title: Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages.
abstract: BACKGROUND
  The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium), several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE) combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established.
  
  
  RESULTS
  Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2.
  
  
  CONCLUSIONS
  A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.
authors.affiliation: Department of Microbiology, School of Pharmacy, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Haneda
authors.forename: Mariko
authors.initials: M
authors.lastname: Sugimoto
authors.forename: Yukie
authors.initials: Y
authors.lastname: Yoshida-Ohta
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Kodera
authors.forename: Masamichi
authors.initials: M
authors.lastname: Oh-Ishi
authors.forename: Tadakazu
authors.initials: T
authors.lastname: Maeda
authors.forename: Satomi
authors.initials: S
authors.lastname: Shimizu-Izumi
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Miki
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kumagai
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Danbara
authors.forename: Nobuhiko
authors.initials: N
authors.lastname: Okada
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D006150:Guanine Nucleotides / Q000172:deficiency*; D006801:Humans; D008264:Macrophages / Q000382:microbiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D050296:Microbial Viability; D008969:Molecular Sequence Data; D009154:Mutation; D040901:Proteomics; D012480:Salmonella Infections / Q000382:microbiology*; D012486:Salmonella typhimurium / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D006150:Guanine Nucleotides; D037521:Virulence Factors; C018957:guanosine 5'-diphosphate-3'-monophosphate