title: Analysis of T4SS-induced signaling by H. pylori using quantitative phosphoproteomics.
abstract: Helicobacter pylori is a Gram-negative bacterial pathogen colonizing the human stomach. Infection with H. pylori causes chronic inflammation of the gastric mucosa and may lead to peptic ulceration and/or gastric cancer. A major virulence determinant of H. pylori is the type IV secretion system (T4SS), which is used to inject the virulence factor CagA into the host cell, triggering a wide range of cellular signaling events. Here, we used a phosphoproteomic approach to investigate tyrosine signaling in response to host-pathogen interaction, using stable isotope labeling in cell culture (SILAC) of AGS cells to obtain a differential picture between multiple infection conditions. Cells were infected with wild type H. pylori P12, a P12Δ CagA deletion mutant, and a P12Δ PAI deletion mutant to compare signaling changes over time and in the absence of CagA or the T4SS. Tryptic peptides were enriched for tyrosine (Tyr) phosphopeptides and analyzed by nano-LC-Orbitrap MS. In total, 85 different phosphosites were found to be regulated following infection. The majority of phosphosites identified were kinases of the MAPK family. CagA and the T4SS were found to be key regulators of Tyr phosphosites. Our findings indicate that CagA primarily induces activation of ERK1 and integrin-linked factors, whereas the T4SS primarily modulates JNK and p38 activation.
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology Berlin, Germany.
authors.forename: Frithjof
authors.initials: F
authors.lastname: Glowinski
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology Berlin, Germany.
authors.forename: Carsten
authors.initials: C
authors.lastname: Holland
authors.affiliation: The Biotechnology Centre of Oslo, University of Oslo Oslo, Norway ; Department of Biosciences, University of Oslo Oslo, Norway.
authors.forename: Bernd
authors.initials: B
authors.lastname: Thiede
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology Berlin, Germany.
authors.forename: Peter R
authors.initials: PR
authors.lastname: Jungblut
authors.affiliation: Department of Molecular Biology, Max Planck Institute for Infection Biology Berlin, Germany.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Meyer
keywords: CagA; H. pylori; SILAC; T4SS; phosphoproteomics; tyrosine signaling
references.citation: Gut. 2001 Jul;49(1):18-22
references.pmid: 11413105
references.citation: Nat Cell Biol. 2002 Aug;4(8):565-73
references.pmid: 12134157
references.citation: Nat Biotechnol. 2009 Jul;27(7):598-9
references.pmid: 19587657
references.citation: Nat Biotechnol. 2008 Dec;26(12):1367-72
references.pmid: 19029910
references.citation: J Biol Chem. 2000 Feb 4;275(5):3629-36
references.pmid: 10652359
references.citation: Nat Biotechnol. 2005 Jan;23(1):94-101
references.pmid: 15592455
references.citation: N Engl J Med. 2002 Oct 10;347(15):1175-86
references.pmid: 12374879
references.citation: Eur J Cell Biol. 2011 Nov;90(11):880-90
references.pmid: 21247656
references.citation: J Proteome Res. 2009 Sep;8(9):4333-41
references.pmid: 19655813
references.citation: Mol Microbiol. 1998 Apr;28(1):37-53
references.pmid: 9593295
references.citation: Cell. 2007 Dec 14;131(6):1190-203
references.pmid: 18083107
references.citation: Nat Rev Cancer. 2002 Jan;2(1):28-37
references.pmid: 11902583
references.citation: Nat Protoc. 2006;1(6):2650-60
references.pmid: 17406521
references.citation: Microbes Infect. 2007 Jun;9(7):838-46
references.pmid: 17533150
references.citation: Proc Natl Acad Sci U S A. 2012 Sep 4;109(36):14640-5
references.pmid: 22908298
references.citation: EMBO J. 2003 Feb 3;22(3):515-28
references.pmid: 12554652
references.citation: Cell Host Microbe. 2012 Jun 14;11(6):576-86
references.pmid: 22704618
references.citation: J Proteome Res. 2011 Apr 1;10(4):1794-805
references.pmid: 21254760
references.citation: Proteomics. 2011 Jul;11(14):2798-811
references.pmid: 21717572
references.citation: Nature. 2007 Oct 18;449(7164):862-6
references.pmid: 17943123
references.citation: Trends Microbiol. 2010 Nov;18(11):479-86
references.pmid: 20863705
references.citation: Biochim Biophys Acta. 2014 Apr;1843(4):715-24
references.pmid: 24418622
references.citation: Mol Cell Proteomics. 2005 Dec;4(12):2010-21
references.pmid: 16249172
references.citation: J Biol Chem. 2003 Feb 7;278(6):3664-70
references.pmid: 12446738
references.citation: J Biol Chem. 2002 Mar 1;277(9):6775-8
references.pmid: 11788577
references.citation: Proc Natl Acad Sci U S A. 2005 Jun 28;102(26):9300-5
references.pmid: 15972330
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2451-6
references.pmid: 18268350
references.citation: Science. 2002 Jan 25;295(5555):683-6
references.pmid: 11743164
references.citation: Nat Cell Biol. 2000 Feb;2(2):62-9
references.pmid: 10655584
references.citation: Cell Host Microbe. 2011 Jun 16;9(6):520-31
references.pmid: 21669400
references.citation: Cell. 2007 Jun 29;129(7):1415-26
references.pmid: 17570479
references.citation: Nat Rev Mol Cell Biol. 2008 Oct;9(10):815-20
references.pmid: 18784729
references.citation: J Biol Chem. 2008 May 16;283(20):13952-63
references.pmid: 18356158
references.citation: Nat Immunol. 2004 Nov;5(11):1166-74
references.pmid: 15489856
references.citation: Mol Cell Proteomics. 2013 Feb;12(2):529-38
references.pmid: 23033477
references.citation: Oncogene. 2008 Nov 24;27(55):7047-54
references.pmid: 19029944
references.citation: Mol Microbiol. 2002 Feb;43(4):971-80
references.pmid: 11929545
references.citation: Helicobacter. 2010 Jun;15(3):163-76
references.pmid: 20557357
references.citation: Chem Cent J. 2008 Jul 18;2:16
references.pmid: 18638390