Unit: Pathogenesis of Mucosal Bacteria
Director: LABIGNE Agnès
The research work undertaken within the Unit is focused on the study of mucosa-associated bacterial pathogens. These include: Helicobacter pylori, the bacterium associated with the formation of inflammatory gastroduodenal diseases (chronic gastritis, peptic ulcer, lymphoma and gastric cancer) in humans; and pathogenic Escherichia coli strains that are associated with diarrhea and extra-intestinal infections (UTI, septicemia, meningitis). Functional and comparative genomics, studies on bacterium-host cell interactions, signalization pathways induced in vitro and in experimental infection models, studies on as well as genotoxic effects due to inflammation, search for prophylactic and therapeutic targets, and epidemiological studies are some of the approaches that are being used to study these mucosal pathogens.
The prevalence of H. pylori infections is very high, with 30% of individuals in developed countries, and as many as 80 to 95% of those in developing countries, infected with the bacterium. H. pylori is responsible for the most common bacterial infection worldwide, and diseases associated with these infections have an important medical and economic impact. Indeed, it is estimated that 10% and 1% of infected individuals develop peptic ulcer disease and gastric neoplesia (adenocarcinoma and MALT lymphoma), respectively. Different projects are currently pursued that aimed at better understanding the adaptation of this bacterium to its unique gastric niche, its biodiversity, and those of the mechanisms that are involved in the genesis of epithelial lesions.
The adaptive response of H. pylori to the gastric environment (Hilde De Reuse with Kerstin Stingl, Marie Thibonnier, Kristine Schauer and Chantal Ecobichon).
Genetics, functional genomics (transcriptomics and proteomics) and animal models are used to investigate the response of H. pylori to the gastric niche. We previously found that acidity is a signal modulating the expression of several virulence factors among which urease and the vacuolating cytotoxin, VacA. Regulatory networks implicate two metal responsive transcriptional regulators (NikR and Fur) and an essential two component response regulator (OmpR-like) as effectors of this response to acidity. The three-dimensional structure of the nickel responsive regulator NikR of H. pylori, and its unique properties were solved thanks to cristallogenesis (collaboration with L. Terradot, ESRF Grenoble) and site-directed mutagenesis. The role of the two carbonic anhydrases (CA-HP) in the adaptive response to acidity was also investigated. These enzymes catalyze the interconversion of carbon dioxide and bicarbonate and have, in other organisms, been involved in pH homeostasis. Single and double CA-HP mutants were constructed and were found to be impaired in their urease-dependent response to low pH, in ammonia production as well as their ability to colonize the mouse gastric mucosa and to induce an inflammatory response. CA thus constitute new important partners in the adaptation of H. pylori to the gastric environment.
Metabolism and role of peptidoglycan (PG) (Ivo G. Boneca with Catherine Chaput and Chantal Ecobichon)
Genetic and biochemical analyses of hydrolases AmiA, Slt, and MltD involved in PG assembly in the periplasmic compartment of H. pylori emphasize their role in cell growth, morphology as well as in the innate immune response. AmiA is involved in cell division of H. pylori and in the morphological transition from the bacillary to the coccoid form of H. pylori. This transition correlates with changes in PG structure. It is blocked in an AmiA deficient mutant and allows the bacterium to escape the Nod1/Nod2 signalization response. Slt is a lytic transglycosylase required for the release of PG fragments during PG turnover. A Slt deficient mutant is impaired in its ability to induce an inflammatory response on epithelial gastric cells. MltD, also identified as a lytic transglycosylase, plays a role in PG remodeling and in a transition phase between a cultivable to a non cultivable status of the bacterium that precedes the morphological transition phase. Deficient mutants in either Slt, MltD, or AmiA are impaired in their ability to colonize the mouse gastric mucosa. Thus, hydrolases might be considered as new putative therapeutic targets to eradicate H. pylori.
The role of peptidoglycan in the innate immune response to bacterial infections in drosophila (collaboration with J. Royet, CNRS Strasbourg for the characterization of "peptidoglycan recognition proteins", PGRPs) and mammals are also part of the studies carried on. In mammals, recognition of PG is sensed via the Nod1 and Nod2 pathways. We showed that during Pseudomonas aeroginosa infections, recognition of PG via the Nod1 signalization pathway is essential for an efficient innate immune response. These analyses are currently pursued within a transversal research program that aims at establishing a link between PG metabolism and innate immune response during the course of infections with various pathogens such as H. pylori, Neisserai meningitides, Yersinia or Listeria.
Study of the host response and of genotoxicity associated with H. pylori infection (Eliette Touati with Jose Ramos Vivas and Valérie Michel, in collaboration with Béatrice Régnault (Plateform Microarrays Genopole, as well as Michel Huerre and Patrick Avé, Histopatholgy).
The global host response to H. pylori infection has been investigated in mice infected for 6 and 12 months, using the Affymetrix® DNA microarrays. Almost 200 genes show a modified expression by the infection, according to the selection criteria, of which 25% are commonly found regulated at 6 and 12 months. They are mainly involved in the immune response, transcription regulation, signaling cascade, transport and rearrangement of cytoskeleton. Experiments are in progress to validate these data and to identify potential candidate genes that might be implicated in the genotoxicity associated to the infection. Indeed, in a previous study, using the Big Blue transgenic mouse model allowing the detection and characterization of mutations in any organ, we showed that chronic infection of mice infected for 6 months had a mutagenic effect, relevant to the induction of oxidative DNA damages, mainly 8-oxo guanines. These lesions are specifically repaired by the DNA glycosylase OGG1. Thus, the influence of a deficiency in OGG1 on the inflammatory and mutagenic host responses to the infection has been investigated in Big Blue OGGI-/- mice. Surprisingly, a less severe inflammation as compared to the wild-type mice and no mutagenic effect were detected at the gastric level. These results strengthen the role of inflammation in the mutagenicity associated to the infection.
Genetic, biochemical and structural analyses of functions associated to DNA repair lesions, recombination mechanisms and replication control in H. pylori (Eliette Touati, Agnès Labigne with Aurélie Mathieu, Chantal Ecobichon, and Anna Pawlik, in collaboration with Pablo Radicella, CEA Fontenay aux Roses and J. Zakrzewska-Czerwinska, Pologne).
The aim of these studies is to characterize biological systems in H. pylori that are distinct from those commonly found in the other bacteria and to study their specificity and the impact of this system on H. pylori biology. As an example, MutS in H. pylori, belongs to the MutS2 family and is not involved in the DNA mismatch repair system that does not exist in H. pylori, and has an anti-recombinogenic activity. The role of MutS in the ability of bacteria to exchange genetic material as a mean to adapt to the host is studied in the mouse model. In the same way, proteins involved in DNA replication control found in E. coli, are absent in H. pylori, though this crucial needs to be controlled. The yeast two-hybrid assay allowed us to identify proteins as putative candidates to control the initiation process, and they are currently studied by biochemical and structural approaches.
Comparative genomics of clinical isolates of H. pylori (Agnès Labigne with Jean-Michel Thiberge in collaboration with the PT2 and PT3 platforms from Genopole)
Comparative genomics carried out with 120 clinical isolates collected in France allowed us to analyze the distribution of 213 non ubiquitous genes in relation to the various associated pathologies (gastritis, peptic ulcer, gastric metaplasia, MALT lymphoma). Cumulative gene distribution within populations of isolates associated with distinct pathologies confirmed some of the correlations previously established between cag-PAthogenicity Island (PAI) composition and H. pylori-associated gastro-duodenal disorders. A hierarchical cluster analysis based on the continuous and normalized values of hybridization allowed us to identify clusters of isolates one of which containing exclusively strains that lack the entire PAI and are MALT lymphoma associated isolates. Strain B38 was chosen as a representative of this cluster based on its genetic characteristics (transformability), and its ability to colonize the mouse and Mongolian gerbil gastric mucosa. The genome of strain B38 is currently sequenced and analyzed to identify properties specifically associated with the isolates belonging to this cluster.
PATHOGENIC ESCHERICHIA COLI
Escherichia coli, the subject of numerous biochemical and genetic studies, is one of the most intensively studied of all microorganisms. Although most strains of this species exist as harmless symbionts in the intestine, they are many pathogenic E. coli strains that cause a variety of diseases in animals and humans. Various projects are pursued in 2005 with a special emphasis on the characterization of mechanisms by which pathogenic bacteria colonize epithelial cells, identification of bacterial factors involved in host infectivity, epidemiological studies, and comparative and functional genomics.
Role of bacterial metabolic factors in host colonization. (Chantal Le Bouguénec with Laurence du Merle, and Vanessa Martinez-Jehanne, in collaboration with partners from a research Transversal program; group leader C. Le Bouguénec).
Preliminary studies suggest that most E. coli isolates causing renal infections are able to use sugars not metabolized by strains of the intestine as carbon sources. In order to identify bacterial factors influencing colonization of the intestine and urinary tract by pathogenic E. coli, we carried a more complete exploration of metabolic traits using phenotypic microarrays. Then, we used allelic exchange to knock out several pathways of interest to allow us to compare in vitro and in vivo the fitness of the mutants and wild-type parental strains by means of competition assays, growth in urine, biofilm formation ability, and in vivo colonization of the mouse intestine and urinary tract. Both colonization of the mucosa and persistence of the bacteria at these sites are evaluated.
Interaction of uropathogenic E. coli with kidney cells. (Chantal Le Bouguénec with Céline Héchard-Lafleuriel, and Laurence du Merle, in collaboration with partners from a research Transversal program; group leader C. Le Bouguénec).
An untreated or recurrent kidney infection can lead to chronic pyelonephritis, scarring of the kidneys and permanent kidney damage. Although several studies have shown that type I fimbriae are involved in the entry of E. coli in the bladder epithelium, the entry of bacteria into the kidney epithelium and its consequences on the severity of the infection have not been studied. We characterized the interaction of uropathogenic strains with collecting duct cells, the first epithelium encountered by bacteria ascending from bladder to kidneys. We showed for the first time that bacteria could invade kidney cells.
A sequencing project for the understanding of commensalism and virulence in E. coli. (Chantal le Bouguénec with Vanessa Martinez-Jehnna, and Laurence du Merle in collaboration with partners from the Coliscope consortium).
We proposed to increase our understanding of E. coli pathogenicity by sequencing and analyzing the genome of a diarrhea-associated isolate belonging to the enteroaggregative E. coli pathotype recognized as an emerging cause of diarrhea in children and adults worldwide.
Keywords: Helicobacter,gastritis, gastric carcinoma, ulcer, lymphoma, MALT, signalization, acidity, peptidoglycan, mucosa, inflammation, comparative genomics, proteomics, regulation, urease, nickel, adhesion, invasion, metabolom, Escherichia coli