Microaerophilic conditions permit to mimic in vitro events occurring during in vivo Helicobacter pylori infection and to identify Rho/Ras-associated proteins in cellular signaling.
Molecular dissection of the mechanisms underlying Helicobacter pylori infection suffers from the lack of in vitro systems mimicking in vivo observations. A system was developed whereby human epithelial cells (Caco-2) grown as polarized monolayers and bacteria can communicate with each other under culture conditions optimal for each partner. Caco-2 cells grown on filter supports were inserted in a vertical position into diffusion chambers equilibrated with air and 5% CO(2) at their basolateral surface (aerophilic conditions) and 5% CO(2), 5% O(2), 90% N(2) (microaerophilic conditions) in the apical compartment. Remarkably, the epithelial polarized layer was stable under these asymmetric culture conditions for at least 24 h, and the presence of Caco-2 cells was necessary to maintain H. pylori growth. In contrast to previous studies conducted with non-polarized Caco-2 cells and other cell lines kept under aerophilic conditions, we found H. pylori-dependent stimulation of cytokine secretion (MCP-1 (monocyte chemoattractant protein-1), GRO-alpha (growth-regulated oncogene-alpha), RANTES (regulated on activation normal T cell expressed and secreted)). This correlated with nuclear translocation of NF-kappaB p50 and p65 subunits. Tyrosine phosphorylation of nine cellular proteins was induced or enhanced; we identified p120(RasGAP), p190(RhoGAP), p62dok (downstream of tyrosine kinases), and cortactin as H. pylori-inducible targets. Moreover, reduction of H. pylori urease expression was observed in adherent bacteria as compared with bacteria in suspension. In addition to mimicking several observations seen in the inflamed gastric mucosa, the novel in vitro system was allowed to underscore complex cellular events not seen in classical in vitro analyses of microaerophilic bacteria-epithelial cell cross-talk.
Cottet
Sandra
S
Division of Immunology and Allergy, R & D Laboratory, Centre Hospitalier Universitaire Vaudois, 1005 Lausanne, Switzerland.
Corthésy-Theulaz
Irène
I
Spertini
François
F
Corthésy
Blaise
B
D018938:Caco-2 Cells; D016764:Cell Polarity; D018925:Chemokines / Q000378:metabolism; D051356:Cortactin; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D020690:GTPase-Activating Proteins; D005753:Gastric Mucosa / Q000378:metabolism; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D016481:Helicobacter Infections / Q000378:metabolism*; D016480:Helicobacter pylori / Q000201:enzymology / Q000254:growth & development; D006801:Humans; D008840:Microfilament Proteins / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012097:Repressor Proteins; D014510:Urease / Q000096:biosynthesis; D020729:p120 GTPase Activating Protein / Q000378:metabolism*; D020666:ras-GRF1
C074560:ARHGAP35 protein, human; C513043:ARHGAP5 protein, human; C495868:CTTN protein, human; D018925:Chemokines; D051356:Cortactin; D004268:DNA-Binding Proteins; C489560:DOK1 protein, human; C074561:GAP-associated protein p62; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008840:Microfilament Proteins; D016328:NF-kappa B; D009687:Nuclear Proteins; D010750:Phosphoproteins; C513039:RASGRF1 protein, human; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; D020729:p120 GTPase Activating Protein; D020666:ras-GRF1; D014510:Urease