Unit: Genomics of Microbial Pathogens

Director: Frank KUNST, Philippe GLASER

We study the evolution and adaptation of three opportunistic bacterial pathogens: Listeria monocytogenes, Streptococcus agalactiae and Legionella pneumophila. Our goal is to understand the interactions between these bacteria, their hosts and their environments by combining comparative genomics, transcriptome analysis, bacterial physiology and genetics. To elucidate the genomic bases of the acquisition of virulence determinants by these pathogens, we study the diversity and evolution of the genera to which they belong.

The species Listeria monocytogenes and the genus Listeria (C. Buchrieser, C. Rusniok, T. Vallaeys, P. Severino, S. Duperrier, P. Glaser)

Listeria monocytogenes is an opportunistic pathogen causing serious food-borne infections (mortality rate up to 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 first complete genome sequence of L. monocytogenes (strain EGD-e), and have compared it to the genome sequence of L. innocua (Glaser et al., 2001). In order to get a better insight into virulence of pathogenic Listeria we have now determined and analyzed the genome sequence of an epidemic strain of L. monocytogenes (serovar 4b). In collaboration with the National Reference Centre for Listeria (directed by P. Martin), we have used these sequence data to design and construct macroarrays carrying 739 genes specific for at least one of the sequenced Listeria strains. Hybridization results obtained for 263 L. monocytogenes strains of different origin and characteristics as well as for 20 strains representing the remaining 5 species of the genus Listeria show that this "Listeria-array" is a promising typing tool allowing distinction of species within the genus Listeria and classification of strains belonging to the species L. monocytogenes. Analyses in progress aim to better characterize the genomic bases of the epidemic strains.

A second aim is to understand the regulatory networks affecting virulence of L. monocytogenes. Thus we use macro-arrays containing all genes of L. monocytogenes EGDe to perform comparative expression profiling of the wild type strains and derived mutants, in which regulatory genes are inactivated. These experiments are performed under various growth conditions, reflecting different environments encountered by Listeria during infection.

The species Streptococcus agalactiae (M. Brochet, E. Couvé, M. Zouine, C. Rusniok, C. Buchrieser, P. Glaser, F. Kunst)

We have recently determined the complete genome sequence of strain NEM316, which caused sepsis in a newborn. Based on this sequence we constructed macroarrays containing PCR products representing the complete genome. In collaboration with the teams of P. Trieu-Cuot (Institut Pasteur) and C. Poyart (Hôpital Cochin), we have studied the genes covRS encoding a two-component regulatory system affecting the expression of a large number of secreted proteins, surface proteins and virulence factors (Lamy et al., 2004). These macroarrays were also used to study biodiversity of strains of the species S. agalactiae of highly diverse origins. This has allowed us to identify a highly conserved "core" genome and plasticity regions located in islands, representing, in certain cases, pathogenicity islands.

The covRS system affects only a subset of virulence genes and surface proteins. To characterize additional regulatory networks affecting the synthesis of these proteins, we have developed an high throughput immunological screening method using high-density membranes. This method allows the identification of mutations affecting the expression of secreted or surface proteins. Using this method, we identified differences in the level of expression of certain proteins among different S. agalactiae strains, probably due to a mechanism of phase variation. Based on these results, candidate proteins for the development of a vaccine will be chosen.

The projects on L. monocytogenes and S. agalactiae are carried out in the framework of a GPH funded by the Institut Pasteur and coordinated by Pascale Cossart " Towards therapeutics against low-GC% Gram-positive bacteria ".

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) has constructed a transposon library of 4000 insertion mutants of N. meningitidis 8013 (serogroup C), and characterized by sequencing the transposon insertion sites of 4000 mutants. We have determined the complete genome sequence of this strain. We are analyzing this sequence by comparing it with the three other genome sequences available for this species. 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.

The species L. pneumophila and the genus Legionella (C. Cazalet, H. Brüggemann, A. Hagman, F. Kunst, P. Glaser, C. Buchrieser)

Legionella is an environmental bacterium that colonizes natural water reservoirs and water circuits. Two species, L. pneumophila and L. longbeachae, are responsible for the majority of legionellose cases worldwide.

We have determined the genome sequence of L. pneumophila strain Paris (3.5 Mb), an endemic and epidemic strain in France, and that of the epidemic strain L. pneumophila Lens, in collaboration with the National Reference Centre for Legionella (directed by Jérôme Etienne), Anjou Recherche-Veolia Water, and the Genopole Institut Pasteur. The two most striking features of Legionella are: 1) a large number of genes encoding proteins similar to eukaryotic proteins. Some of these proteins may modulate host functions; 2) a very high genome plasticity as 9 - 13 % of the genomes are specific for each strain (Cazalet et al., 2004).

In order to characterize the diversity present among different Legionella species, 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 comparison of these partial sequences with those of the L. pneumophila strains has revealed a considerable diversity among Legionella species : for example, only 50 % of L. pneumophila and L. longbeachae genes are orthologous.

Based on these genome data we are developing a genome-based epidemiology project, in collaboration with the National Reference Centre for Legionella and the Anjou Recherche to identify virulence factors, to develop a molecular typing tool and to exploit genomic features from strains isolated from patients to predict the risk following contamination.

European networks

The Unit participates in the establishment of two networks, recently accepted by the European Commission. The Network of Excellence " Europathogenomics ", coordinated by J. Hacker (Univ. Würzburg, Germany) aims to create a scientific impetus in the field of functional genomics of pathogenic bacteria, to favour collaborations and to propose training for postdoctoral researchers. The goal of ERA-NET is the coordination of research efforts of member states to lead to a structured European research on bacterial and fungal pathogens for humans.

Keywords: genomics, comparative genomics, evolution, transcriptome, microbial pathogens, virulence, regulations

Activity Reports 2004 - Institut Pasteur

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