title: 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.
abstract: Helicobacter pylori (H. pylori) is a ε-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-α 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Proteomics, UFZ, Helmholtz-Centre for Environmental Research Leipzig, 04318 Leipzig, Germany; Department of Neuroproteomics, DZNE - German Center for Neurodegenerative Diseases, 81377 Munich, Germany.
authors.forename: Stephan A
authors.initials: SA
authors.lastname: Müller
authors.affiliation: Research Center for Infectious Diseases (ZINF), University of Würzburg, Würzburg, 97082, Germany.
authors.forename: Sandy R
authors.initials: SR
authors.lastname: Pernitzsch
authors.affiliation: Department of Proteomics, UFZ, Helmholtz-Centre for Environmental Research Leipzig, 04318 Leipzig, Germany.
authors.forename: Sven-Bastiaan
authors.initials: SB
authors.lastname: Haange
authors.affiliation: Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, VA 23284, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Uetz
authors.affiliation: Department of Proteomics, UFZ, Helmholtz-Centre for Environmental Research Leipzig, 04318 Leipzig, Germany; Department of Metabolomics, UFZ, Helmholtz-Centre for Environmental Research Leipzig, 04318 Leipzig, Germany; Aalborg University, Department of Biotechnology, Chemistry and Environmental Engineering, Aalborg University, DK-9000 Aalborg, Denmark.
authors.forename: Martin
authors.initials: M
authors.lastname: von Bergen
authors.affiliation: Research Center for Infectious Diseases (ZINF), University of Würzburg, Würzburg, 97082, Germany.
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Sharma
authors.affiliation: Department of Proteomics, UFZ, Helmholtz-Centre for Environmental Research Leipzig, 04318 Leipzig, Germany; Department of Bioanalytics, University of Applied Sciences and Arts of Coburg, 96450 Coburg, Germany. Electronic address: stefan.kalkhof@ufz.de.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kalkhof
mesh_terms: D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D016480:Helicobacter pylori / Q000378:metabolism*; D007553:Isotope Labeling / Q000379:methods*; D040901:Proteomics / Q000379:methods*
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins
keywords: Cell morphology; Helicobacter pylori; Quantitative proteomics; SILAC