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  Director : LABIGNE Agnès (alabigne@pasteur.fr)


  abstract

 

The research work undertaken within the Unit is focussed 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; enterohepatic Helicobacter spp involved in chronic intestinal inflammation; 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 and on the genotoxic effects of inflammation, the search for prophylactic and therapeutic targets, and epidemiological studies are some of the approaches that are being used to study these mucosal pathogens.



  report

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Helicobacter bacteria

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 carcinoma, respectively.

Several approaches are being developed in parallel to study these infections: i) a fundamental approach that is aimed at a better understanding of the mechanisms allowing the bacterium to colonize, persist and to cause lesions within the gastric mucosa; ii) an applied approach involving epidemiological (studies of bacterial transmission within families) and clinical (immune response to infection, treatment follow up, diagnosis) studies.

Adaptation of H. pylori to the gastric environment in relation to virulence(Stéphanie BURY-MONE, and Hilde DE REUSE)

H. pylori is able to survive and multiply in a particularly hostile acidic environment: the human stomach. Its exceptional ability to resist and adapt to the stress encountered during colonization of its host is intimately related to its virulence. This bacteria has developed novel mechanisms of acid resistance. These depend, in part, on the production of high levels of ammonia, which allows the bacteria to maintain an intracellular pH close to neutrality. Ammonia is also involved in the genesis of lesions. We are studying different pathways of ammonia production in H. pylori. The first depends on urea hydrolysis by urease and on the UreI protein, a novel type of acid-activated urea channel. We have recently shown heterologous complementation between UreI and a urea transporter from Yersinia pseudotuberculosis, homologous to eukaryotic urea channels. The second pathway depends on the activity of two aliphatic amidases. These enzymes are also found in other Helicobacter species able to colonize the stomach and have almost certainly been acquired through horizontal transfer. Our present investigations are focused on the identification of new factors involved in the mechanism of acid resistance in H. pylori using mutagenesis of individual candidate genes and functional genomics (transcriptome). Multiple genes whose expression was found to be acid-regulated were identified. We are examining the role of the products of some of theses genes in resistance to acidity in vitro and in colonization of a mouse model with mutant strains. These studies should lead to the identification of new factors involved in H. pylori acid resistance, some of which might be important for its virulence.

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 and the GEFH, Groupe d'Etude Francophone des Helicobacter)

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 (gastritis, peptic ulcer disease, MALT lymphoma, intestinal metaplasia). Statistical analyzes of the hybridization results allowed us to cluster isolates originating from patients with the same disorder.

Functional analyses: assigning the essential character of a selection of genes (Chantal ECOBICHON, 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 316 genes that were individually analyzed, 44 have already been identified as such. Of the 210 ubiquitous, and H. pylori-specific genes, 155 have been analyzed and 15 were shown to be essential and thus 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, their ability to rescue a suicide plasmid, as well as to colonize mice were 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.

H. pylori-induced genotoxicity at the gastric mucosa in a transgenic mouse model. (Eliette TOUATI, Valérie MICHEL, Jean-Michel THIBERGE and Agnès LABIGNE, in collaboration with Michel HUERRE, PTR coordinated by E.TOUATI)

The "Big Blue" transgenic mouse model was first demonstrated to be a reliable and useful tool to quantify the genotoxic effects of H. pylori infections on the gastric mucosa. At 6 months post-infection, the gastric mutant frequency was induced 4-fold in H. pylori-infected samples, as compared to those from non-infected animals. The analysis of the mutation spectra shows the induction of events known to result from oxidative DNA damage (GC->TA and AT->CG transversions). Concomitantly, the expression of nitric oxide synthase (iNOS), responsible for a high production of NO•, is induced. Using this model, we would like to analyze early events leading to carcinogenesis, and to determine the role of bacterial, host and environmental risk factors involved in gastric carcinogenesis due to H. pylori infection.

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 to have a functional peptidoglycan metabolic pathway. We are interested in characterizing the few peptidoglycan hydrolases (AmiA, Slt, MltD) 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 of great importance. We have characterized single mutants in hydrolase-encoding genes, as well as the corresponding recombinant proteins, to confirm the function of these enzymes. AmiA has a major role in the late stages of bacterial cell division, while the Slt protein is essential for growth under certain specific conditions. To our knowledge, this is the first demonstration of the essential nature of hydrolases for bacterial growth.

The PyloriGene database: update of the 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, (iii) the detection of errors in the physical annotation of the genome. Errors in physical annotation could be attributed to sequencing errors, problems in the attribution of translational start codons, and allowed the identification of new genes regulated by slipped-strand mispairing. Functions were predicted for some unknown proteins and new functions were reassigned to already annotated proteins.

Innate immune recognition and responses to H. pylori infection in gastric epithelial cells (Catherine CHAPUT, Ivo G. BONECA, Richard FERRERO in collaboration with Jérôme VIALA, Dana PHILPOTT, as part of a "PTR"). H. pylori isolates that possess the "cag" pathogenicity island induce pro-inflammatory responses in gastric epithelial cells. The aim of the project is to investigate the mechanism by which epithelial cells are able to recognise and respond to the extracellular pathogen, H. pylori. To this end, we seek to identify both the pathogen-associated molecule(s) and cognate host receptor(s) responsible for the induction of pro-inflammatory responses in these cells.

The role of macrophages in the maintenance of chronic inflammation due to gastric H. pylori infection (Alain Gobert and Richard FERRERO). Although professional phagocytes appear to play a role in the chronic inflammatory lesions associated with H. pylori infection, the interactions between the bacterium and these cells is poorly understood. We have shown that H. pylori bacteria, and their components, induce the production of pro-inflammatory cytokines and nitric oxide (NO) in stimulated murine macrophages. While both responses appear to be dependent on the activation of the transcription factor NF-kappaB, the signalling pathways leading to either cytokine or NO synthesis are distinct. Investigations are currently underway to determine the pathways leading to the production of these two types of inflammatory mediators. An important objective of the work is to identify the bacterial factor(s) that are released or secreted by H. pylori bacteria, and which mediate IL-6 synthesis in macrophages.

Modulation of intestinal inflammatory responses by Helicobacter spp. (Nadia CHAOUCHE and Richard FERRERO). Helicobacter spp that naturally colonise the intestinal tracts of mice are responsible for the formation of chronic inflammatory lesions with similarities to inflammatory bowel disease (IBD) in humans. For the first time, we have characterised the pro-inflammatory responses induced by both pathogenic and commensal Helicobacter spp in a murine intestinal cell line. Several of these bacterial species were also found to modify the barrier function properties of polarised cell layers. Future work will address the mechanisms by which these bacteria are able to modulate the expression of host cytokine and immune mediator functions in intestinal epithelial cells.

PATHOGENIC ESCHERICHIA COLI

Several bacterial species cause intestinal and extraintestinal diseases in humans, but pathogenic Escherichia coli strains account for most of these infections. According to the WHO, E. coli strains are the major cause of bacterial diarrhea worldwide. E. coli accounts for as much as 90 % of all urinary tract infections (UTI) in non-hospitalized patients. E. coli strains are also the second most common cause of neonatal meningitis, and are a frequent cause of septicemia.

In addition to their role in human diseases, pathogenic E. coli strains are also etiological factors in a number of animal diseases. Our goal is to better understand the pathogenic processes leading to the development of the various types of mucosal infections. A major aspect of the research concerns the characterization of mechanisms by which pathogenic E. coli strains colonize epithelial cells and promote cell injury. We have also performed comparative genomic and proteomic studies to identify bacterial factors specific to pathogenic E. coli and are involved in the development of tools for molecular epidemiology.

Identification and characterization of virulence factors in diarrhea-associated enteroaggregative E. coli (Christine BERNIER, Maryline DELATTRE, and Chantal LE BOUGUENEC)

Enteroaggregative E. coli (EAEC) are recognized as an emerging cause of diarrhea in children and adults worldwide. EAEC strains are heterogeneous with respect to both symptoms developed by infected hosts and virulence genes. The aim of our studies was to explore this diversity and to increase our understanding of the genetic basis of virulence factors in EAEC by identifying new bacterial fimbriae implicated in the interaction with host cells. We identified the aggregative adhesion fimbriae type III (AAF-III) and studied its implication in both adhesion to, and internalization into, epithelial cells by the bacteria. Internalization of EAEC strains into epithelial cells may be the key step for the development of persistent infections. The study of EAEC invasive properties showed a low but significant level of internalization into epithelial cells for some strains. We also identified components of the three AAF fimbriae that are related to bacterial invasins previously described in our laboratory.

The epidemiological significance of the EAEC virulence markers identified to date will be evaluated by testing large collections of strains. One limitation of such studies is the lack of probes for the detection of all adhesins that generate the aggregative pattern. An EAEC-sensitive probe that was developed by comparing all the AAF-encoding operons should facilitate progress in this direction.

Identification and characterization of pathogenicity islands in pathogenic E. coli (Juana ORDONEZ, and Chantal LE BOUGUENEC)

In pathogenic E. coli, virulence-associated genes are usually clustered on the chromosome in pathogenicity islands (PAIs). Identification and study of PAIs are useful to characterize new virulence genes. The possible association of the afa-1 operon with a PAI has been investigated in a strain isolated from a Brazilian child with diarrhea.

Characterization of human and animal E. coli isolates (Laurence du MERLE, and Chantal LE BOUGUENEC)

Similarities have been found between isolates from patients and animals with extraintestinal infections (in collaboration with J.P. Girardeau, INRA, Theix), and phylogenetic relationships have been studied between pathogenic E. coli isolates belonging to the various pathotypes (in collaboration with Erick Denamur, INSERM U458).

Comparative genomics of pathogenic and non pathogenic E. coli strains (Laurence du MERLE, Christine BERNIER, and Chantal LE BOUGUENEC in collaboration with Anne Marie Gilles, Evelyne Turlin, Pasteur institutes of Dakar and Bangui, and Cantacuzène institute, Bucarest — P.T.R. coordinated by C. LE BOUGUENEC)

Knowledge of the pathogenic or non-pathogenic status of an isolate may be of use to clinicians for diagnosis, especially in cases of opportunistic pathogens. Isolation of an E. coli strain from a clinical specimen does not, by itself, demonstrate that the isolate is pathogenic, since commensal strains of E. coli can cause infections when the host is compromised. The aim of our study was to identify genes encoding functions that are conserved in pathogenic strains but are absent in commensal E. coli, and to use these data to develop new diagnostic tools. Comparative proteome and metabolome analyses of pathogenic and commensal E. coli strains have been initiated. A large number of pathogenic isolates belonging to various pathotypes have been shown to metabolize a sugar (deoxyribose) that is not fermented by commensal E. coli K-12. The genes encoding this function were identified and found to be conserved among pathogenic strains. The acquisition of deoxyribose metabolizing genes by pathogenic E. coli isolates conferred an evolutionary fitness advantage to the strains. Genetic and bacteriological assays to identify deoxyribose-positive E. coli strains have been developed.

Keywords: Helicobacter , gastritis , gastric carcinoma , ulcer , lymphoma , signalization , acidity , peptidoglycan , mucosa , inflammation , genomics , proteomics , gene regulation , urease , nickel , adhesion , Escherichia coli



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  ONDET Maxence (mondet@pasteur.fr) DE REUSE Hilde, IP (hdereuse@pasteur.fr)

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

LABIGNE Agnès, IP, (alabigne@pasteur.fr)

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

TOUATI Eliette, IP, (etouati@pasteur.fr)

BERNIER-FEBREAU Christine, PhD student

BONECA Ivo, Postdoc

BOUCHET Soizic, DEA student

BURY-MONE Stéphanie, PhD student

CHAOUCHE Nadia, Postdoc

CHAPUT Catherine, PhD student

CONTRERAS DE CAMACHO Monica, PhD student

DELATTRE Maryline

GISMERO ORDONEZ Juana (University of Sao Paulo, Brazil)

GOBERT Alain, Postdoc

KAOUKAB Abdelmoughit

PINTO Alicia Viviana (University of Buenos Aires, Argentina)

SKOULOUBRIS Stéphane

CHEVALIER Catherine, Engineer INSERM

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

ECOBICHON Chantal, Technician (ceco@pasteur.fr)

MICHEL Valérie, Technician (vmichel@pasteur.fr)

ONDET Maxence, Secretary (mondet@pasteur.fr)

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


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