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, 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 compared the genome sequences of L. monocytogenes (strain EGD-e) and L. innocua (Glaser et al., 2001) and, more recently, we have analyzed the genome sequence of an epidemic strain of L. monocytogenes (serovar 4b) in order to get a better insight into virulence of pathogenic Listeria and their relations with non pathogenic species. In collaboration with the German consortium PathoGenoMics, we have determined the nucleotide sequences of representatives of the four remaining species of Listeria. The knowledge of the genomes of all species of the genus will provide us with a unique set of genomic data concerning the evolution of Listeria and the acquisition of virulence traits by pathogenic Listeria species.

We study, in collaboration with the National Reference Centre for Listeria (Institut Pasteur), the diversity of the species L. monocytogenes and the specific features of clinical isolates by hybridization with macroarrays carrying genes specific for at least one of the sequenced Listeria strains. Hybridization results obtained for 300 Listeria strains of different origins and characteristics show that these arrays are 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 specificity of the epidemic strains compared to environmental ones.

To understand the regulatory networks controlling virulence of L. monocytogenes, we use macro-arrays containing all genes of L. monocytogenes EGDe to study the expression profiles 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. This tool will also allow us to study the specificity of expression profiles of clinical isolates compared to environmental isolates.

The species Streptococcus agalactiae

(M. Brochet, E. Couvé, S. van Baarle, C. Rusniok, C. Buchrieser, P. Glaser, F. Kunst)

After the determination of the complete genome sequence of strain NEM316, which has caused sepsis in a newborn, we are studying the genomic diversity of 75 strains of human and animal origin. For this purpose, we have combined multilocus sequence typing (MLST), analysis of variable loci by sequencing, serotyping, and comparative genomics by hybridization using DNA arrays. These studies have confirmed the mosaic structure of the S. agalactiae genome with a highly conserved "core" genome and a set of variable islands. Virulence genes associated with these islands are present in the majority of these isolates, suggesting that these islands were present at the origin of the S. agalactiae species, and were implicated in the acquisition of these virulence genes. We have also identified six gene families, encoding secreted or surface proteins, presenting variable alleles in the strains studied. The analysis of these allelic combinations showed a large diversity and the possibility of allelic exchanges that could take place during interaction of the bacterium with the host immune system.

This study also revealed the important genomic homogeneity of the "hypervirulent" clone ST17 containing a specific set of surface proteins. The origin of this clone is thus probably recent. These genomic data constitute a first step to the understanding of the hypervirulence of this clone.

We combine to this genomic approach a functional analysis of the expression of genes involved in the interaction with the host by means of whole genome DNA arrays and antibodies hybridization to high-density clone arrays.

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

The species L. pneumophila and the genus Legionella

(C. Cazalet, H. Brüggemann, A. Hagman, M. Jules, S. Duperrier, 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 legionellosis cases worldwide.

After the determination of the genome sequences of two isolates, L. pneumophila "Paris" and L. pneumophila "Lens", in collaboration with the National Reference Centre (CNR) for Legionella (directed by Jérôme Etienne), Veolia Water, and the Genopole Institut Pasteur, we have undertaken biodiversity and postgenomic studies.

In order to analyze the genomic diversity within the genus Legionella, we are sequencing the genome of L. longbeachae, the second cause of legionelloses in Australia and New Zealand. Comparative genomics of these pathogenic species should allow to identify virulence genes important for human infection and to elucidate mechanisms of pathogenicity of these bacteria. The final goal is to understand the genomic specificity of human pathogens as compared to non pathogenic Legionella strains.

To get a profound knowledge of the diversity present within the genus L. pneumophila, we have extended the genomic analysis to a set of 300 clinical and environmental isolates of the species L. pneumophila having different epidemiological and phenotypic characteristics, by means of DNA-arrays based on available genomic information (in collaboration with the CNR for Legionella and Veolia Water). This array appears to be a powerful typing tool, discriminating, for example, between various isolates of the "Paris" strain cluster. This analysis will be combined to functional studies to identify new virulence factors and to validate the possibility to assess the risk associated with a contamination by a genome-based analysis.

L. pneumophila has a typical bi-phasic lifestyle comprising an extracellular invasive phase and an intracellular replicative phase that is not infectious. The cellular differentiation allowing alternation between these two states is a central process in the multiplication cycle of this bacterium. To understand which, are the strategies developed by L. pneumophila to adapt to and to survive in a water environnement (amoeba, biofilm), we have developed microarrays carrying genes specific to the three sequenced strains of L. pneumophila (in collaboration with the CNR for Legionella and the Genopole Institut Pasteur). In vivo transcriptome studies in the Acanthamoeba castellanii model revealed extensive gene expression changes during the biphasic lifecycle of L. pneumophila. The gene expression levels of all genes of strains Paris, Lens and Philadelphia were compared in the two phases, revealing increased levels of expression of 547 genes and 456 genes in an in vivo time course experiment. Interestingly, numerous virulence genes are expressed in the transmissive phase, the regulon affecting flagellar biosynthesis, many regulators, genes affecting sugar catabolism and many genes of unknown function. This study points to putative novel virulence genes, and will allow us to better understand the regulatory systems of the L. pneumophila lifecycle.

Finally, a metabolic reconstruction has been undertaken to better understand the relation between virulence and metabolism, and to identify the nature of the nutrients preferentially utilized in-vitro and in-vivo (in the amoeba).

European networks

The Unit is responsible for the French section of the Network of Excellence " Europathogenomics ", coordinated by J. Hacker (Univ. Würzburg, Germany) and for ERA-NET " Pathogenomics ". The network of excellence aims to create a scientific impetus in the field of functional genomics of pathogenic bacteria, to favor 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, regulation


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