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; 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.
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.
Presence of Active Aliphatic Amidases in Helicobacter species able to colonize the stomach (Stéphanie BURY-MONE and Hilde DE REUSE)
Ammonia production is of great importance for the gastric pathogen Helicobacter pylori as a nitrogen source, as a compound protecting against gastric acidity, and as a cytotoxic molecule. In addition to urease, H. pylori possesses two aliphatic amidases responsible for ammonia production: AmiE, a classical amidase, and AmiF, a new type of formamidase. Both enzymes are part of a regulatory network consisting of nitrogen metabolism enzymes, including urease and arginase. We examined the role of the H. pylori amidases in vivo by testing the gastric colonization of mice with H. pylori SS1 strains carrying mutations in amiE and/or amiF and in coinfection experiments with wild-type and double mutant strains. A new cassette conferring resistance to gentamicin was used in addition to the kanamycin cassette to construct the double mutation in strain SS1. Our data indicate that the amidases are not essential for colonization of mice. The search for amiE and amiF genes in 53 H. pylori strains from different geographic origins indicated the presence of both genes in all these genomes. We tested for the presence of the amiE and amiF genes and for amidase and formamidase activities in eleven Helicobacter species. Among the gastric species, H. acinonychis possessed both amiE and amiF, H. felis carried only amiF, and H. mustelae was devoid of amidases. H. muridarum, which can colonize both mouse intestine and stomach, was the only enterohepatic species to contain amiE. Phylogenetic trees based upon the sequences of H. pylori amiE and amiF genes and their respective homologs from other organisms as well as the amidase gene distribution among Helicobacter species are strongly suggestive of amidase acquisition by horizontal gene transfer. Since amidases are found only in Helicobacter species able to colonize the stomach, their acquisition might be related to selective pressure in this particular gastric environment.
Characterization of the roles of NikR, a nickel-responsive pleiotropic autoregulator of Helicobacter pylori (M. CONTRERAS, Jean-Michel THIBERGE, and Agnès LABIGNE, in collaboration with Marie-Andrée MANDRANT - INSA, Villeurbanne)
The Helicobacter pylori genome contains a gene (HP1338 or nikR) that encodes a nickel-dependent regulator that is homologous to the Escherichia coli nickel-responsive regulator, NikR. The H. pylori nikR product acts as a pleiotropic metal-dependent regulator. We constructed a non-polar isogenic mutant deleted for the nikR gene. NikR was essential for the survival of H. pylori in the presence of high nickel and cobalt ion concentrations in vitro. We screened a DNA macroarray for genes that were differentially expressed in parental and nikR-deficient H. pylori strains grown in the presence of excess nickel. We found that H. pylori NikR mediates the expression of nickel-activated and -repressed genes. In the presence of excess nickel, NikR activated the transcription of ureA-ureB (HP72-73), nixA (HP1077), copA2 (HP1072), hpn (HP1427) and hpn-like (HP1432) genes and repressed the expression of genes encoding proteins involved in ferric iron uptake and storage [pfr (HP0653), fur (HP1027), frpB4 (HP1512), exbB/exbD (HP1339-1340), ceuE (HP1561)], motility [cheV (HP616), flaA (HP0601), flaB (HP0115)], stress responses [hrcA-grpE-dnaK (HP111-110-109)] and encoding outer-membrane proteins [omp11(HP0472), omp31 (HP1469), omp32 (HP1501)]. Slot blot DNA/RNA hybridization experiments using RNA from three independent bacterial cultures confirmed the transcriptome data for 10 selected genes. The results of gel shift experiments using purified native NikR, beta-galactosidase assays with the region between nikR and the exbB/exbD divergent operon, and the study of exbB gene expression using a gentamicin/apramycin reporter gene in H. pylori indicated that NikR is an autorepressor that binds to this intergenic region and also controls the expression of the exbB/exbD/tonB operon, which provides energy for ferric iron uptake. Thus, as previously suggested for Fur in H. pylori, NikR appears to be a global regulator of the metabolism of some divalent cations within a highly complex regulated network.
DNA repair in H. pylori (Catherine CHEVALIER and Agnès LABIGNE, in collaboration with Pablo RADICELLA from CEA, Fontenay aux Roses)
Helicobacter pylori elicits an oxidative stress during host colonization. This oxidative stress is known to cause lesions in the host DNA. We addressed the question as to whether the pathogen DNA is subject to lethal or mutational damage by the host-generated oxidative response. H. pylori Hpnth mutants unable to repair oxidized pyrimidines from the bacterial DNA were generated. H. pylori strains lacking a functional endonuclease III (HpNth) showed elevated spontaneous and induced mutation rates and were more sensitive than the parental strain to killing by exposure to oxidative agents or activated macrophages. Although under laboratory conditions the Hpnth mutant strain grows as well as the wild-type strain, in a mouse infection the stomach bacterial load gradually decreases while the population in the wild-type strain remains stable, showing that endonuclease III deficiency reduces the colonization capacity of the pathogen. This was observed during the course of monoinfections and coinfections between the parental and the mutated strains. These results show that the host effectively induces lethal and premutagenic oxidative DNA adducts on the H. pylori genome.
Genotoxicity induced by H. pylori infection in mouse and risk factors (Eliette TOUATI, Valérie MICHEL, Jean-Michel THIBERGE, Agnès LABIGNE, in collaboration with Michel HUERRE and Patrick AVE)
In this study, we have shown that H. pylori infections lead to an induction of mutagenic events at the gastric epithelial cells. Using " Big Blue " mice transgenic system that allows the detection of mutations in any organs, a 4-fold increase of the gastric mutant frequency was observed after 6 months of infection as compared to non-infected mice. The analysis of the mutation spectra shows the induction of transversions GC->TA and AT->CG, resulting in part from oxidative DNA damages. The mutagenic effect is observed concomitantly with the induction of a severe gastritis, and underlined the role of the inflammatory host response in the observed genotoxicity. After long term infection of 12 months, an induction of gastric epithelial cell proliferation and apoptosis are observed and could account for the absence of mutagenic effect at this time point. Our main objectives now are to identify and to determine the mechanism of action of potential risk factors implicated in the events responsible for such a genotoxic effect leading to the initiation of the gastric precancerous processes. These effectors are likely from various origins, bacterial, host but also environmental factors could be implicated. We have analysed the influence of high-salt diet known to be associated in the population with a high gastric cancer incidence. No synergistic effect with the infection was observed, concerning the induction of the gastric mutant frequency.
Helicobacter pylori heat shock protein 60 mediates interleukin-6 production by macrophages via a toll-like receptor (TLR)-2-, TLR-4-, and myeloid differentiation factor 88-independent mechanism. (Alain GOBERT and Richard FERRERO).
Helicobacter pylori has been reported to induce interleukin-6 (IL-6) production in monocytes/macrophages and in chronically inflamed gastric tissues. The mechanism by which H. pylori induces IL-6 production in macrophages, however, has not been investigated. To identify the H. pylori factor responsible for this activity, we fractionated soluble proteins from H. pylori strain 26695 by ion exchange and size exclusion chromatography and screened the fractions for IL-6-inducing activity on RAW 264.7 macrophages. A single protein was purified and identified by mass spectrometry as H. pylori heat shock protein 60 (HSP60). Consistent with the observed IL-6-inducing activity of H. pylori HSP60, soluble protein extracts of H. pylori 26695 and SS1 strains that were depleted of this protein by affinity chromatography had dramatically reduced IL-6-inducing activities. The immunopurified HSP60 stimulated IL-6 production in macrophages. When stimulated with H. pylori HSP60 or intact bacteria, peritoneal macrophages from mice deficient in Toll-like receptor (TLR)-2, TLR-4, TLR-2/TLR-4, and myeloid differentiation factor 88 produced the same amount of IL-6 than macrophages from wild-type mice, demonstrating the independence of H. pylori HSP60 responses from these signaling molecules. H. pylori HSP60-induced IL-6 mRNA expression, and NF-kappaB activation in RAW 264.7 cells was abrogated in the presence of MG-132, a proteasome inhibitor. In contrast, inhibitors of protein kinase A or C, mitogen-activated protein kinase kinase, and phosphoinositide 3-kinase had no effect on IL-6 mRNA levels. This study demonstrates the induction of innate immune responses by H. pylori HSP60, thereby implicating this highly conserved protein in the pathophysiology of chronic gastritis.
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.
Study of peptidoglycan metabolism in Helicobacter pylori (Ivo G. BONECA and 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 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 beta-lactam antibiotics. Understanding how bacteria respond to antibiotic challenge and modulate peptidoglycan synthesis is therefore of great importance.
Peptidoglycan structure and penicillin resistance in Neisseria meningitidis (Ivo G. BONECA, in collaboration with Aude ANTIGNAC and Muhamed-Kheir TAHA)
We have structurally characterized the composition of Neisseria meningitidis peptidoglycan by combining HPLC analysis and mass spectrometry of muropeptides. Penicillin intermediate resistance in meningococci has been shown to be mediated by mosaic alleles of the penA gene coding the meningococcal PBP2. Comparison of the peptidoglycan of susceptible and intermediate-resistant clinical strains showed a direct correlation between resistance and accumulation of pentapeptides, suggesting a potential D,D-carboxypeptidase for PBP2.
The role of peptidoglycan in innate immunity (Ivo G. BONECA, in collaboration with Stéphane GIRARDIN, Philippe SANSONETTI and Dana PHILPOTT)
We were interested in determining how intra-cellular bacteria are sensed by epithelial cells. Results by the group of Dana Philpott, showed that this recognition in epithelial cells was NOD1 dependent. NOD1 together with NOD2, a Chron's disease susceptibility gene, belong to a large family of intracellular pattern recognition receptors (PRR) for which it was not known the bacterial product(s) sensed. We have shown that both PRRs, NOD1 and NOD2, respond to peptidoglycan by with different specificity. NOD2, which is specific to monocytes, macrophages and dendritic cells, is the receptor for the muramyl dipeptide (MDP). MDP has been extensively studied for its adjuvant properties. However, until now its receptor had never been identified. NOD2 mediates recognition of the peptidoglycan of every bacterial species tested due to the ubiquitous nature of MDP in all bacteria. On the other hand, NOD1 seems to be specific for Gram negative bacteria. This specificity is based on the recognition of muramyl tripeptide carrying at the third position a diaminopimelic amino acid common to most medically important Gram-negative pathogens but absent from a variety of Gram-positive bacteria which carry a lysine residue at the same position.
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. The WHO recognized that E. coli strains are the major cause of bacterial diarrhea worldwide. The frequency of bacterial urinary tract infections (UTI) is second only to that of bacterial respiratory infections. E. coli accounts for as much as 90 % of all UTIs in non-hospitalized patients. E. coli strains are also the second most common cause of neonatal meningitis, and are frequently responsible for septicemia. 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 are involved in the development of tools for molecular epidemiology. A recent additional objective is to perform comparative genomic and proteomic studies to identify bacterial factors specific to pathogenic E. coli and that are absent or not produced by commensal E. coli strains.
Characterization of the AfaD-like family of invasins (Laurence du MERLE, and Chantal LE BOUGUENEC)
Pathogenic E. coli strains carrying afa operons are frequently associated with establishment of persistent diarrhea and of chronic or recurrent extra-intestinal infections in human and animals. We explored the internalization processes used by Afa strains to enter eukaryotic host cells and attempted to determine the role of the AfaD invasins during the interaction of pathogenic bacteria with epithelial cells. We demonstrated that the characteristics of the AfaD-mediated entry of bacteria are consistent with the hypothesis that intracellular bacteria can form a reservoir in epithelial cells for subsequent infection cycles. Indeed, once in the cytoplasm of the epithelial cells, the bacteria formed inclusions in which they were able to survive to at least 72 h. Internalization assays using polystyrene beads coated with purified recombinant AfaD proteins demonstrated that this invasin mediates entry into cells derived from various tissues (intestine and urothelium) that are targets for Afa strains. We also identified for the first time a cellular receptor for the AfaD invasins. We showed that these proteins recognize cellular adhesion molecules (eta 1 integrins) present in mucosal epithelial cells, as a receptor. All together these data strongly suggested that the AfaD invasins participate in the formation of bacterial reservoirs during the development of both intestinal and urinary tract infections.
Comparative genomics of pathogenic and non pathogenic E. coli strains (Laurence du MERLE, Christine BERNIER, Soizic BOUCHET, and Chantal LE BOUGUENEC, in collaboration with Anne-Marie GILLES, Evelyne TURLIN, Arnaud FONTANET, Pasteur Institutes of Dakar and Bangui, and Cantacuzène Institute, Bucarest - P.T.R. coordinated by C. LE BOUGUENEC)
The aim of our study is to identify genes encoding functions that are conserved in pathogenic strains and to use these data to develop new diagnostic tools. DNA regions encoding metabolic functions such as carbohydrate transport and utilization are frequently detected in pathogenicity islands. However, little is known about the substrates and functional analyses of these regions. We identified and characterized the deoK operon coding for use of deoxyribose from a pathogenicity island of an E. coli sepsis isolate. Analysis of the sequence of the deoK operon from different E. coli isolates demonstrated its high level of conservation in E. coli and suggests that the deoK operon from E. coli originated from Salmonella enterica via a two step process of acquisition. We developed genetic and bacteriological assays to identify deoK-positive strains and showed that the deoK operon is widespread among extra-intestinal and intestinal pathogenic E. coli. We also demonstrated that the use of deoxyribose by clinical isolates increases their competitiveness, strongly suggesting a role of this biochemical characteristic in host infectivity. An important objective of the work is to determine other biochemical traits carried by specific islands from pathogenic E. coli strains, that will not only increase our understanding of pathophysiological processes during E. coli infections but also permit to develop tests, easy to perform and selective, for the identification of pathogenic E. coli strains. Comparative proteome and metabolome analyses of pathogenic and commensal strains, as well as determination of the nucleotide sequence of pathogenicity islands, have been initiated
Keywords: Helicobacter , gastritis , gastric carcinoma , ulcer , lymphoma , signalization , acidity , peptidoglycan , mucosa , inflammation , genomics , proteomics , gene regulation , urease , nickel , adhesion , invasion , metabolome , Escherichia coli