Unit: Pathogenesis of Mucosal Bacteria
Director: LABIGNE Agnès
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; 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.
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 maltoma), respectively.
Different projects are currently pursued in different areas of fundamental or applied research: gene expression profiling of the adaptive response to acidity; nitrogen, nickel ion and peptidoglycan metabolisms; comparative and functional genomics; studying role of inflammatory responses in host adaptation; damages induced by persistent infection; DNA repair functions in H. pylori. Emphasis will be given underneath to those projects that have reached completion.
The adaptive response of H. pylori to the gastric environment - (Stéphanie BURY-MONE, Marie Thibonnier, Kerstin Stingl and Hilde De Reuse)
For pathogenic bacteria, survival to the stress encountered during colonization of their host is intimately linked to their virulence. H. pylori has the exceptional particularity to be able to resist to extreme acidity (in the gastric lumen) and, subsequently to efficiently adapt to moderate acidity, a condition encountered in the stomachal mucus layer during colonization. Functional genomics were used to analyze the response of H. pylori to moderate acidity, We compared the transcriptome of strain 26695 grown at neutral pH or at pH5 and found that acidity is a signal modulating the expression of several virulence factors. The genes encoding urease and two aliphatic amidases were found to be upregulated at low pH. Our results demonstrate that H. pylori, adaptation to moderate acidity is closely linked to its capacity to produce large amounts of intracellular ammonia. 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. We found numerous genes encoding functions involved in cell wall, membrane synthesis and metal metabolism were acid-regulated. Finally, we observed that NikR and/or Fur are necessary for efficient colonization of the mouse sstomach by H. pylori.
Study of peptidoglycan (PG) metabolism in H. pylori (Ivo G. Boneca, Catherine Chaput)
We are interested in analyzing the role of peptidoglycan hydrolases in growth, morphology and tolerance to antibiotics. Three potential hydrolases, Slt, MltD and AmiA were identified from the genome sequence: AmiA is involved in H. pylori cell division as well as in its morphological change from bacillary to coccoïd shape. This transformation is associated with the modification of the peptidoglycan structure, modifiction that no longer occurs in the amiA mutant. AmiA is the first H. pylori genetic determinant found to be associated with the morphological shape transition (bacillary to coccoïd). Slt is involved in the release of PG fragments; we have shown that a mutant deficient in Slt is modified in its ability to induce pro-inflammatory responses on gastric epithelial cells. MltD, despite a similar activity, seems to mainly play a role in remodeling PG during the stationary growth phase and during a stage that precedes the morphological shape transition, characterized by the production of non-cultivable forms of H. pylori.
The role of peptidoglycan (PG) in innate immunity (Catherine Chaput and Ivo G. Boneca)
In collaboration with several research groups, we demonstrated that recognition of pathogenic bacteria is achieved via the recognition of PG motives. In Drosophila, this recognition is dependent upon the presence of PGRPs (PeptidoGlycan Recognition Proteins). In collaboration with Julien Royet (CNRS; Strasbourg) and Bruno Lemaître (CNRS, Gif sur Yvette) we were able to identify the smallest motives that are recognized via the IMD pathway which is specific for Gram-negative bacteria. We also demonstrated that recognition of Gram-positive bacteria by the Toll receptor involved the recognition of PG motives and requires the participation of two PGRPs, PGRP-SA and SD. In mammalians, PG is sensed by NOD1 and NOD2 signalization pathways. In contrast to what was thought up to now, TLR2 is not a receptor for PG. In collaboration with Dana Philpott, we showed that H. pylori, an extracellular pathogen, is sensed by the intra-cellular Nod1 receptor. This recognition is dependent upon the synthesis of a type IV secretion apparatus that permits the translocation of PG fragments of H. pylori into the epithelial cells.
DNA repair in H. pylori (Aurélie Mathieu, Chantal Ecobichon, Eliette Touati and Agnès Labigne in collaboration with Pablo RADICELLA from CEA-Fontenay aux Roses)
The aim of that study is the analysis of the DNA repair systems of H. pylori and their involvement in the important genetic variability of the various H. pylori strains. The MutS protein encoded by ORF Hp0621 belongs to the mutS2 family. It is not involved in mismatch repair but inhibits homologous and homeologous recombination. The role of MutS2 in the ability of bacteria to exchange genetic material as a mean to adapt to environmental variations is currently investigated.
Genotoxicity associated with H. pylori infection (Valérie Michel, José Ramos-Vivas and Eliette Touati, collaboration with Michel Huerre and Patrick Avé)
The main goals of our studies are i) the determination of early events promoting precancerous lesions at the gastric level due to H. pylori infection; ii) the identification of risk factors responsible for such events. Using the " Big blue " transgenic mouse model allowing the detection of mutation in any organ, a mutagenic effect induced by the H. pylori infection at the gastric level has been previously evidenced. The analysis of the mutation spectra shows the induction of transversions GC->TA and AT->CG, resulting in part from oxidative DNA damages, among them 8oxo-guanines. These lesions are specifically repaired by the DNA glycosylase OGGI. The influence of the ogg deficiency on the mutagenic and inflammatory host response to the infection is actually investigated. Not only free radicals produced during the inflammatory host response, but also nitro/nitrosamine compounds mainly issued from bacterial metabolism and diet are able to contribute to the genotoxicity associated with the infection. Experiments are in progress in mouse but also in vitro to characterize DNA adducts and genetic instability events and to determine the implicated mechanisms.
Comparative genomics of H. pylori clinical isolates (Jean-Marie Thiberge, Sébastien Breurec and Agnès Labigne in collaboration with plateform PT2 of Genopole, & GEFH, Groupe d'Etude Français des Helicobacter)
Comparative genomics allowed us to classify the coding sequences of H. pylori strain 26695, within two categories: ubiquitous genes that defined the genome core of H. pylori, and a set of 213 non-ubiquitous or strain specific genes. We thus wanted to examine on a large scale the genetic diversity among 120 independent clinical isolates from patients with duodenal ulcer (27), chronic gastritis (33), intestinal metaplasia (17), and gastric extranodal marginal zone B-cell lymphoma of MALT-type (43) by comparative genomic hybridization. Each isolate was found to have a unique hybridization profile allowing a specific typing of the isolate. Multidimensional analysis on continuous values allowed us to visualize clusters of isolates associated with the same pathology. A hierarchical cluster analysis based on the most discriminative genes from principal component alnalysis led to the identification of one cluster containing 77% of the MALT lymphoma isolates whether or not harboring the cag pathogenicity island. These results indicate that bacterial genome contributes at determining the outcome of the H. pylori infections. This is relevant for MALT Lymphoma associated strains for which the common set of genes playing a role in determining the evolution of the disease has now to be identified from the genome sequence of a representative isolate of this strain cluster.
Intrafamilial transmission and evolutionary mechanisms leading to genetic diversity in H. pylori (HP) (Josette Raymond, Jean-Michel Thiberge andAgnès Labigne in collaboration with C. Dauga from the Génopole)
The aim of the study was to demonstrate intrafamilial transmission and mechanisms involved in genome evolution through the molecular study of multiple independent isolates of HP originating from family members. The family consists of the 2 parents and 4 siblings suffering from recurrent upper gastrointestinal disorders. Ten individual clones from antrum and from fundus were isolated from each family member, and chromosomal DNA was extracted. Two housekeeping genes (hspA and glmM) were sequenced and their sequences were compared with those of 131 unrelated strains. The multiple alignments of the hspA and glmM sequences revealed 11 different alleles for hspA and 6 for glmM.. For each clone representative of a unique combination of the two hspA-glmM alleles, chromosomal DNA was hybridized to a DNA array containing 213 non-ubiquitous genes of HP, isolate 26695. Based on DNA hybridization quantification, normalization, and establishment of a hierarchical classification of the various family isolates, only three scaffold-strains (S1, S2, S3) were found to be circulating within the family. S1 was found in the father and child-1; S2 in the mother and child-1, -3 and -4; S3 was exclusively found in child-2. These results demonstrated that strains had been transmitted both between parents and children and between siblings. Based on the complete genome comparison, three scaffold-strains were found to circulate within the family; several mechanisms took place to enhance strain diversity: genetic drift via individual mutations, intragenic recombinations, and interstrain recombinations.
PATHOGENIC ESCHERICHIA COLI
Several bacterial species cause intestinal and extra-intestinal diseases in humans, yet pathogenic Escherichia coli strains account for most of these infections. E. coli consists of both non-pathogenic strains, found in the normal intestinal flora of humans and animals, and pathogenic strains, which are the leading cause of a wide variety of infectious diseases by colonizing different tissues. Although, there is some virulence trait overlap between various pathogenic strains, each pathotype possesses a unique combination of virulence traits that results on a distinctive pathogenic mechanism. Our goal is to better understand the pathogenic processes allowing the development of the various types of mucosal infection. A major aspect of our research concerns the characterization of mechanisms by which pathogenic E. coli strains colonize epithelial cells and promote cell injury. We also developed genomic and proteomic studies to compare pathogenic and non-pathogenic E. coli.
Characterization of the deoK operon coding for use of deoxyribose by E. coli strains. (Laurence du Merle, Samira Baghaz and Chantal Le Bouguénec, in collaboration with partners from a Research Transversal Program; group leader C. Le Bouguénec).
We previously described a pathogenicity island in the sepsis-associated E. coli AL862 isolate. Nucleotide sequence analysis of this region identified the deoK operon coding for use of deoxyribose as a sole carbon and energy source. We studied the distribution of this operon among a collection of pathogenic and non-pathogenic E. coli strains. The 1152 E. coli isolates screened in this investigation were from three countries: Senegal, Romania, and Central African Republic. We demonstrated the association of deoK with strains that are true (or potential) pathogenic isolates and showed that use of deoxyribose by clinical isolates increases their competitiveness, strongly suggesting a role for this biochemical characteristic in host infectivity.
Structural analysis of Afa adhesins. (Laurence du Merle, and Chantal Le Bouguénec, in collaboration with S. Matthews (Imperial College, London) and S. Lea (Oxford University).
afa operons that are carried by E. coli strains associated with both intestinal and urinary tract infections, encodes adhesins and invasins. To better understand bacteria-epithelial cells interactions mediated by these Afa proteins, we determined the architecture of the adhesive sheath at the bacterial surface. Using the three-dimensional structure of the AfaE adhesin, we have constructed a robust atomic resolution model that reveals the structural basis for assembly by donor strand complementation, and for the architecture of capped surface fibers. The structure of AfaE, combined with binding studies, has allowed the mutual interaction surfaces of AfaE and its cellular receptor, decay-accelerating factor, to be defined. High resolution studies of the AfaE adhesin and its interaction with chloramphenicol may provide the start point for the design of novel antimicrobial compounds because, in contrast to other chloramphenicol-protein complexes, where binding of the antibiotic involves the benzene ring, the interactions seen here are focused on the chorines.
Pathogenesis of ascending pyelonephritis. (Christelle Chaudray and Chantal Le Bouguénec).
Preliminary results strongly suggested that various pathophysiological mechanisms are involved in the development of pyelonephritis and that some E. coli isolates are able to invade epithelial kidney cells.
Keywords: Helicobacter, gastritis, gastric carcinoma, ulcer,lymphoma, signalization, acidity, peptidoglycan, mucosa, inflammation, genomics, proteomics, gene regulation, urease, nickel, adhesion, invasion, metabolome, Escherichia coli