Stable isotope labeling by amino acids in cell culture based proteomics reveals differences in protein abundances between spiral and coccoid forms of the gastric pathogen Helicobacter pylori

被引:13
作者
Mueller, Stephan A. [1 ,2 ]
Pernitzsch, Sandy R. [3 ]
Haange, Sven-Bastiaan [1 ]
Uetz, Peter [4 ]
von Bergen, Martin [1 ,5 ,6 ]
Sharma, Cynthia M. [3 ]
Kalkhof, Stefan [1 ,7 ]
机构
[1] Helmholtz Ctr Environm Res Leipzig, UFZ, Dept Prote, D-04318 Leipzig, Germany
[2] DZNE German Ctr Neurodegenerat Dis, Dept Neuroprote, D-81377 Munich, Germany
[3] Univ Wurzburg, Res Ctr Infect Dis ZINF, D-97082 Wurzburg, Germany
[4] Virginia Commonwealth Univ, Ctr Study Biol Complex, Richmond, VA 23284 USA
[5] Helmholtz Ctr Environm Res Leipzig, UFZ, Dept Metabol, D-04318 Leipzig, Germany
[6] Aalborg Univ, Dept Biotechnol Chem & Environm Engn, DK-9000 Aalborg, Denmark
[7] Univ Appl Sci & Arts Coburg, Dept Bioanalyt, D-96450 Coburg, Germany
关键词
Helicobacter pylori; SILAC; Cell morphology; Quantitative proteomics; ALPHA-INDUCING PROTEIN; EPITHELIAL-CELLS; NUTRITIONAL-REQUIREMENTS; QUANTITATIVE PROTEOMICS; STAPHYLOCOCCUS-AUREUS; GNOTOBIOTIC PIGLETS; CHEMOTAXIS RECEPTOR; STATIONARY-PHASE; GENE-EXPRESSION; DEFINED MEDIUM;
D O I
10.1016/j.jprot.2015.05.011
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
Helicobacter pylori (H. pylori) is a epsilon-proteobacterium that colonizes the stomach of about half of the world's population. Persistent infections have been associated with several gastric diseases. Mainly rod- or spiral shaped but also coccoid H. pylori forms have been isolated from mucus layer biopsies of patients. It is still being debated whether the coccoid form can be transformed back into the spiral form or whether this morphology is a result of bacterial cell death or persistence. We established stable isotope labeling by amino acids in cell culture (SILAC) for quantitative proteomics of H. pylori and applied it to investigate differences between the spiral and the coccoid morphology. We detected 72% and were able to relatively quantify 47% of the H. pylori proteome. Proteins involved in cell division and transcriptional and translational processes showed a lower abundance in coccoid cells. Additionally, proteins related to host colonization, including CagA, the arginase RocF, and the TNF-alpha inducing protein were down-regulated. The fact that outer membrane proteins were observed at higher abundances might represent a mechanism for immune evasion but also preserves adherence to host cells. The established protocol for relative protein quantification of H. pylori samples offers new possibilities for research on H. pylori. Biological significance Our study shows that SILAC can be employed to study protein abundance changes in H. pylori. We have chosen to establish SILAC for H. pylori because it facilitates fractionation on both, protein and peptide level and thus enables deep proteome coverage. Furthermore, SILAC allows robust and highly accurate protein quantification. The manuscript includes a detailed description of the applied method, suggestions for further improvement as well as a practical application. The investigation of differences between the coccoid and infectious spiral morphology of H. pylori with SILAC revealed the regulation of proteins that are involved in host colonization, motility, cell division as well as transcriptional and translational processes. The data will help molecular biologist to focus on relevant pathways that were found to be regulated in response to morphological changes. Furthermore, the application of SILAC offers new possibilities to study the biology of H. pylori. It enables to monitor protein abundance changes in response to certain stimuli such as oxygen stress or antibiotics. Moreover, SILAC raises the possibility to study co-cultures of host cells and H. pylori on protein level. Additionally, pulsed SILAC experiments enable the quantification of protein turnover. (C) 2015 Published by Elsevier B.V.
引用
收藏
页码:34 / 45
页数:12
相关论文
共 109 条
  • [21] Labeling of the Pathogenic Bacterium Staphylococcus aureus with Gold or Ferric Oxide-Core Nanoparticles Highlights New Capabilities for Investigation of Host-Pathogen Interactions
    Depke, Maren
    Surmann, Kristin
    Hildebrandt, Petra
    Jehmlich, Nico
    Michalik, Stephan
    Stanca, Sarmiza E.
    Fritzsche, Wolfgang
    Voelker, Uwe
    Schmidt, Frank
    [J]. CYTOMETRY PART A, 2014, 85 (02) : 140 - 150
  • [22] Monitoring of changes in the membrane proteome during stationary phase adaptation of Bacillus subtilis using in vivo labeling techniques
    Dreisbach, Annette
    Otto, Andreas
    Becher, Doerte
    Hammer, Elke
    Teumer, Alexander
    Gouw, Joost W.
    Hecker, Michael
    Voelker, Uwe
    [J]. PROTEOMICS, 2008, 8 (10) : 2062 - 2076
  • [23] Colonization of gnotobiotic piglets by Helicobacter pylori deficient in two flagellin genes
    Eaton, KA
    Suerbaum, S
    Josenhans, C
    Krakowka, S
    [J]. INFECTION AND IMMUNITY, 1996, 64 (07) : 2445 - 2448
  • [24] MOTILITY AS A FACTOR IN THE COLONIZATION OF GNOTOBIOTIC PIGLETS BY HELICOBACTER-PYLORI
    EATON, KA
    MORGAN, DR
    KRAKOWKA, S
    [J]. JOURNAL OF MEDICAL MICROBIOLOGY, 1992, 37 (02) : 123 - 127
  • [25] Pseudogene Recoding Revealed from Proteomic Analysis of Salmonella Serovars
    Feng, Ye
    Chien, Kun-Yi
    Chen, Hsiu-Ling
    Chiu, Cheng-Hsun
    [J]. JOURNAL OF PROTEOME RESEARCH, 2012, 11 (03) : 1715 - 1719
  • [26] Systematic mutagenesis of the Helicobacter pylori cag pathogenicity island:: essential genes for CagA translocation in host cells and induction of interleukin-8
    Fischer, W
    Püls, J
    Buhrdorf, R
    Gebert, B
    Odenbreit, S
    Haas, R
    [J]. MOLECULAR MICROBIOLOGY, 2001, 42 (05) : 1337 - 1348
  • [27] p21-activated kinase 1 activates the nuclear factor κB (NF-κB)-inducing kinase-IκB kinases NF-κB pathway and proinflammatory cytokines in Helicobacter pylori infection
    Foryst-Ludwig, A
    Naumann, M
    [J]. JOURNAL OF BIOLOGICAL CHEMISTRY, 2000, 275 (50) : 39779 - 39785
  • [28] Helicobacter pylori possesses two CheY response regulators and a histidine kinase sensor, CheA, which are essential for chemotaxis and colonization of the gastric mucosa
    Foynes, S
    Dorrell, N
    Ward, SJ
    Stabler, RA
    McColm, AA
    Rycroft, AN
    Wren, BW
    [J]. INFECTION AND IMMUNITY, 2000, 68 (04) : 2016 - 2023
  • [29] Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics
    Geiger, Tamar
    Wisniewski, Jacek R.
    Cox, Juergen
    Zanivan, Sara
    Kruger, Marcus
    Ishihama, Yasushi
    Mann, Matthias
    [J]. NATURE PROTOCOLS, 2011, 6 (02) : 147 - 157
  • [30] Tip-α (hp0596 gene product) is a highly immunogenic Helicobacter pylori protein involved in colonization of mouse gastric mucosa
    Godlewska, Renata
    Pawlowski, Marcin
    Dzwonek, Artur
    Mikula, Michal
    Ostrowski, Jerzy
    Drela, Nadzieja
    Jagusztyn-Krynicka, Elzbieta K.
    [J]. CURRENT MICROBIOLOGY, 2008, 56 (03) : 279 - 286