Unit: Bacterial Molecular Genetics

Director: Stewart COLE

Comparative and functional genomic approaches are being used to further our understanding of tuberculosis and leprosy. New targets for therapy and potential subunit vaccine candidates have been identified and are currently being characterised. Genomics has now been extended to include Mycobacterium ulcerans, an emerging pathogen responsible for Buruli ulcer.

Search for new targets enabling the development of powerful inhibitors of the initiation and/or establishment processes of the pathogenic clostridia in different infectious sites, is the main objective of our research on these anaerobic bacteria. To the prospect, we are working on toxin regulation and secretion, the main virulence factors of these organisms, and the characterisation of the immunogenic capacity of Choline Binding membrane Proteins (CBP).

Functional genomics of the Mycobacterium tuberculosis complex: Secretion and virulence factors (Priscille Brodin, Marien de Jonge, Caroline Demangel, Brigitte Saint-Joanis, Roland Brosch, Stewart Cole)

The members of the Mycobacterium tuberculosis complex, M. tuberculosis, M. bovis, M. bovis BCG, M. canettii, M. africanum and M. microti share great genetic similarity. However, they have a different host spectrum. Results of comparative genomic analyses, demonstrated that there are at least 14 regions of difference (RD1-14) that are present in M. tuberculosis H37Rv but absent from BCG. PCR analysis showed that most of the RD regions absent from BCG were also missing from other strains of M. bovis, indicating that some of these variable regions reflect the evolutionary divergence of M. tuberculosis and M. bovis rather than genomic modifications that were introduced during the attenuation process of BCG. In contrast, there was only one region, RD1, absent from vaccine strains but present in all tested virulent isolates. This region represents one of the most interesting genomic loci of the tubercle bacilli because it appears to be simultaneously involved in enhancing virulence while its encoded proteins ESAT-6 and CFP-10 can induce protective immune responses.

The detailed analysis of this region is ongoing in our laboratory. Some of the aims of this project are to construct an unmarked M. bovis BCG::RD1 vaccine suitable for use in phase 1 trials and to learn more about the function of ESAT-6 secretion system. We also study the function of a putative Twin arginine translocation (TAT) system, which appears to be functional in M. tuberculosis.

Post-genomic analysis of Mycobacterium leprae (Nadine Honoré, Marc Monot, Romulo Araoz)

Comparative genomics of different M. leprae isolates revealed exceptional genome stability in the leprosy bacillus. The extensive reductive evolution previously described must have occurred therefore before the spread of the disease. The discovery of very rare point mutations (S.N.P) and their polymorphism studies have allowed us to retrace the worldwide dissemination of leprosy. The disease seems to have originated in either Eastern Africa or the Near East and spread with successive human migrations.

The second part of our research on leprosy is the establishment of a sensitive and reliable immunodiagnostic test which would permit the detection of all infected patients. Only rapid detection of the infectious patients will control the disease. Thanks to genomics and bio-informatics 17 potential antigens have been defined. After production and purification of these proteins, we have tested their antigenicity in patients from endemic countries : Korea, Mali and Bangladesh. The first results are very promising.

Genomics of Mycobacterium bovis and M.bovis BCG Pasteur (Thierry Garnier, Wafa Frigui, Stewart Cole)

The genome sequence of M. bovis has now been fully assembled and annotated. The M. bovis genome comprises 4345492 base pairs and encodes 3952 proteins. Intergenomic comparisons have been performed between the M. bovis and M. tuberculosis genomes in two ways; firstly, at the nucleotide level which identifies SNPs (Single Nucleotide Polymorphisms) and secondly, at the genomic level.These two complementary approaches give us the opportunity to identify genes responsible for host specificity and to explain the differences found in the metabolic pathways between the two species.

The genome sequence of the vaccine strain M. bovis BCG Pasteur has been completed. Composed of 4375192 bases, its annotation as well as bioinformatic analysis is currently in progress. Extensive analysis of the sequence of this organism with those of M. bovis and M. tuberculosis will improve our understanding of the genetic basis of the attenuation of BCG.

In order to obtain comprehensive knowledge of the tuberculosis complex members, we have initiated the genome sequencing of another vaccine strain, M. microti.

Genomic and post-genomic analysis of Mycobacterium ulcerans (Emmanuelle Coutanceau, Caroline Demangel, Laurent Marsollier, Gilles Reysset)

Mycobacterium ulcerans causes severe, chronic, necrotizing lesions of subcutaneous fat tissues. It is the causative agent of Buruli ulcer, a disease that, since the 1980s, has become a very significant cause of morbidity throughout the world, particularly in the countries of central and West Africa. Buruli ulcer is considered as the third most common mycobacterial disease of immunocompetent individuals, after tuberculosis and leprosy. M. ulcerans produces a macrolide toxin called mycolactone, which is highly cytotoxic and may also have immunosuppressive properties. Unlike other mycobacterial pathogens, M. ulcerans appears to be primarily extracellular. However, experimental infection of animal models has demonstrated that the parasite is transiently intracellular early in the infection. Numerous approaches (genomic, post genomic and immunological) are undertaken to better understand the biology and the virulence of M. ulcerans, in order to find effective prophylactic and therapeutic treatments.

The M. ulcerans genome sequencing project has provided major information about the physiology of the bacterium, as well as the mechanism of production of mycolactone. We have found that the M. ulcerans genome contains a chromosome and a circular 174-kb plasmid, pMUM001, bearing a cluster of genes encoding giant enzymes, the polyketide synthases (PKSs), the biological function of which is to produce mycolactone. This is the first example of plasmid-mediated virulence in mycobacterial species, and the emergence of M. ulcerans as a pathogen most likely reflects the recent acquisition of pMUM001 by horizontal transfer.

Epidemiological studies have shown that infection with M. ulcerans is related to swampy areas of tropical countries. It has been demonstrated that infected aquatic insects were able to transmit M. ulcerans to laboratory mice by biting, and that after experimental infection of the insects, M. ulcerans was located in their salivary glands. It is thought that after ingestion of food (small fishes, mollusks) bearing M. ulcerans, circulating phagocytic cells present in the hemolymph of predator insects ingest the bacteria, transport them to secondary sites such as the salivary glands, where M. ulcerans actively multiplies without causing disease. Fascinatingly, only strains that produce mycolactone colonize the water bug Naucoris cimicoides whereas isogenic mutants deficient for mycolactone biosynthesis are eliminated. This implies that mycolactone confers a selective advantage to the bacterium, possibly by enabling it to withstand the innate immune response and to multiply within insect phagocytic cells.

Regulation of toxinogenesis in Clostridia (Bruno Dupuy, Susana Matamouros, Ana Maceira Antunes)

The pathogenicity capacity of Clostridium species responsible for human and animal diseases (antibiotic associated diarrhea and pseudomembranous colitis (Clostridium difficile), gas gangrene (Clostridium perfringens), botulism (Clostridium botulinum) and tetanus (Clostridium tetani) is mainly due to the large production of extracellular toxins. We showed that alternative RNA polymerase sigma factors are required for activation of toxin gene expression. Thus TcdR, UviA, BotR and TetR regulate expression of the toxins A and B in C. difficile, the bacteriocin in C. perfringens, the botulinum neurotoxin and the tetanus neurotoxin, respectively. These sigma factors have some similarities with members of the extracytoplasmic function (ECF) family but are sufficiently different in structure and function to leave open the question of their phylogenetic relationship. We showed that these sigma factors are sufficiently similar so that they are able to substitute for each other. Thus it appears that a common molecular mechanism, involving a new subgroup of the sigma70 family of RNA polymerase sigma factor (Group 5 or TcdR subfamily), seems to control the production of important toxins in several major pathogenic clostridium species.

In all cases examined, the same regulatory effects observed by environmental stimuli on toxin expression (carbon sources, nitrogen sources, temperature, etc…), regulate expression of their sigma factors. Interestingly, these sigma factors are not sufficient for their own expression suggesting that additional factor(s) are required. Thus, the environmental regulation of the toxin synthesis would be mediated via modulation of the factor expression. In C. difficile several regulatory proteins are currently being studied such as homologs of B. subtilis CcpA and CodY, the principal mediator of glucose repression and the global regulator of genes induced by general nutrient limitation, respectively. Moreover, we are also working on two homologs of the two-component regulatory systems VirS/VirR of C. perfringens, which regulate directly or indirectly several toxins, and we are testing the idea that C. difficile TcdC, whose gene belongs to the pathogenicity locus, is an anti-sigma factor.

Standard therapy against Clostridium infections is treatment with antibiotics. As a consequence, resistant strains are observed and the antibiotic therapy often causes relapse of C. difficile disease. The search for new targets to enable the development of strong inhibitors of the initiation and/or establishment processes of these organisms during infection, led us to work on the holin like proteins, potentially involved in the toxin secretion, and on the pathogenicity and the immunogenic capacity of four CBP membrane proteins (Choline Binding Protein). These CBP belong to a small family of proteins that have affinities to choline residues.

Keywords: Functional genomics, Mycobacterium tuberculosis complex, leprosy, Buruli ulcer, Clostridia

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