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 in our laboratory on M. tuberculosis strains received from several hospitals allowed to elucidate several cases of hypothesis of transmission between patients at hospital or in the community, and hypotheses of laboratory contamination. A molecular epidemiological study conducted in the Brest area identified contact settings and risk factors which have not been detected by conventional epidemiological investigation. Our laboratory is one of the co-coordinators of the WHO network for the surveillance of antituberculous drug resistance in Europe. In 2000, we participated to the national survey led by the Institut de Veille Sanitaire on infections due to M. xenopi. We participated to the WHO writing committee for the guidelines for drug susceptibility testing for second-line anti-tuberculous drugs.
Study of linear plasmids (Corinne Le Dantec, Véronique Vincent, Mathieu Picardeau)
We previously described the first linear plasmids in mycobacteria. The elements have an invertron structure, the telomeres of which have terminal inverted repeats and covalently attached proteins. We investigated the internal replication region of the smallest identified plasmid, pCLP, a 25 kb element. The region shows sequence identity with the replication regions of mycobacterial circular plasmids, indicating that these plasmids of distinct topology are related. Homology with linear plasmids from other actinomycetes is less, suggesting that the linear plasmids in actinomycetes may not have a common ancestor. pCLP, which replicates in slowly- (e.g. Mycobacterium bovis BCG) as well as in rapidly-growing mycobacterial species (e.g. M. fortuitum, M. smegmatis), may be useful for the introduction of genes in various species of mycobacteria. Moreover, pCLP represents a new compatibility group and may be used in co-transformation experiments with other mycobacterial vectors.
Genetic diversity of Mycobacterium africanum (Cristina Gutierrez-Perez, Véronique Vincent)
M. africanum encompasses tubercle bacilli strains, mostly isolated from patients from sub-saharian african countries, with heterogeneous phenotypes suggesting that the species could be defined as a continuum between M. tuberculosis and M. bovis. Molecular characterisation, based on several independent markers, confirmed the validity of the taxon and showed a specific pattern, distinct from both M.tuberculosis and M. bovis. Strains are subtyped in 3 subtypes, two homogeneous and one heterogeneous, with neither particular geographical link nor specific feature for a phenotypic identification. The homogeneity of the M. africanum sub-groups prevents an accurate strain typing otherwise efficiently performed in M. tuberculosis for the differentiation of non epidemiologically related strains.
Immune responses against tuberculosis
The immune protection against tuberculosis can be achieved only by prior vaccination with a living vaccine. The attenuated Mycobacterium 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, Anne-Marie Balazuc, 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 intragastric administration was as efficient as oral route to induce potent specific immune responses in mice.
Microencapsulation of the bacteria would be used to minimize the side effects of inadvertent pharyngeal inoculation, but such microencapsulation is not easy to perform. At the opposite 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.
Presence of the mannose residues on the Apa protein is essential for the antigenicity of the molecules (Félix Romain, Cynthia Horn, 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.
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)
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.