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  Director : Véronique Vincent (vvincent@pasteur.fr)


  abstract

 

New genetical and immunological tools have been described in the laboratory : characterization of the different species of tubercle bacilli, 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, Nathalie Barilone, Cyril Depozat)

Our laboratory is a National Reference Center and is implied in the surveillance of tuberculosis and other mycobacterial infections. The surveillance of multi-drug resistant tuberculosis, i.e. difficult to treat cases due to strains resistant to both major drugs isoniazide and rifampicin, is achieved by a systematic strain typing. Molecular typing shows narrow transmission groups, usually including only two patients, indicating an efficient patient management and transmission control. However, chronic cases contribute to the extension of the disease within the community. These data are merged in an European network for multi-drug resistant tuberculosis.

Our laboratory is member of the WHO network of supranational laboratories and one of the European co-coordinators of the WHO network for the surveillance of global antituberculous drug resistance. Our laboratory participated to surveys led by national tuberculosis programmes in Madagascar and Djibouti. In Djibouti the antituberculous treatment is efficient in AIDS patients and directly observed treatment prevents resistance. A large study of multi-drug resistant strains is currently performed in the Institut Pasteur network with institutes in Algeria, Morocco, Central Africa, Cameroon, Ivory Coast, Madagascar, Viet-Nam and Russia. The first phase of the study shows a high prevalence in Russia and Viet-Nam of the Beijing family, characterized by a high genetic homogeneity and correlated with accumulation of resistance. Ancestral type strains are mainly detected in Madagascar, a characteristic which may be related to South East Asia immigration.

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

In collaboration with S. Cole ‘s laboratory, we evaluated the epidemiological use of new molecular markers for typing strains of tubercle bacilli. The genomic regions deleted along with bacilli evolution are specific markers useful for the identification of M. tuberculosis, M. bovis, M. africanum and other speices of tubercle bacilli. A study of strains close to the common ancestor is currently performed.

Phylogeny of M. marinum and M. ulcerans (Cristina Gutierrez-Perez, Véronique Vincent)

M. marinum is the agent of benigne cutaneous infections and M. ulcerans the agent of necrotizing ulcers endemic in Sub-saharian Africa and in Australia. The two species are phylogenetically close and differential identification may be difficult. The repeated sequence IS2404, specific for M. ulcerans, was detected in a strain with phenotypic properties and lipid composition closely related to M. marinum. This strain may represent a possible missing link between M. marinum and M. ulcerans as only few IS2404 are present versus more than a hundred copies in M. ulcerans.

Mycobacterial biodiversity (Corinne Le Dantec, Véronique Vincent)

The search of mycobacteria in water distribution system reveals a various flora with very high chlorine resistance, 100 and more than 300 times more resistant than E. coli. Temperature increase and low pH result in a better inactivation. Study of the mycobacterial flora in treatment plants shows that mycobacteria are able to colonize granular activated carbon and enter distribution systems. However, mycobacterial species recovered in internal networks differ from species recovered from treatment plants suggesting the importance of biofilms in the colonization of internal networks.

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.

Keywords: Tuberculosis, Epidemiology, Molecular typing, Immunodominant antigen, Mannosylation, Environmental mycobacteria



  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, Institut Pasteur, Professeur, gmarchal@pasteur.fr

Véronique Vincent, Institut Pasteur, Chef de Laboratoire, vvincent@pasteur .fr

Maria-Cristina Gutierrez Perez, Institut Pasteur, Chargée de recherches, crisgupe@pasteur .fr

Stéphane Flamant

Abdelmajid Snouber

Helmi Merdassi

Samiya Al Zadjali

Mabrouka Saidani

Malika ifticene

Parissa Farnia

Fatma Galem

Corinne Le Dantec

Céline Louveau

Mohammad Abolhassani

Sara Irène Ngo Niobe-Eyangoh

Stéphane Flamant

Abdelmajid Snouber

Helmi Merdassi

Samiya Al Zadjali

Mabrouka Saidani

Félix Romain

Pierre Chavarot

Cyril Depozat

Eddie Maranghi

Maria de Moura Goncalves

Pascale Pescher

Nathali Barilone

Anne Varnerot

Sylviane Wolkom


Activity Reports 2002 - Institut Pasteur
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