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  Director : COLE Stewart (stcole@pasteur.fr)


  abstract

 

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 M. ulcerans, an emerging pathogen responsible for Buruli ulcer.

The pathogenesis of Clostridium difficile and C. perfringens is due to powerful toxins and in C. difficile their synthesis shown to be controlled by a novel sigma factor.



  report

cale

Functional genomics of mycobacteria (Stewart Cole, Roland Brosch, Nadine Honoré)

A number of variable regions were identified in the genomes of members of the M. tuberculosis complex using microarrays. Some of these regions are probably associated with the attenuation of the vaccine strains, M. bovis BCG and Mycobacterium microti, and various phenotypic differences. This hypothesis is being tested experimentally using gene knock-ins and knock-outs. Isoniazid (INH) is a powerful anti-tuberculous agent whose toxicity results from its transformation into acyl radicals during a reaction catalysed by catalase-peroxidase, KatG. The virulence of selected isoniazid-resistants clinical isolates was assessed in an animal model of tuberculosis. This work revealed that the most frequently-encountered mutation, katGSer315Thr, had no significant effect on the virulence of M. tuberculosis but resulted in high level drug resistance. Ethionamide (ETA), a second-line drug, is a structural analogue of INH which inhibits cell wall synthesis after activation. On studying the ETA resistance of Mycobacterium leprae, mutations were found in the ethA gene whose product transforms the drug into a toxic agent by S-oxidation.

Genome sequence analysis of Mycobacterium ulcerans (Timothy Stinear)

Mycobacterium ulcerans is an emerging pathogen that causes Buruli ulcer, a chronic, necrotic skin lesion in humans, and has rapidly emerged as an important cause of morbidity around the world. The prevalence of Buruli ulcer throughout West Africa appears to have increased dramatically since the late 1980s. Buruli ulcer is considered the third most common mycobacterial disease of non-immunocompromised persons after tuberculosis and leprosy. M. ulcerans is unlike other mycobacterial pathogens in that it appears to maintain an extracellular location during infection and produces a macrolide toxin, mycolactone. The time has now come to apply the genomic approach to M. ulcerans as this will elucidate the epidemiology, biology and pathogenesis of this important emerging pathogen, and enable new prophylactic and therapeutic approaches to be conceived.

The project is progressing well and is on schedule. To date a BAC library has been constructed and terminal sequencing of 300 clones completed. This library provides the scaffold for assembly of the genome from the smaller DNA fragments of the shotgun library. This library contains approximately 28,000 clones and has yielded 47,000 good sequence reads, representing a theoretical 6-7 fold genome coverage. Assembly and gap-filling are now in progress, and the annotation phases of the project will commence shortly. In parallel, we have screened the BAC library for clones with the potential to encode the type 1 polyketide synthase required for mycolactone production and isolated two suitable candidates.

The Mycobacterium bovis genome sequencing project (Thierry Garnier, Karin Eiglmeier)

This work has been performed in collaboration with the Pathogen Genome Sequencing Unit at the Sanger Centre, directed by Bart Barrell, and Glyn Hewinson at the United Kingdom's Veterinary Laboratories Agency. The strain of M. bovis that is being studied (AF2122/97 spoligotype 9) was responsible for herd breakdown in Devon in the UK and has been isolated from lesions in both cattle and badgers.

The strategy being followed to sequence the M. bovis genome comprises two approaches. At first, sequences were obtained different genome shotgun libraries and assembled into contigs. In parallel, a bacterial artificial chromosome library (BAC) library of M. bovis was constructed and the end-sequences of its clones were determined. By comparison with the genome of M. tuberculosis H37Rv, taking advantage of the high degree of sequence identity between M. bovis and M. tuberculosis ( >99.9%), it was possible to position the BAC clones, resulting in a set representing almost the entire M. bovis chromosome. The BAC clone framework permitted the positioning of the shotgun sequence contigs and enabled the complete genome sequence to be obtained. Its analysis is currently underway.

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 and to identify and characterise 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. Mutagenesis experiments using transposon Tn916 led to the identification of a number of genes involved in this response and some of them have been investigated further. We found that two ORFs, orthologous to the ydaD and ycdF genes of Bacillus subtilis, were essential for efficient resistance to oxidative stress and both may be involved in the generation of NADPH. Other genes were also shown to participate for the oxidative stress response, such as the genes encoding an atypical iron sulphur prismane protein, a ß-glucuronidase, and an ATP-dependant RNA helicase.

In a separate study, the search for new targets to enable the development of strong inhibitors of the initiation and establishment processes of these organisms during infection, led us to work on the functional organisation of a small family of proteins that have an affinity to choline residues. We have demonstrated that these CBP proteins (Choline Binding Protein) are present in all toxinotypes of C. perfringens strains and are cell membrane associated. The role in the pathogenicity and the immunogenic capacity of all CBP protein are currently being investigated.

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 in response to environmental changes. We have shown that TxeR, like its UviA homologue which controls the bacteriocine expression of C. perfringens (strain CPN50) belongs to the new alternative sigma factor (Class V).

Finally, results obtained from a study in collaboration with a team from St Antoine on metronidazole resistance in C.difficile, suggest a novel mechanism of resistance, different from that shown in Bacteroides fragilis, occurring through antibiotic inactivation which we are currently trying to identify. It was also shown that all the clinical strains studied in the laboratory could be differentiated by their MLS and quinolone resistance phenotype, their plasmid profiles and by the RAPD technique, suggesting that these different metronidazole resistant strains did not have a clonal origin



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  publications

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  personnel

  Office staff Researchers Scientific trainees Other personnel
 

FARGUES Anne-Marie (fargues@pasteur.fr)

COLE Stewart, IP (professeur, stcole@pasteur.fr)

GARNIER Thierry, IP, (chargé de recherche, tgarnier@pasteur.fr)

EIGLMEIER Karin , IP (chargé de recherche, kei@pasteur.fr)

BROSCH Roland, IP (chargé de recherche, rbrosch@pasteur.fr)

REYSSET Gilles, IP (chef de laboratoire, greysset@pasteur.fr)

DUPUY Bruno, IP (chargé de recherche, bdupuy@pasteur)

BRODIN Priscille, chercheur contractuel

PYM Alexander, postdoc. (left the research unit on 15 November 2001)

STINEAR Timothy, postdoc.

MARMIESSE Magali (graduate student)

HONORE Nadine, ingénieur (nhonore@pasteur.fr)

SAINT-JOANIS Brigitte, ingénieur (bsjoanis@pasteur.fr)

BRIOLAT Valérie, technicienne (vbriolat@pasteur.fr)

GRONDIN Sophie, technicienne temporaire (sgrondin@pasteur.fr)


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