Unit: Genomics of Microbial Pathogens
Director: Frank KUNST, Philippe GLASER
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, Neisseria meningitidis and Legionella pneumophila.
Genome analysis of the genus Listeria (C. Buchrieser, C. Rusniok , P. Garrido, 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 ruminant pathogen), and four non-pathogenic relatives : L. innocua, L. seeligeri, L. welshimeri and L. grayi. We have sequenced and analyzed the genome sequence of L. monocytogenes strain EGD-e, and compared this sequence to that of L. innocua In order to get insight into the gene flux and the diversity within the genus Listeria we partially sequenced the genome of an epidemic strain, L. monocytogenes (serovar 4b). In collaboration with the Laboratoire de Référence des Listeria (directed by P. Martin), we have used these sequence data to construct macroarrays carrying 409 genes specific for at least one of the three Listeria strains. The hybridization results obtained for 93 L. monocytogenes strains of different origin and features as well as 20 strains representing all species of the genus Listeria 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. Furthermore, we identified 18 specific markers for the species L. monocytogenes and markers of each subgroup of this species that allow now the development of rapid strain typing tools. The knowledge of the distribution of different genes, such as genes encoding surface proteins probably involved in virulence and adaptation of L. monocytogenes, will be the basis for the study of virulence differences among L. monocytogenes strains (Doumith et al., In press). The German consortium " Pathogenomics " is sequencing the genomes of L. seeligeri and L. welshimeri. In collaboration with this consortium, we are sequencing the genomes of L. ivanovii and L. grayi. The availability of the genome sequences of each species of the genus Listeria will provide us with a comprehensive knowledge of the complete Listeria genus and will serve as a model for the study of the evolution of bacterial genomes.
Genome analysis of Photorhabdus luminescens (E. Duchaud, C. Rusniok, F. Kunst)
Photorhabdus luminescens is a commensal of nematodes 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 analysis of the complete genome sequence of 5.6 megabases has been published (Duchaud et al. 2003). In collaboration with the Laboratoire de Pathologie Comparée (INRA, Montpellier), the Unité de Génétique des Génomes Bactériens (Institut Pasteur) and the company Bayer CropScience we have identified numerous genes encoding toxins. Two patent applications have been deposited (PCT/FR02/00483 and PCT/FR03/01238).
Genome analysis of Streptococcus agalactiae (M. Zouine, E. Couvé, C. Rusniok, C. Buchrieser, P. Glaser, 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 (Institut Pasteur), we have determined the complete genome sequence of strain NEM316, which caused sepsis in a new-born.
We recently constructed macroarrays containing PCR products representing the complete genome of S. agalactiae NEM316. In collaboration with the teams of P. Trieu-Cuot (Institut Pasteur) and C. Poyart (Hôpital Cochin), we have studied the effect of culture conditions in relation to the infectious process (pH, oxidative stress, anaerobiosis, growth in the presence of human serum) and the effect of a knockout mutation of the covR gene encoding a transcription regulator of virulence genes. The use of these macroarrays to study biodiversity of the S. agalactiae species has allowed us to identify a highly conserved "core" genome and plasticity regions located in islands, representing in certain cases pathogenicity islands.
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. 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.
Genome analysis of the genus Legionella (C. Cazalet, P. Glaser, C. Buchrieser)
Legionella is an environmental bacterium that colonizes natural water reservoirs and water circuits. L. pneumophila is the causative agent of legionellosis which represents a public health problem with 5% of the patients with community-acquired pneumonia admitted to hospitals and cases of nosocomiales infections. The number of registered cases increases by about 30 % per year : 1020 cases have been reported in France during the year 2002. The L. pneumophila strains seems to be more virulent than strains from other Legionella species, since it is the causative agent of 90% of the legionellosis cases. Among the fifteen L. pneumophila serogroups, sérogroupe 1 isolates are associated with 80% of the cases.
We have started a genome-based epidemiology project, in collaboration with the Centre National de Référence des Legionella (Team INSERM E320, headed by Jérôme Etienne) and the Company Anjou Recherche. In collaboration with C. Bouchier (Génopole Institut Pasteur), we are sequencing the genome of the L. pneumophila strain "Paris", that is endemic in France and causes 10% of the legionellosis cases. To analyze the diversity among L. pneumophila species and to establish a correlation with virulence, we have partially sequenced the genome of a L. longbeachae strain, the second cause of legionelloses in Australia, and the genome of a non-pathogenic relative, L. anisa. The preliminary comparisons have revealed a considerable diversity among Legionella species.
These genome data will be used for the development of macro- or microarrays for the genomic characterization of Legionella isolates. We aim to identifiy virulence factors, to develop a typing tool and to exploit genomic features of clinical isolates to predict the risk associated with a contamination.
Keywords: genomics, comparative genomics, evolution, transcriptome, microbial pathogens, virulence