Unit: Bacterial Molecular Genetics
Director: COLE Stewart
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.
The pathogenesis of Clostridium difficile and Clostridium perfringens is due to powerful toxins and in C. difficile their synthesis shown to be controlled by novel sigma factors.
Functional genomics of mycobacteria (Stewart Cole, Roland Brosch, Priscille Brodin, Caroline Demangel, Brigitte Saint-Joanis)
Mycobacterium tuberculosis, the etiological agent of human tuberculosis shares more than 99,9 % sequence similarity with other members of the so-called M. tuberculosis complex, which comprises Mycobacterium africanum, Mycobacterium canettii, Mycobacterium microti, Mycobacterium bovis and M. bovis BCG. A number of regions of difference (RD1-RD14) that range in size between 2 and 13 kb were identified in the genomes of the members of the M. tuberculosis complex by various comparative genome analyses. The study of presence or absence of certain RD regions in 100 strains from the M. tuberculosis complex has allowed to redefine the phylogenetic relationship among the members of the complex. These results contradict the often reported hypothesis that M. bovis is the progenitor of M. tuberculosis. This investigation further has identified some important markers (TbD1, RD9, RD4) for the rapid identification of the tubercle bacilli. Some other regions like RD1 are probably associated with the attenuation of the vaccine strains, M. bovis BCG and M. microti, the vole bacillus. This hypothesis has been tested experimentally using gene knock-ins of several regions absent from BCG and M. microti. Interestingly only complementation with the RD1 region, which contains genes coding for important T-cell antigens, resulted in increased virulence in immune-compromised mice. Immune-competent mice, however were able to control the infection with the BCG::RD1 and M. microti::RD1 strains, but increased persistence and immunogenicity was noted. This finding opens new perspectives for the development of more efficient anti-tuberculosis vaccines, a strategy which is presently investigated in our laboratory.
However, BCG::RD1 remains much less virulent than M. tuberculosis and its persistence is well controlled by immunocompetent hosts. This approach has also revealed the existence of a secretion machinery which is necessary for the export of ESAT-6 and for induction of Th1-type specific cellular immune responses. The immunogenicity of BCG::RD1 has been increased and the resulting level of protection against disseminated tuberculosis and its pathology have been substantially improved relative to BCG alone. Similar results have been obtained following immunisation by M. microti::RD1 whose virulence and persistence are inferior to those of BCG::RD1. The construction of BCG::RD1 and M. microti::RD1 consequently represents an important advance in the development of anti-tuberculosis vaccines with better efficacy, which we are currentlty trying to further optimize.
The post-genomic analysis of Mycobacterium leprae (Nadine Honoré, Marc Monot, Romulo Araoz) :
Molecular methods may be the key to understanding the epidemiology of leprosy. We have developed DNA microarrays based on the whole genomeof M. leprae. Our aim is to try and find genetic differences among
M. leprae strains from Africa, Asia and America.
The control of leprosy would also be improved if rapid diagnostic tests were available. Comparative genomic analyses allowed us to identify 40 candidate genes that may encode M. leprae-specific antigens. The recombinant expression of these genes is currently underway and preliminary results indicate that some of the products show seroreactivity with sera from leprosy patients.
Genomics of Mycobacterium bovis and M. bovis BCG Pasteur (Thierry Garnier, Stewart Cole)
The genome sequence of M. bovis has now been fully assembled and annotated. The M. bovis genome comprises 4345492 base pairs and encode 3952 genes. 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 sequencing and analysis of the genome of the vaccine strain M. bovis BCG Pasteur is in the final stage. Comparison between this genomic sequence and those of M. bovis and M. tuberculosis will improve our comprehension of the genetic basis of the attenuation of BCG.
Determination of the complete genome sequence of Mycobacterium ulcerans (Timothy Stinear, Peter Leadlay)
Mycobacterium ulcerans causes severe, chronic, necrotizing lesions of subcutaneous fat. 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, but particularly in the countries of central and West Africa. Buruli ulcer is considered the third most common mycobacterial disease of immunocompetent individuals, after tuberculosis and leprosy. Unlike other mycobacterial pathogens, M. ulcerans produces a macrolide toxin called mycolactone and appears to remain extracellular during infection. We are adopting the genomic approach to better understand the epidemiology and the pathogenesis of this important disease and to find effective prophylactic and therapeutic treatments.
The M. ulcerans genome sequencing project is nearing completion but has already provided much information about the mechanism of production of mycolactone, macrolide toxin, responsible for the massive tissue destruction seen in Buruli ulcer. 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) whose biological role is to produce mycolactone. This is the first example of plasmid-mediated virulence in a Mycobacterium, and the emergence of M. ulcerans as a pathogen most likely reflects the recent acquisition of pMUM001 by horizontal transfer.
Investigation of the pathogenicity in Clostridia (Gilles Reysset, Bruno Dupuy)
Our work on the pathogenicity of toxinogenic strains of Clostridia (C. perfringens and C. difficile) aims to understand mechanisms of the regulation of the major enterotoxins and to identify and characterize new virulence factors that may be implicated in the pathogenic behaviour of these two organisms.
C. perfringens is responsible for conditions ranging from mild food poisoning to necrotic enteritis or gas gangrene. It seems that adaptation to oxidative stress is an important factor in the pathogenesis of this ubiquitous organism. Analysis of the whole genome sequence of the strain 13 shows that C. perfringens possesses genes potentially encoding (i) a classical Mn-superoxide dismutase, (ii) an alkyl peroxide reductase, and (iii) two superoxide reductase systems potentially catalyzing the reduction of O2- to H2O2 without the production of oxygen, such as those of sulfate-reducing bacteria. In addition, there are three genes encoding potential non-heme rubrerythrin proteins, which may act as the terminal component of NADH peroxidase, catalyzing the reduction of hydrogen peroxide to water. The catalytic properties of the products from all these genes, and the role of each of them in the oxidative stress response in C. perfringens, were assessed by complementation experiments in Escherichia coli strains impaired in either superoxide dismutase, alkyl peroxidase or calalase activity. Transcriptional analysis of these genes was also performed to investigate their regulation in C. perfringens.
In the separate study, the search for new targets to enable the development of strong inhibitors of the initiation and establishment processes of C. perfringens during infection, led us to work on the pathogenicity and the immunogenic capacity of four CBP proteins (Choline Binding Protein). These CBP belong to a small family of proteins that have affinities to choline residues. They are present in all toxinotypes of C. perfringens strains and are cell membrane associated.
The pathogenic capacity of C. difficile, the organism responsible for pseudomembranous colitis and most antibiotic-associated diarrhoeas, is mainly due to the large production of two major toxins, Tox A and Tox B. The transcription of the tox genes is regulated through the txeR gene product, an alternative RNA sigma factor, that directs transcription from the tox promoters and its own promoter in response to growth phase and nutrition. We have shown that UviA, a protein homologous to TxeR, regulates as a sigma factor, transcription of a bacteriocin gene in C. perfringens. TxeR and UviA are the first described members of a new family of alternative sigma factors (Class V).
Keywords: Functional genomics , Mycobacterium tuberculosis complex , leprosy , Buruli ulcer , Clostridia