pierre Chavarot, Gilles Marchal)

The oral route was used by Calmette in 1921 for initial BCG vaccination but was abandoned due to its deleterious effects such as suppurative cervical adenitis. We demonstrated that BCG translocation across the oropharyngeal area after oral ingestion is not critical to the induction of a protective immunity against a virulent challenge with M. tuberculosis. The rectal route for BCG vaccination would be easy to use under field conditions, inexpensive and safe. Various animal species (mice, guinea pigs and macaques) have been immunized with lyophilized BCG by rectal and parenteral routes. The induced specific immune responses were similar. Similar levels of protection against a virulent challenge assessed 6 months after immunization were observed for the parenteral or the rectal routes of immunization in mice immunized as adults or as new-borns.

Presence of the mannose residues on the Apa protein is essential for the antigenicity of the molecules (Félix Romain, Pascale Pescher, Gilles Marchal)

The 45/47 kDa antigen complex (Apa) present in culture filtrate of BCG, M. tuberculosis or M. bovis has been identified and isolated based on its ability to interact mainly with T lymphocytes and/or antibodies induced by immunization with living bacteria. The protein is glycosylated ; mannose residues are convalently linked on defined threonines. The chemical or enzymatic removal of the mannose residues was performed. The deglycosylated antigen was 30 to 100 fold less active than native molecules in eliciting delayed type hypersensitivity reactions in guinea pigs immunized with BCG or in stimulating in vitro specific T lymphocytes.

The recombinant Apa molecules expressed in Mycobacterium smegmatis was 4 to 10 fold less potent than the native proteins in correlation with the glycosylation pattern. When expressed in E. coli, the recombinant protein was nearly non antigenic in relation with the absence of glycosylation. The role of mannose residues in the constitution of the T cell epitope is presently under study with the help of chemically synthesized glycopeptides.

Role of Mycobacterium tuberculosis Copper Zinc superoxide dismutase (Olivier Dussurget, Pascale Pescher, Gilles Marchal)

Superoxide dismutase (SOD) play an important role in protection against oxidative stress and have been demonstrated to contribute to the pathogenicity of many bacterial species. M. tuberculosis and M. bovis null mutants have been constructed. The ability of such superoxide dismutase strains to resist oxidative stress has been only slightly decreased in vitro. The mycobacterial CuZn SOD was demonstrated to do not contribute to the pathogenicity of M. tuberculosis.

Use of the 45/47 kDa (Apa) antigen for the diagnosis of active tuberculosis (Cynthia Horn, Gilles Marchal)

Cellular immunity: Peripheral blood lymphocytes were collected from sensitised (PPD+) subjects or from tuberculous patients. The lymphocytes were in vitro stimulated with PPD or with the 45/47 kDA (Apa) antigen. Only the lymphocytes obtained from persons suffering from tuberculosis were stimulated by the defined antigen, at the opposite the response of cells obtained from the two groups were similar toward PPD.

Antigens detection: In an attempt to develop a tuberculosis diagnostic tool, monoclonal antibodies directed against the 45/47 kDa (Apa) antigen and the molecule itself have been used in a collaborative work performed at Institut Pasteur de Madagascar by Vohangy Rosolofo and Suzanne Chanteau. Pathological fluids like pleural effusion liquid or cerebrospinal fluid which contain very few living bacteria were found positive for the presence of the specific antigen.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

Laurence Hay (1/2 temps)

Martine Mercier (1/2 temps)

Gilles Marchal

Véronique Vincent

Maria-Cristina Gutierrez Perez

Danièle Mathieu

Minh Ly Ho Thi

Kouassi Raymond N’Guessan

Stéphane Flamant

Mabrouka Saidani

Corinne Le Dantec

Céline Louveau

Mohammad Abolhassani

Sara Irène Ngo Niobe-Eyangoh

Félix Romain

Pierre Chavarot

Cyril Depozat

Eddie Maranghi

Maria de Moura Goncalves

Pascale Pescher

Jean Sautereau

Anne Varnerot

Sylviane Wolkom


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