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



Our main research interests are  the study of the diversity and evolution of pathogenic microorganisms in relation to virulence and the study of global gene expression using macro- or microarays. The studied organisms are the pathogenic bacteria Listeria monocytogenes, Listeria ivanovii, Streptococcus agalactiae, Photorhabdus luminescens, and Neisseria meningitidis.



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

Listeria monocytogenes is a bacterium causing serious food-borne infections (mortality rate of 30 %) The clinical signs of listeriosis are most frequently meningitis, abortion and neonatal infections. The genus Listeria comprises two pathogenic species, L. monocytogenes and L. ivanovii (a bovine pathogen), and four non-pathogenic relatives : L. innocua, L. seeligeri, L. welshimeri et L. grayi. The genome sequence (2.94 megabases) of L. monocytogenes strain EGD-e has been completed by a consortium of 10 laboratories coordinated by P. Glaser and P. Cossart (Institut Pasteur, Unité des Interactions Bactéries-Cellules). We compared this sequence to that of L. innocua whose sequence was determined in our laboratory (P. Glaser et al., 2001. Science, 294: 849-852 ; brevets FR n°0004629 et FR n° 0012697). In order to get insight into the gene flux and the diversity within the genus Listeria we partially sequenced an epidemic strain of L. monocytogenes (serovar 4b) and — in collaboration with the German consortium " Pathogenomics " - we are sequencing the genomes of L. ivanovii and L. grayi. To understand the molecular basis of virulence of L. monocytogenes and certain particularly virulent strains, we extend these genome analyses to a large number of isolates. In collaboration with the Laboratoire de Référence des Listeria (directed by P. Martin), we have constructed macroarrays carrying genes absent in at least one of three Listeria strains (L. monocytogenes EGDe, 4b and L. innocua). The hybridization results obtained for more than one hundred strains of different origin and features showed that it is a diagnostic and strain typing tool allowing distinction of species within the genus Listeria and classification of strains belonging to the species L. monocytogenes in five groups, and several sub-groups. These results allowed us to identify specific markers for each subgroup as well as genes probably involved in virulence.

In collaboration with the Unité des Interactions Bactéries Cellules (directed by P. Cossart) and the Genopole Institut Pasteur we are studying global gene expression of L. monocytogenes using macroarrays. In particular, we have studied the targets of PrfA, a global virulence regulator of L. monocytogenes. The effect of the prfA mutation is more complex than initially expected. PrfA controls directly or indirectly three classes of genes. Analysis of our data led to the following hypotheses: 1) the presence of cellobiose in the culture medium prevents activation of Listeria monocytogenes virulence genes, confirming the results obtained in other laboratories that cellobiose could be considered as a signal indicating non-infectious conditions of growth; 2) PrfA apparently acts as both an activator and a repressor; 3) a relation exists between two regulons in L. monocytogenes, the PrfA and sigma B regulon (Milohanic et al., 2002. Mol. Microbiol., in press).

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

Photorhabdus luminescens is a commensal of a nematode and an insect parasite. This bacterium is both a model to study host-parasite interactions, and a potential industrial exploitable bacterium because of its capacity to synthesize numerous toxins (insecticides, bacteriocides and fungicides) and to secrete numerous enzymes. The genome sequence of 5.6 megabases has been determined 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. Three patent applications have been deposited (PCT FR n° 0200483 ; FR n° 0204798 et FR n° 0204799). A manuscript describing the analysis of this genome is in preparation.

Genome analysis of Streptococcus agalactiae (M. Zouine, E. Couvé, C. Rusniok, C. Buchrieser, P. Glaser and F. Kunst)

Streptococcus agalactiae is a causative agent of neonatal infections, including 85 % of meningites of the newborn (<2 months) and 10 % of infants (2 months — one year). In collaboration with the team of P. Trieu Cuot (Faculté de Médecine Necker), we have determined the complete genome sequence of strain NEM316, which caused sepsis in a new-born. This genome has a size of 2.2 Mb and contains 2136 predicted genes. Interestingly, we identified 14 genomic regions resembling pathogenicity islands. Most of the known or putative virulence genes are located on these pathogenicity islands. Therefore the genome organization of S. agalactiae is different from that found in other Streptococci, such as S. pneumoniae or S. pyogenes, but is reminiscent of that found for pathogenic E. coli. Genome analysis also identified a great number of genes coding for surface proteins, which are potentially interesting for the development of a vaccine (Glaser et al. 2002. Mol. Microbiol. 45 : 1499-1513 ; patent PCT FR n° 0201460).

We recently constructed macroarrays containing PCR products representing the complete genome of S. agalactiae NEM316. In collaboration with the team of P. Trieu-Cuot (Faculté de Médecine Necker), these macroarrays are used to study the effects of different culture conditions (pH, oxidative stress, anaerobiosis, growth in the presence of human serum) and different mutations in regulatory genes on global transcription to evaluate the importance of these conditions and these regulatory genes for virulence.

Genome analysis of Neisseria meningitidis (C. Rusniok, C. Buchrieser, E. Couvé, P. Glaser)

Neisseria meningitidis is the etiological agent of meningitis. Vladimir Pelicic (Faculté de Medecine Necker-Enfants Malades) has constructed a transposon library of 4000 insertion mutants of N. meningitidis 8013 (serogroup C). The transposon insertion sites of 4000 mutants have been characterized by sequencing. We are determining the complete sequence of this strain in collaboration with C. Bouchier (Génopole, Institut Pasteur). The complete genome sequence in combination with the 4000 mutants characterized and positioned on the chromosome will be a powerful tool for identifying virulence genes and for the phenotypic characterization of these genes.

Keywords: genomics, comparative genomics, evolution, arrays, microbial pathogens, virulence, bioinformatics

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  Office staff Researchers Scientific trainees Other personnel
  DULIEU Isabelle (idulieu@pasteur.fr)

Luchier Françoise (fluchier@pasteur.fr)

BUCHRIESER Carmen, IP (Chargé de Recherche IP,cbuch@pasteur.fr)

GLASER Philippe, IP (Chef de Laboratoire IP,pglaser@pasteur.fr)

KUNST Frank, CNRS, IP (DR2 CNRS, Chef de Laboratoire IP,fkunst@pasteur.fr)

VALLAEYS Tatiana, INRA (CR1,tvallaey@pasteur.fr)

CAZALET Christel, CNRS (Ph. D. Student)

DUCHAUD Eric, IP (Postdoctoral fellow,educhaud@pasteur.fr)

FEURER Carole, IP (Predoctoral fellow, Master’s degree Univ. Cork, Ireland)

MILOHANIC Eliane, (Postdoctoral fellow,emilohan@pasteur.fr)

ZOUINE Mohamed, IP (Postdoctoral fellow,mzouine@pasteur.fr)

CHETOUANI Farid, IP (Ingénieur,fchetou@pasteur.fr)

COUVÉ Elisabeth, IP (Technician,ecouve@pasteur.fr)

DOUALOT Harry, IP (Laboratory assistant)

RUSNIOK Christophe, IP (Technician,rusniok@pasteur.fr)

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