title: Proteomic analysis of Salmonella enterica serovar typhimurium isolated from RAW 264.7 macrophages: identification of a novel protein that contributes to the replication of serovar typhimurium inside macrophages.
abstract: To evade host resistance mechanisms, Salmonella enterica serovar Typhimurium (STM), a facultative intracellular pathogen, must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM pathogenesis, we have isolated STM cells from RAW 264.7 macrophages at various time points following infection and used a liquid chromatography-mass spectrometry-based proteomic approach to detect the changes in STM protein abundance. Because host resistance to STM infection is strongly modulated by the expression of a functional host-resistant regulator, i.e. natural resistance-associated macrophage protein 1 (Nramp1, also called Slc11a1), we have also examined the effects of Nramp1 activity on the changes of STM protein abundances. A total of 315 STM proteins have been identified from isolated STM cells, which are largely housekeeping proteins whose abundances remain relatively constant during the time course of infection. However, 39 STM proteins are strongly induced after infection, suggesting their involvement in modulating colonization and infection. Of the 39 induced proteins, 6 proteins are specifically modulated by Nramp1 activity, including STM3117, as well as STM3118-3119 whose time-dependent abundance changes were confirmed using Western blot analysis. Deletion of the gene encoding STM3117 resulted in a dramatic reduction in the ability of STM to colonize wild-type RAW 264.7 macrophages, demonstrating a critical involvement of STM3117 in promoting the replication of STM inside macrophages. The predicted function common for STM3117-3119 is biosynthesis and modification of the peptidoglycan layer of the STM cell wall.
authors.affiliation: Pacific Northwest National Laboratory, Richland, Washington 99354, USA. liang.shi@pnl.gov
authors.forename: Liang
authors.initials: L
authors.lastname: Shi
authors.forename: Joshua N
authors.initials: JN
authors.lastname: Adkins
authors.forename: James R
authors.initials: JR
authors.lastname: Coleman
authors.forename: Athena A
authors.initials: AA
authors.lastname: Schepmoes
authors.forename: Alice
authors.initials: A
authors.lastname: Dohnkova
authors.forename: Heather M
authors.initials: HM
authors.lastname: Mottaz
authors.forename: Angela D
authors.initials: AD
authors.lastname: Norbeck
authors.forename: Samuel O
authors.initials: SO
authors.lastname: Purvine
authors.forename: Nathan P
authors.initials: NP
authors.lastname: Manes
authors.forename: Heather S
authors.initials: HS
authors.lastname: Smallwood
authors.forename: Haixing
authors.initials: H
authors.lastname: Wang
authors.forename: John
authors.initials: J
authors.lastname: Forbes
authors.forename: Philippe
authors.initials: P
authors.lastname: Gros
authors.forename: Sergio
authors.initials: S
authors.lastname: Uzzau
authors.forename: Karin D
authors.initials: KD
authors.lastname: Rodland
authors.forename: Fred
authors.initials: F
authors.lastname: Heffron
authors.forename: Richard D
authors.initials: RD
authors.lastname: Smith
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Squier
mesh_terms: D000818:Animals; D001483:Base Sequence; D027682:Cation Transport Proteins / Q000378:metabolism; D002460:Cell Line; D015964:Gene Expression Regulation, Bacterial; D008264:Macrophages / Q000378:metabolism / Q000382:microbiology*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods; D012481:Salmonella Infections, Animal / Q000378:metabolism; D019779:Salmonella enterica / Q000378:metabolism* / Q000472:pathogenicity; D014357:Trypsin / Q000494:pharmacology; D000099173:Natural Resistance-associated Macrophage Protein 1
chemical_list: D027682:Cation Transport Proteins; D014357:Trypsin; D000099173:Natural Resistance-associated Macrophage Protein 1