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  Director : LABIGNE Agnès (alabigne@pasteur.fr)


  abstract

 

The research undertaken within the Unit is aimed at studying two classes of mucosa-associated, bacterial pathogens. These are: Helicobacter pylori, the bacterium associated with the genesis of inflammatory gastroduodenal diseases (chronic gastritis, peptic ulcer, and cancer) in humans; and pathogenic Escherichia coli strains that are associated with diarrhea and urinary tract infections. Functional and comparative genomics, as well as studies on bacterium-host cell interactions, are among the various approaches developed within the different projects.



  report

cale

Helicobacter bacteria

Comparative genomics: studies on gene distribution in H. pylori clinical isolates by hybridization on high density nylon membranes (Jean Michel THIBERGE and Agnès LABIGNE in collaboration with the genomic platform of Institut Pasteur)

Gene distribution studies of the 1590 ORFs identified in the sequenced genome of H. pylori strain 26695 revealed 242 ORFs to be non-ubiquitous among 28 clinical isolates from patients of different ethnic origins. High density nylon membranes containing these 242 non-ubiquitous ORFs, as well as 50 ubiquitous ORFs, were prepared and validated. These membranes are currently being used in a multi-centric study to type clinical isolates in relation to different gastroduodenal disorders and geographical origins.

Functional analyses: assigning the essential character of a selection of genes (Chantal ECOBICHON, Catherine CHEVALIER and Agnès LABIGNE)

Two genomic libraries were constructed in E. coli: the first of these consists of individual recombinant plasmids corresponding to the 1590 ORFs, which were cloned and stored as an ordered library, while the second library contains a pool of plasmid constructs in which each ORF has been disrupted to saturation by the insertion of a transposable element. Using these genetic tools, we are attempting to identify, via a global approach, those genes that are ubiquitous as well as essential for the viability of the bacterium. Of the 200 genes that were analyzed, 35 have already been identified as such. Proteins encoded by ubiquitous, essential and H. pylori-specific genes represent potential targets for new therapeutic agents.

DNA repair in H. pylori (Catherine CHEVALIER and Agnès LABIGNE in collaboration with Pablo RADICELLA from CEA-Fontenay aux Roses)

This program seeks to identify and characterize the DNA repair systems of H. pylori, as well as to evaluate their impact upon the intrinsic genomic variability found in this bacterial species. Genes predicted to be associated with DNA repair functions were inactivated. Hyper- or hypo-mutator activities of the constructed single or double mutants were measured and their ability to colonize mice was tested. H. pylori EndoIII glycosylase (HP0585) was shown to dramatically impair the ability of the bacterium to persist during chronic infection, indicating that the bacterium is exposed to a deleterious oxidative stress during infection, while the annotated EndoIII glycosylase (HP0602) was found to be an atypical 3-methyladenine DNA glycosylase.

Role of inflammation in the induction of mutations in the gastric mucosa as measured in the " Big Blue " transgenic mouse model (Jean-Michel THIBERGE and Agnès LABIGNE in collaboration with Eliette TOUATI, Maurice HOFNUNG and Michel HUERRE — P.T.R. coordinated by E. TOUATI)

This transgenic model was first demonstrated to be a reliable and useful model to quantify the genotoxic effect of H. pylori infections on the gastric mucosa. The induced mutations were mainly found to be of the transversion type, mutations known to be induced during oxidative stress. Such a model should allow us to quantify the genotoxic effects associated with various bacterial or environmental factors in order to gain a better understanding of the carcinogenic processes associated with certain H. pylori infections.

Using H. pylori as a model organism to study peptidoglycan metabolism (Ivo G. BONECA, Catherine CHAPUT)

Compared to other bacteria, H. pylori seems to have a minimal number of genes required for a functional peptidoglycan metabolic pathway. We are interested in characterizing the few peptidoglycan hydrolases and the three potential synthetases (PBPs 1, 2 and 3) involved in the last periplasmic enzymatic steps. These are of great interest due to their central role in the mode of action of ß-lactam antibiotics. Understanding how bacteria respond to antibiotic challenge and modulate peptidoglycan synthesis is therefore relevant to the development of new therapeutic molecules.

The PyloriGene database: an updated annotation of the two sequenced H. pylori genomes (Ivo G. BONECA, Hilde DE REUSE Agnès LABIGNE)

We are interested in setting up a highly informative genomic database on H. pylori (comprising physical and biological annotation as well as bibliographic references). The update of the annotation takes into account (i) recent data on H. pylori (ii) information on other organisms and also (iii) the detection of errors on the physical annotation of the genome. This allowed us to predict some functions for unknown proteins or to reassign new functions to already annotated proteins.

The mechanisms of acid resistance of H. pylori (Stéphanie Bury-Moné, Hilde DE REUSE )

The virulence of H. pylori is related to its exceptional ability to survive in the extremely hostile environment of the stomach. This bacterium has developed novel mechanisms to survive under acid conditions. These depend in part on the production of large amounts of ammonia, which allow the bacteria to maintain a constant intracellular pH. Different pathways of ammonia production in H. pylori have been examined, in particular those dependent on urea hydrolysis or on the activity of two paralogous amidases. The properties of UreI, a novel type of acid-activated urea channel essential for gastric colonization, are further investigated. Residues of UreI involved in its activity or acid-activation were recently identified in H. pylori. The study of the amidases has been extended to the analysis of their distribution among bacterial species belonging to the Helicobacter genus. Mutagenesis of candidate genes, as well as post-genomic approaches, are presently being used to identity new factors involved in the mechanisms of acid resistance in H. pylori.

Modulation of host immune responses by Helicobacter bacteria (Jean Christophe BAMBOU, Richard FERRERO in collaboration with Dana PHILPOTT, Sylvie MEMET, Patrick AVE).

The aim of the work is to study the interactions between the bacteria of the genus Helicobacter and the host immune system. It was previously found that H. pylori clinical isolates which were poor inducers of proinflammatory responses in epithelial and macrophage cell lines were better adapted to colonize mice. Moreover, mouse-adapted variants, which were capable of infecting animals with bacterial loads 2 log-fold higher than those of the respective parental clinical isolates were affected in their capacity to induce pro-inflammatory responses host cells. Parallel studies in Igk-lacZ transgenic mice showed that, during the acute phase of infection, Helicobacter bacteria inhibited the activation of the transcription factor NF-kB in the gastric mucosa of the animals. Thus, the findings suggest that gastric helicobacters are able to negatively modulate the inflammatory responses so as to establish an infection in the host. Future investigations will seek to address the molecular mechanisms involved in the modulation of the host immune system by bacteria belonging to the Helicobacter genus.

 

PATHOGENIC ESCHERICHIA COLI

Pathogenic Escherichia coli are associated with a great variety of infections in humans and animals. Our research concerns the identification of virulence factors with the goal to better understand the pathogenic processes allowing the development of various types of mucosal infections. The reservoir of pathogenic E. coli is the intestinal tract of mammals. A major aspect of our work concerns the characterization of the mechanisms associated with colonization and persistance of a subgroup of pathogenic E. coli, those encoding afimbrial adhesins (AFA).

Molecular typing of AfaE adhesins (Laurence DU MERLE and Chantal LE BOUGUENEC)

afa operons are expressed by pathogenic strains that cause various type of infections. We have developed PCR assays to study 1) the role of afa strains as etiological agents in the development of human septicemia, and 2) the association between the AfaE subtype and the site of the infection (intestinal versus extra-intestinal).

Identification of cellular receptors for AfaD invasins (Laure PLANCON, Nathalie POIDEVIN Christine BERNIER and Chantal LE BOUGUENEC)

Internalization of afa strains into epithelial cells may be the key step for the development of persistent infections. To better understand this phenomenon, we conducted experiments to identify the cellular receptor(s) of the invasins. We demonstrated that the AfaD-III and AfaD-VIII invasins, which share only 45 % identity, recognize the b1 integrin subunit as a receptor.

Identification and characterization of fimbriae implicated in the interaction of diarrhea-associated strains with epithelial cells (Christine BERNIER et Chantal LE BOUGUENEC)

Enteroaggregative E. coli (EAEC) are recognized as an emerging cause of diarrhea worldwide. We constructed a genomic library from a strain isolated from a HIV-infected patient presenting persistent diarrhea. Using this library, we 1) identified a new pathogenicity factor implicated in the interaction (adhesion and invasion) of the bacteria with the host, and 2) defined an efficient genetic tool for the identification of EAEC strains.

Identification of opportunistic, diarrhea-associated pathogens (Laurence DU MERLE, Christine BERNIER and Chantal LE BOUGUENEC with the "Groupe d'étude des infections diarrhéiques" of the International Institut Pasteur Network). We participated in studies that demonstrated the role of enteroadherent strains of E. coli and Klebsiella pneumoniae in chronic diarrhea, hemorrhagic colitis, uremic syndromes, and pseudomembranous colitis in Africa.



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
 

ONDET Maxence (mondet@pasteur.fr)

LABIGNE Agnès, IP (alabigne@pasteur.fr)

LE BOUGUENEC Chantal, IP (clb@pasteur.fr)

DE REUSE Hilde, IP (hdereuse@pasteur.fr)

FERRERO, Richard, IP (rferrero@pasteur.fr)

BAMBOU Jean-Christophe, DEA student

BERNIER Christine, PhD student

BONECA Ivo, postdoc

BRINGER Marie-Agnès

BURY Stéphanie, PhD student

CHAPUT Catherine, engineer student in ndustrial biology

COLLAND Frédéric (Hybrigenics)

CONTRERAS DE CAMACHO Monica, PhD student

DE FINANCE Nancy

GISMERO ORDONEZ Juana (University of Sao Paulo, Brasil)

PINTO Viviana (University of Buenos Aires, Argentina)

PLANÇON-ARNOULD Laure, postdoc

POIDEVIN Nathalie, DEA student

STAM Mark

CHEVALIER Catherine (Engineer INSERM, cchevali@pasteur.fr)

DU MERLE Laurence (Technician, dlaur@pasteur.fr)

ECOBICHON Chantal (Technician, ceco@pasteur.fr)

THIBERGE Jean-Michel (Technician, jmthiber@pasteur.fr)

TROUBADOUR Pascale (Technician, ptrouba@pasteur.fr) (1/2 poste)

ONDET Maxence (Secretary, mondet@pasteur.fr) (1/2 poste)


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