Helicobacter pylori is a bacterial pathogen colonizing the stomach of about half of the world's human population. Infection by H. pylori is chronic and can evolve from gastritis to severe pathologies such as peptic ulcers and gastric cancer accounting for more than half a million deaths per year worldwide. H. pylori is till now the only bacteria to be recognized as a type 1 carcinogenic agent. In addition to this public health issue, H. pylori is of particular interest because it is the only microorganism to persistently colonize the human gastric mucosa, its unique niche, which makes it an excellent model organism to address fundamental questions related to its adaptation to such an hostile acidic environment.

Our research focuses on the pathogenesis of this gastric pathogen by studying both the bacterial virulence factors and the host response. First, we aim at identifying and characterizing the H. pylori factors and mechanisms that make this bacterium a successful and persistent pathogen in an hostile ecological niche. Second, we investigate the host response to H. pylori focusing on mechanisms related to the induction of genetic instabilities and epigenetic alterations, known as early events in the developmentof gastric cancer lesions.

Recent studies

Coupled amino acid deamidase-transport systems essential for Helicobacter pylori colonization.

H. pylori produces significant amounts of ammonia by strong Gln and Asn hydrolysis activities. We found that two enzymes, L-asparaginase (AnsB) and gamma-glutamyltranspeptidase (GGT) are highly active periplasmic deamidases in H. pylori, producing ammonia and Asp or Glu from Asn and Gln, respectively. GGT of H. pylori was previously shown to activate apoptosis and purified Hp-GGT induces the production of IL8 by the host cell. We found that GltS and DcuA are the sole H. pylori transporters for Glu and Asp, respectively. Uptake of Gln and Asn strictly relies on indirect pathways following prior periplasmic deamidation into Glu and Asp. Importantly, each of the four components of these novel coupled deamidase-transport systems (DcuA, GltS, AnsB and GGT) is individually crucial for animal colonization.

We propose a model in which these two non-redundant systems participate in H. pylori virulence by depleting gastric or immune cells from protective amino acids such as Gln and/or producing toxic ammonia close to the host cells.

Nickel trafficking and NikR regulation in H. pylori

Nickel is the co-factor of two metallo-enzymes of H. pylori crucial for colonization, hydrogenase and urease (required for acid resistance). We previously identified a novel TonB-dependent mechanism for energized nickel uptake through the outer membrane. We are now investigating the role of several nickel-binding proteins that are unique to H. pylori and predicted to be important for nickel homeostasis. HspA, a homolog of the GroES co-chaperonine that contains a unique His-rich C-terminal extension was shown to be involved in intracellular nickel detoxification and plays a novel role as a specialized chaperone for hydrogenase maturation. In H. pylori, expression of genes involved in nickel transport, storage and delivery is controlled by a Ni²⁺-dependent transcriptional regulator, HpNikR that presents unique characteristics (i) it is a pleiotropic regulator and, (ii) depending on its target gene, it acts either as an activator or a repressor. In collaboration with I. Michaud-Soret (CEA) and L. Terradot (ESRF), we characterized the role of the HpNikR nickel-binding sites for DNA binding in vitro and for in vivo regulation and we validated mechanistic hypothesis for these functions based on the crystal structures obtained for four selected HpNikR mutants.

Genotoxicity associated with H. pyloriinfection

Our studies provided the first direct evidence that long term chronic infection by H. pylori induces a mutagenic effect with an impairment of DNA repair systems at the gastric mucosa level, associated with inflammation. Mitochondrial DNA (mtDNA) mutations are found in various tumors including gastric cancer. We recently described the induction of mtDNA mutations in vitro in H. pylori-infected gastric epithelial cells and in chronically infected mice. Our data support the notion that the genotoxic activity associated with H. pylori infection contributes to early events in the gastric neoplastic process.

Consequences of H. pylori infection on mitochondria

Mitochondria are strategic targets for pathogens. In collaboration with M. Ricchetti (Institut Pasteur) we recently demonstrated that H. pylori infection of gastric epithelial cells results in a clustering of mitochondria in the perinuclear space associated with the induction of their biogenesis. Experiments are in progress to define the role of mitochondria dysfunction in the gastric pathogenicity associated to H. pylori infection.

Influence of DNA hypermethylation on the host response to H. pylori infection

Epigenetic silencing of gene expression by promoter CpG hyper-methylation plays a key role in the early steps of carcinogenesis. We demonstrated that H. pylori infection induces DNA hyper-methylation in the promoter regions of the USF1 and USF2 transcription factors, that is associated with their down-regulation. These factors are pleiotropic regulators suggested to be tumor suppressor genes. Decrease of USF1 and USF2 was also observed in gastric metaplasia of chronically infected mice and confirmed in biopsies from gastric cancer patients. Our data indicate that diminished levels of USF1 and USF2 might play a role in the induction of gastric malignancies during H. pylori infection.

Keywords: Helicobacter pylori – stomach - gastritis - gastric cancer – peptic ulcer – MALT lymphoma – regulation - urease - nickel – resistance to acidity – inflammation – genotoxicity - cancer detection markers – proteomics – mouse model

Leduc, D., Gallaud, J., Stingl, K. and De Reuse, H. (2010) Coupled amino acid deamidase-transport systems essential for Helicobacter pylori colonization.Infection and Immunity 78: 2782-2792.PMID: 20368342

Bussière, F.I., Michel, V., Mémet, S., Avé, P., Vivas, J.R., Huerre, M. and Touati, E. (2010) H. pylori-induced promoter hypermethylation downregulates USF1 and USF2 transcription factor gene expression.Cellular Microbiology 12:1124-1133.PMID: 20180799

Bahlawane, C., Dian, C., Muller, C., Round, A., Fauquant, C., Schauer, K., De Reuse, H., Terradot, L. and Michaud-Soret, I. (2010)Structural and mechanistic insights into Helicobacter pylori NikR activation.Nucleic Acids Research 38: 3106-3118.PMID: 20089510

Machado, A.M., Figueiredo, C., Touati, E., Máximo, V., Sousa, S., Michel, V., Carneiro, F., Nielsen, F.C., Seruca, R., and Rasmussen, L.J. (2009)Helicobacter pylori infection influences genetic stability of nuclear and mitochondrial DNA. Clinical Cancer Research, 15 : 2995-3002.PMID: 19383819

Stingl, K., Schauer, K., Ecobichon, C., Labigne, A., Lenormand, P., Rousselle J-C., Namane, A. and De Reuse, H. (2008)In vivo interactome of Helicobacter pylori urease revealed by tandem affinity purificationMolecular and Cellular Proteomics 7:2429-41.PMID: 18682379