A major focus of the work in the unit during the last year has been the development of postgenomic approaches in H. pylori; to study the biodiversity of clinical isolates, and to create tools that should permit further functional analyses on the bacterium in the future. The ultimate goal is to identify those genes that are common to all H. pylori isolates (ubiquitous), and to classify these as either essential, nonessential or conditionally essential genes. In addition to these systematic postgenomic studies, more targeted research programs were pursued (studies concerning nitrogen metabolism: urea transport, characterization of amidase and formamidase, gene expression and regulation) or initiated (DNA repair in H. pylori). To better understand the nature of proinflammatory responses to Helicobacter infection, physiopathological studies were conducted using experimental infection models in mice and in tissue culture assays.
Use of high density nylon membranes to study the biodiversity of clinical isolates from various geographical origins (Jean-Michel THIBERGE and Agnès LABIGNE)
The aim of this work was to investigate the distribution of the 1590 ORFs of the H. pylori genome (strain 26695) among isolates originating from patients with various ethnic origins, and suffering from the following gastroduodenal disorders: gastritis (G), dyspepsia (D), duodenal ulcer (DU), gastric ulcer (GU). Chromosomal DNA was prepared from 28 clinical isolates, including strains 26695 (DU) and J99 (G) which were used as controls, the SS1 (DU) mouse-adapted strain, as well as 24 isolates from Europe, Hong Kong, Bangui or Dakar. These DNA samples were used as probes in DNA/DNA hybridizations performed against the whole set of the H. pylori ORFs, generated by gene amplification and spotted on nylon membranes (in collaboration with Eurogentec). The results allowed us to identify two subsets of ORFs: one subset of non-ubiquitous ORFs which can be used for the molecular typing of the isolates, as well as one subset of ubiquitous H. pylori-specific ORFs that deserve further functional investigations.
Systematic ordered cloning and mutagenesis of the 1590 ORFs of the H. pylori genome (Chantal ECOBICHON, Catherine CHEVALIER, Denis BAYLE and Agnès LABIGNE)
Two genomic libraries were constructed in Escherichia coli. The first one consists of the individual recombinant plasmids corresponding to the 1590 ORFs of H. pylori strain 26695, cloned into an H. pylori suicide vector and stored as an ordered library in a 96-well format. The second one corresponds to the cloned ORFs from library I, in which each individual ORF was disrupted to saturation by the insertion of a transposon. These libraries are basic tools for the future development of systematic and precise analyses of the genome: library I permits the reamplification of any ORF of the genome to prepare home-made high density membranes containing subsets of ORFs. Each disrupted ORF of library II can be introduced by natural transformation in H. pylori to knock out ORFs of specific interest, or to systematically analyze ORFs for functionality (essential/nonessential/conditionally essential).
DNA repair in H. pylori (Catherine CHEVALIER and Agnès LABIGNE in collaboration with Pablo RADICELLA 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. The set of genes that were predicted by gene annotation to be associated with DNA repair functions has been listed. Systematic gene inactivation studies allowed us to confirm the predicted functions for certain of these genes. Nevertheless, several essential DNA repair activities could not be identified in silico. In this instance, biochemical approaches (conducted at the CEA) allowed us to confirm their existence in H. pylori, and led to the purification of enzymes and to the identification of the corresponding encoding ORF. This resulted in the identification of an atypical 3-methyladenine DNA glycosylase encoded by HP0602, which was initially annotated as an EndoIII enzyme.
Ammonia metabolism in H. pylori (Hilde de REUSE, Stéphanie BURY, Stéphane SKOULOUBRIS)
H. pylori has developed an unusual nitrogen metabolism, which is well adapted to an environment rich in ammonia. We are interested in the different pathways of ammonia production in H. pylori, either by urease-catalyzed hydrolysis of urea, or through the activity of two paralogous amidases. These two enzymes, AmiE and AmiF, hydrolyse short-chain aliphatic amides to produce ammonia and the corresponding organic acid. We showed that these enzymes have different substrate specificities since AmiE is an aliphatic amidase while AmiF is a novel type of formamidase. In addition, these two sulphydryl-enzymes have conserved active sites. The H. pylori amidases thus constitute exemplary paralogues since these enzymes seem to have evolved to achieve enzymatic specialization after ancestral gene duplication. AmiE and AmiF are both involved in H. pylori nitrogen metabolism, their production is regulated by the activity of two other enzymes of this pathway, urease and arginase.
Resistance to acidity in H. pylori (Hilde de REUSE, Stéphanie BURY, Stéphane SKOULOUBRIS)
Urease activity is essential to allow H. pylori to resist to the extremely low pH values of the stomach. We are studying a membrane protein of H. pylori, UreI, necessary for the rapid degradation of urea into ammonia by urease. UreI is required for resistance to acidity in vitro and this process has been quantified by measuring the production of ammonium ions. It was observed that the UreI protein is specifically activated when the extracellular pH is below 5. We also showed that UreI is essential for the colonization of the mouse gastric mucosa. UreI presents the characteristics of an urea transporter and it was shown by others that this protein behaves like a urea-channel when expressed in Xenopus oocytes. The H. pylori UreI protein is thus a novel type of H+-gated urea channel perfectly suited for an adapted emergency response to the acid gastric environment.
Biological validation of protein-protein interaction: identification of an anti-sigma in H. pylori (Hilde de REUSE in collaboration with Hybrigenics)
H. pylori flagellar motility constitutes an essential colonization factor. Data from the protein-protein interaction map (two-hybrid, see above) revealed the interaction between an unknown protein, HP1122, and the sigma 28 transcription initiation factor, known to regulate the expression of flagellar genes in many bacteria. Regions of interaction between these two proteins were defined and it was shown that HP1122 specifically inhibits the interaction between RNA-polymerase and the sigma 28 factor. The HP1122 protein thus presented the characteristic of an anti-sigma 28 regulator, also designated FlgM. The role of HP1122 in flagellar gene expression has been confirmed in H. pylori by electron microscopic analysis of flagella synthesis in wild-type and mutant strains, and by RNA slot blot studies. The two-hybrid analysis thus identified an anti-sigma 28 protein with weak homology to other FlgM proteins, and demonstrated the existence of a temporally regulated biosynthesis of flagellar appendages in H. pylori.
NikR, a major regulator in H. pylori (Monica CONTRERAS and Agnès LABIGNE)
We previously showed that H. pylori encoded an homolog of NikR, a nickel responsive repressor of E. coli. Using a differential transcriptome analysis of the parental and NikR knock-out strain, several genes were identified as putatively regulated by NikR under conditions of nickel overload or depletion. To further confirm that the expression of the identified genes was indeed regulated by NikR in H. pylori, slot blot experiments were performed. For some of the genes, NikR was shown by the electrophoretic mobility shift assay (EMSA) to directly bind to their promoter region. NikR appears as a major regulator in response to divalent cation concentration in H. pylori.
Role of inflammation in the induction of mutations in the gastric mucosa as measured in the "Big Blue" mouse model (Jean-Michel THIBERGE, Agnès LABIGNE, in collaboration with Eliette TOUATI, Maurice HOFNUNG and Michel HUERRE)
The aim of this work was to evaluate whether the "big blue" transgenic mouse model was useful for the quantification of the genotoxic effects of various parameters known to play a role in gastric carcinogenesis, such as chronic infection with specific strains of H. pylori, and diet. C57BL/6 transgenic mice containing the lacZ reporter gene, integrated into the genome of each cell of the organism, were infected with the H. pylori SS1 strain, or with Helicobacter felis, bacterium that colonizes the gastric mucosa of mice and which has more pronounced pro-inflammatory properties than H. pylori. A genotoxic chemical compound (R7000) was used as a positive control in these experiments. So far, the model has been established and long-term infections (6 and 12 months) are currently on-going.
Adaptation to the murine host and induction of pro-inflammatory responses by H. pylori clinical isolates (Richard FERRERO, Djilali BELAID, Pascale TROUBADOUR, in collaboration with Dana PHILPOTT)
A relatively small number of H. pylori isolates have been reported to colonize the gastric mucosa of mice. To investigate the factors associated with H. pylori adaptation to this host, a selection of recent clinical isolates were used to inoculate mice. The ability of strains to colonize these animals was associated negatively with the presence of the gene cagA; a marker for the Cag pathogenicity island (Cag PAI), which is responsible for more severe inflammatory lesions in H. pylori-infected individuals, via the production of NF-kB-dependent factors. The presence of a functionally active Cag PAI in each of the cagA+ strains was confirmed in in vitro assays using a gastric epithelial cell line, in which the activation of NF-kB was determined by EMSA and gene reporter assays, as well as by the detection of IL-8 in cell culture supernatants. The findings suggest that H. pylori strains that are poor inducers of pro-inflammatory responses in gastric epithelial cells are better adapted to colonize the murine host. Moreover, we showed that H. pylori Cag PAI+ isolates that had been passaged in vivo, and which colonized mice with higher bacterial loads, were able to down-modulate the production of Cag PAI-dependent proinflammatory factors by as yet unknown mechanisms.
NF-KB transcriptional activation in vivo (Richard FERRERO, Jean-Christophe BAMBOU, in collaboration with Sylvie MEMET and Patrick AVE)
Although numerous workers have studied the activation by H. pylori of NF-kB complex formation in gastric epithelial cells in vitro, very little is currently known regarding the activation of this transcription factor during host infection. To this end, transgenic mice (supplied by S. Mémet), in which the lacZ reporter gene was transcriptionally fused to an NF-kB-responsive element, were infected with different Helicobacter isolates. Preliminary data from these studies demonstrated an increase in the numbers of cells in which NF-kB was activated within the gastric mucosa, during an Helicobacter infection in mice. These cells have been presumptively identified as belonging primarily to the epithelial and endothelial cell lineages.
T cell responses in the genesis of inflammatory responses induced by chronic Helicobacter infection (Richard FERRERO, in collaboration with Michel HUERRE and Patrick AVE)
Mice with chronic H. felis infection develop severe inflammatory lesions, resembling the T helper-1 (Th-1) phenotype associated with H. pylori-mediated chronic gastritis. Studies in BALB/c mice which have a default Th-2 phenotype, however, suggested that the cellular responses involved with Helicobacter-induced inflammation were not restricted to a Th-1 type. To test this hypothesis, IFN-g-/- BALB/c mice, which are affected in their ability to mount Th-1 responses, as well as wild-type animals, were each infected with H. felis. At 7 months post-infection, IFN-g-/- mice displayed dominant Th-2 type cellular responses, as characterized by the production of increased levels of IL-4 by splenocytes and increased mucosal antibody responses, when compared to both uninfected IFN-g-/- and infected wild-type animals. IFN-g-/- mice also had increased numbers of polymorphonuclear cell infiltrates in their gastric mucosa. These findings suggest that Th-2 type cellular responses play a role in the induction of inflammatory lesions associated with chronic Helicobacter infection.
PATHOGENIC ESCHERICHIA COLI (Chantal LE BOUGUENEC)
In human beings, several bacterial species are responsible for the development of intestinal as well as extraintestinal pathogenic processes, but without a doubt, pathogenic E. coli species account for the majority of such infections. E. coli strains are recognized by the WHO as the major cause of bacterial diarrhea worldwide. Urinary tract infections (UTI) are one of the major nonendemic types of infectious disease; these affect up to 1% of the female adult population, and in 80% of cases, E. coli is the causative agent. Finally, E. coli species are the second most common etiological cause of neonatal meningitidis, and are also frequently responsible for septicemia. In addition to their role in human diseases, pathogenic E. coli species also play a major role in animals. Colibacilloses are common and have an enormous economic impact due to mortality and morbidity during intensive breeding. Although a large number of virulence factors have been identified over the last 20 years, and have allowed specific pathogenic traits to be attributed to pathogenic E. coli species, there is still a need for a better understanding of the pathogenic processes associated with certain E. coli infections. 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). We are currently investigating their genetic determinism and the architecture and stability of the genetic material associated with their expression in clinical isolates.
Genetic support of the afa operons responsible for adhesion (Lila LALIOUI).
We first demonstrated that operons responsible for the production of afimbrial adhesine (afa operons) are distributed among pathogenic E. coli species associated with infections both in humans and animals. We showed that the afa operons belonged to mobile genetic elements, a finding which might contribute to the dissemination of these operons among largely distributed E. coli isolates. In addition, certain afa genes have been shown to belong to large pathogenicity islands; one of these PAIs, harboring the afa-8 operon, has been characterized at the molecular level. Of the afa operon family (afa-1 to afa-8), afa-8 is one of the most widely distributed among E. coli isolates associated with extraintestinal infections, both in humans and animals.
Study of the adhesive AFA structure (Laurence du Merle in collaboration with Antoine TOUBERT, INSERM U396).
The adhesive structure encoded by the afa operons present on the bacterial cell surface consists of the combination of two proteins AfaE and AfaD. The respective roles of these two proteins during the process of interaction with eucaryotic cells has been elucidated through genetic and ultrastructural analyses of recombinant E. coli strains expressing the AFA adhesive structure. AfaE is responsible for the adhesion of the bacteria to the epithelial cells and recognizes the SCR3 domain of the complement regulator known as decay-accelerating factor (DAF or CD55). The binding of AfaE to CD55 induces signal transduction that promotes a process of "capping" of receptor molecules associated with the stimulation of an innate immune response.
Internalization of AFA-encoding bacteria (Sandrine LE FRIEC, Laure PLANCON, Laurence du MERLE, Christine BERNIER).
Bacteria expressing the afa operon can be internalized within epithelial cells. This process is mediated by the AfaD invasin. So far, the available data suggest that internalization of bacteria expressing AFA takes place not only in vitro but also in vivo and might play a major role in the development and fate of acute or persistent infections. Clinical isolates expressing AFA, once internalized, can survive up to 72 hours without multiplying within the cells. Recent studies have shown that ß1-integrin was involved in the internalization process of strains expressing AfaD. The AfaD invasin is the prototype of a new invasin family distributed among several adhesion operons harbored by different types of pathogenic E. coli, all involved in persistent infections.
Persistent diarrhea associated with pathogenic E. coli (Christine BERNIER)
In addition to the molecular studies on the Afa system, the group is also involved, in collaborative projects with various Pasteur Institutes, on the pathogenic potential of E. coli strains associated with new diseases, such as persistent diarrhea in HIV-seropositive patients, and on the respective roles of bacterial virulence factors and those of the host, in the pathophysiology of these infections. Recently, we developed a new diagnostic tool for the detection of enteroaggreagative E. coli which will allow the study of the distribution of these strains among isolates associated with persistent diarrhea.