Comparative mycobacterial genomics(Stewart Cole, Karin Eiglmeier, Thierry Garnier)
In recent years, we have determined and analysed the complete genome sequences of M. tuberculosis H37Rv and M. leprae, and established relational databases to present the findings. The genome sequences of the cattle pathogen, M. bovis, and the vaccine strain, M. bovis BCG Pasteur, are currently in the final stages and their comparison should enable us to understand the mechanism of attenuation of BCG, and to design improved tuberculosis vaccines. An extreme case of reductive evolution was uncovered by comparative genomic studies of the closely related tubercle and leprosy bacilli. Over 50% of the M. leprae genome, which is 1.1 Mb smaller than that of M. tuberculosis (4.4 Mb), is occupied by pseudogenes that still have functional counterparts in the tubercle bacilli. This massive gene loss explains in part our inability to culture M. leprae in the laboratory and accounts for its exceptionally long generation time of 14 days. Comparative genomics has not only defined the minimum gene set for an intracellular pathogenic mycobacterium but identified around 100 unknown proteins that are confined to the genus. These proteins are of interest in terms of both drug target discovery and vaccine development and will be the object of extensive functional and structural studies.
Mycobacterium ulcerans, an emerging pathogen that causes Buruli ulcer,a chronic, necrotic skin lesion in humans, has rapidly emerged as an important cause of morbidity around the
world. Buruli ulcer is the third most common mycobacterial disease after tuberculosis and leprosy, and no vaccine or treatment, other than surgery, exists. Unlike other mycobacteria, M. ulcerans remains extracellular during infection and produces a macrolide toxin, mycolactone. Genomics is currently being used to understand the epidemiology of the disease, to provide new therapeutic leads and to test the possibility that M. ulcerans is derived from M. marinum, an intracellular pathogen of fish and humans, commonly isolated from aquatic environments worldwide.
Comparative and functional genomics of the M. tuberculosis complex(Roland Brosch, Stewart Cole)
In addition to M. tuberculosis, M. bovis and BCG, the M. tuberculosis complex comprises M. africanum, M. microti and M. cannettii. Although these species show >99.93% DNA sequence identity, they differ widely in their host range with M. tuberculosis being confined to humans, while M. bovis infects a wide variety of mammals. Furthermore, their ability to cause human disease varies considerably as BCG and M. microti are both safe live vaccines. To elucidate the molecular basis for these differences, various array and mapping technologies have been used to probe genomic diversity. This led to the identification of variable loci that were altered by deletion, insertion or duplication events, and enabled a degree of relatedness based on gene content to be established in which M. tuberculosis is closer to the last common ancestor of the complex members than the others, and BCG is the most divergent member. Blocks of genes that have been deleted during the evolution of BCG have been reintroduced and their effect on phenotype and virulence are being assessed.
Drug resistance in M. tuberculosis and M. leprae(Stewart Cole, Nadine Honoré, Brigite Saint-Joanis)
The susceptibility of M. tuberculosis to isoniazid (INH) results from enzymatic activation of this front-line drug by catalase-peroxidase (KatG), a major virulence factor. The katG gene is situated downstream of furA, encoding a ferric uptake regulator, and the genes are co-transcribed. Their respective roles in the INH susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a D
(furA-katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid and full virulence was restored, indicating that furA regulates transcription of both genes. When furA alone was introduced into the D
(furA-katG) mutant, virulence was partially restored, demonstrating that FurA regulates genes, other than katG, that are involved in pathogenesis. Mutants lacking KatG overproduce alkyl hydroperoxide reductase, AhpC, an enzyme that may detoxify activated INH. This dodecameric enzyme has been purified and its crystal structure resolved by the Unité de Biochimie Structurale.
Investigation of the pathogenicity and antibiotic resistance 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 but also to identify and characterise new virulence factors that could 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(es) is an important factor in the pathogenesis of this ubiquitous organism. Mutagenesis experiments using transposon Tn 916 led to the identification of a number of genes involved in this response and are currently being investigated. Also, the search for new targets to enable the development of strong inhibitors of the intiation and / or establishment processes of these organisms during infection, led us to work on the functional organisation of four CBP proteins (Choline Binding Protein), that belong to the small family of proteins that have an affintiy to choline residues. These proteins are homologous to PspA, a membrane protein of Streptococcus pneumoniae which is immunogenic as well as being a virulence factor.
The pathogenic capacity of C. difficile, the organism responsible for pseudomembranous colitis and most antibiotic-associated diarrheas, is mainly due to the large production of two major toxins, Tox A and Tox B. We have shown that transcription of the tox genes is regulated through a novel mechanism which requires the txeR gene product. The latter belongs to the alternative sigma factors family and acts on the expression of toxins A and B in response to envrìronmental changes. We are currently trying to find out if Vir R, the activator of the two component system VirR / Vir S, is capable of modulating the expression of toxins A and B.
Finally, results obtained from a study in collaboration with a team from the Hôpital Saint-Antoine on metronidazole resistance in C .difficile, suggest a novel mechanism of resistance, different from that shown in Bacteroides fragilis, occuring through antibiotic inactivation which we are trying to identify.