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Infection and Immunity, August 2009, p. 3227-3233, Vol. 77, No. 8
0019-9567/09/$08.00+0 doi:10.1128/IAI.00063-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Proteomic Investigation of the Time Course Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica
,
Liang Shi,1*
Saiful M. Chowdhury,1
Heather S. Smallwood,1
Hyunjin Yoon,2
Heather M. Mottaz-Brewer,1
Angela D. Norbeck,1
Jason E. McDermott,1
Therese R. W. Clauss,1
Fred Heffron,2
Richard D. Smith,1 and
Joshua N. Adkins1*
Pacific Northwest National Laboratory, Richland, Washington 99352,1 Oregon Health and Science University, Portland, Oregon 972392
Received 17 January 2009/ Returned for modification 6 March 2009/ Accepted 26 May 2009
To investigate the extent to which macrophages respond to Salmonella infection, we infected RAW 264.7 macrophages with Salmonella enterica serotype Typhimurium and analyzed macrophage proteins at various time points following infection by using a global proteomic approach. A total of 1,006 macrophage and 115 Salmonella proteins were identified with high confidence. Most of the Salmonella proteins were observed in the late stage of the infection time course, which is consistent with the fact that the bacterial cells proliferate inside RAW 264.7 macrophages. The peptide abundances of most of the identified macrophage proteins remained relatively constant over the time course of infection. Compared to those of the control, the peptide abundances of 244 macrophage proteins (i.e., 24% of the total identified macrophage proteins) changed significantly after infection. The functions of these Salmonella-affected macrophage proteins were diverse, including production of antibacterial nitric oxide (i.e., inducible nitric oxide synthase), production of prostaglandin H2 (i.e., cyclooxygenase 2), and regulation of intracellular traffic (e.g., sorting nexin 5 [SNX5], SNX6, and SNX9). Diverse functions of the Salmonella-affected macrophage proteins demonstrate a global macrophage response to Salmonella infection. Western blot analysis not only confirmed the proteomic results for a selected set of proteins but also revealed that (i) the protein abundance of mitochondrial superoxide dismutase increased following macrophage infection, indicating an infection-induced oxidative stress in mitochondria, and (ii) in contrast to infection of macrophages by wild-type Salmonella, infection by the sopB deletion mutant had no negative impact on the abundance of SNX6, suggesting a role for SopB in regulating the abundance of SNX6.
* Corresponding author. Mailing address for Liang Shi: Microbiology Group, Pacific Northwest National Laboratory, 902 Battelle Blvd., MSIN: P7-50, Richland, WA 99352. Phone: (509) 376-4834. Fax: (509) 372-1632. E-mail: liang.shi{at}pnl.gov. Mailing address for Joshua N. Adkins: Biological Separation and Mass Spectrometry Group, Pacific Northwest National Laboratory, 902 Battelle Blvd., MSIN: K8-98, Richland, WA 99352. Phone: (509) 371-6583. Fax: (509) 371-6546. E-mail: Joshua.Adkins{at}pnl.gov
Published ahead of print on 15 June 2009.
Supplemental material for this article may be found at http://iai.asm.org/.
Infection and Immunity, August 2009, p. 3227-3233, Vol. 77, No. 8
0019-9567/09/$08.00+0 doi:10.1128/IAI.00063-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
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