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  Director : Frank KUNST, Philippe GLASER (fkunst@pasteur.fr, pglaser@pasteur.fr)



The main themes of our laboratory are : 1) the study of the diversity and evolution of pathogenic microorganisms in relation to virulence ; 2) the study of global gene expression by the use of macro- or microarays. The studied organisms are : five pathogenic bacteria (Listeria monocytogenes , Listeria ivanovii, Streptococcus agalactiae, Photorhabdus luminescens, Neisseria meningitidis), and a pathogenic fungus (Aspergillus fumigatus).



Genome analysis of the genus Listeria (C. Buchrieser, P . Glaser, E. Milohanic, C. Rusniok , T. Vallaeys)

Listeria monocytogenes is a bacterium causing serious food-borne infections (mortality : 30 %), whose clinical signs are most frequently meningitis, abortion and neonatal infections. The genome sequence (2.94 megabases) of strain EGD-e of L. monocytogenes has been completed by a consortium of 10 laboratories which has been coordinated by us together with P. Cossart (Institut Pasteur, Laboratoire des Interactions Bactéries-Cellules). In our laboratory we also have completed the genome sequences of other bacteria of the genus Listeria : the complete sequence of L. innocua (a non-pathogenic bacterium closely related to L. monocytogenes), a partial sequence of L. monocytogenes 4b (an epidemic strain), and — in collaboration with the German consortium " Pathogenomics " - the nearly complete sequence of L. ivanovii (an animal pathogen). Specific features of L. monocytogenes EGD-e, deduced from the analysis of its genome, have been described in a recent paper (P. Glaser et al., 2001. Science, 294 : 849-852). It contains a large number of proteins of the following families : surface proteins containing an LPXTG motif, internalins, sugar transport systems, proteins showing similarities to Bacillus subtilis competence proteins and regulators of the Crp/Fnr family. The comparison of these genome sequences - with the help of the software FindTarget developed in our laboratory — is used to identify potential virulence genes, and more generally, to understand the pathogenicity of L. monocytogenes and its ability to contaminate food. Furthermore, we are using a comparative genomics approach based on DNA arrays to characterize clinical and environmental isolates of Listeria.

For the characterization of the biodiversity of the genus Listeria, and in particular that of the species L. monocytogenes, we use a comparative genomics approach with macroarrays. Specific genes for L. innocua, for the strains EGD-e and 4b (CLIP80459) of L. monocytogenes have been identified. In collaboration with the Laboratoire de Référence des Listeria (Institut Pasteur), directed by P. Martin, PCR fragments corresponding to these spécific genes have been deposited on high density membranes and hybridized with total ADN from other isolates of Listeria : two additional strains of L. innocua, two strains of L. ivanovii, two strains of L. seeligeri, L. welshimeri and L. grayi as well as eighty strains of L. monocytogenes, isolated from patients, the food chain or the environment. This study aims to find a correlation between epidemiology and genomic data, in order to distinguish between potentially virulent and non-virulent strains.

Finally, with the help of the Genopole Institut Pasteur, a study of the transcriptome of L. monocytogenes has been undertaken, in particular studies concerning targets of the regulatory gene prfA (E. Milohanic and C. Buchrieser) and those of sigma factor rpoN (Y. Hechard, S. Arous and C. Buchrieser).

Genome analysis of Photorhabdus luminescens (E. Duchaud, F. Kunst)

Photorhabdus luminescens is a commensal of a nematode and an insect parasite. This bacterium is both a model for the study of host-parasite interactions, and a potential industrial bacterium because of its capacity to synthetize numerous toxins (insecticides, bacteriocides and fungicides) and to secrete numerous enzymes. At present, the genome of 5.6 megabases has been assembled into 2 contigs, and has been nearly entirely annotated. In collaboration with the Laboratoire de Pathologie Comparée (INRA, Montpellier) and the company Aventis CropScience we have identified numerous genes encoding toxins. A patent application has been deposited.

Genome analysis of Streptococcus agalactiae (P. Glaser, C. Rusniok, M. Zouine)

Streptococcus agalactiae is a group B Streptococcus. Neonatal infections caused by Streptococci constitute an important health problem. The frequency of occurrence is 2.5 per 1000 births, with a mortality rate in the range of 4 - 10%, and cases of meningitis leading to neurological aftereffects in 25 - 50 % of the cases. We have assembled this genome of about 2 megabases into 3 contigs. The availability of the complete genome sequences of two related Streptococci, S. pneumoniae and S. pyogenes, allows us to carry out a comparative genomics study.

A transcriptome analysis of S. agalactiae, in different culture conditions (pH, oxidative stress, anaerobiosis, growth in the presence of human serum), has recently been started to evaluate the importance of these conditions for virulence.

Participation in genome sequencing of Aspergillus fumigatus (E. Couvé, P. Glaser)

In the framework of the Aspergillus fumigatus genome project and for transcriptome analysis, we have sequenced 9000 genomic tags of this fungus causing invasive aspergillosis. DNA arrays are prepared to carry out studies of global gene expression in collaboration with J.-P. Latgé (Unité des Aspergillus), and to identify new targets to combat this fungus. Recently, an international project aimed at the sequencing of the Aspergillus fumigatus genome has been set up with the participation of the Sanger Centre (UK), the University of Manchester (UK), The Institute for Genomic Research (TIGR, USA), the Universities of Madrid and Salamanca (Spain), and the Institut Pasteur (Unité des Aspergillus and our laboratory).

Genome analysis of Neisseria meningitidis

Neisseria meningitidis is the aetiological agent of meningitis. Our project aims to determine the genome sequence of strain 8013 which presents the following advantages : 1) it is transformable ; 2) in-vitro and in-vivo infection models are available. This project, carried out in collaboration with the laboratory of X. Nassif (Faculté de Medecine Necker-Enfants Malades) and the Unité des Neisseria (Institut Pasteur) directed by J. M. Alonso, aims to identify virulence genes with the help of a library of mutants obtained by transposition.

Development of bioinformatics tools (L. Frangeul, F. Chetouani)

To facilitate the analysis of genome sequences, three informatics software tools have been developed in our laboratory : " GMP-Tool-box (GMPTB) " by Lionel Frangeul, " Findtarget " and " Difftool " by Farid Chetouani. The software GMPTB allows to undertake the annotation of a genome before the obtention of the complete sequence. At the end of the " shotgun " phase of a bacterial genome project, the assembled sequences are usually distributed in several hundred contigs. During the closure phase, the contigs are linked, diminishing their number and modifying the position of the coding sequences within the linked contigs. GMPTB allows to find easily the coding sequences from the previous assembly due to the creation of an IPF file (" individual protein file ") which contains the coding sequence. This tool allows to save time since the user can start annotating the genome - a relatively slow process — before the final assembly of the genome.

Findtarget is a search engine, based on the Blast similarity search tool (F. Chetouani et al. 2001. Microbiology 147 : 2643-2649). Findtarget allows the rapid and automated detection of proteins present in a set of genomes but absent in another set. We currently use this tool for comparative genomics searches to identify potential virulence genes among the genes which are present in L. monocytogenes but absent in L. innocua.

DiffTool is a resource to explore protein clusters computed from a sequence database. The package provides a clustering tool to construct a database of protein families according to sequence similarity criteria extracted from BlastP outputs, and a web interface to query the cluster database. For each protein cluster, it includes access to sequences, to alignments and to phylogenetic trees. A subtractive genome analysis tool allows to select protein families present in a set of genomes but absent in another set.

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  Office staff Researchers Scientific trainees Other personnel

Frank KUNST (e-mail : fkunst@pasteur.fr)

Philippe GLASER (e-mail : pglaser@pasteur.fr)

Carmen BUCHRIESER, researcher Institut Pasteur (e-mail : cbuch@pasteur.fr)

Philippe GLASER, researcher Institut Pasteur (e-mail : pglaser@pasteur.fr)

Frank KUNST, researcher CNRS (e-mail : fkunst@pasteur.fr)

Tatiana VALLAEYS, researcher INRA (e-mail :tvallaey@pasteur.fr)

Eric DUCHAUD, Postdoc (e-mail : educhaud@pasteur.fr)

Carole FEURER, Engineer (e-mail : cfeurer@pasteur.fr)

Eliane MILOHANIC, Postdoc (e-mail : emilohan@pasteur.fr)

Mohamed ZOUINE, Postdoc (e-mail : mzouine@pasteur.fr)

Farid CHETOUANI, Engineer (e-mail : fchetou@pasteur.fr)

Fabien CHEVALIER, Technician

Elisabeth COUVÉ, Technician (e-mail :ecouve@pasteur.fr)

Harry DOUALOT, Laboratory assistant

Christophe RUSNIOK, Technician (e-mail :rusniok@pasteur.fr)


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