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*
- Liang Shi‡1,
- Joshua N. Adkins‡,
- James R. Coleman‡,
- Athena A. Schepmoes‡,
- Alice Dohnkova‡,
- Heather M. Mottaz‡,
- Angela D. Norbeck‡,
- Samuel O. Purvine‡,
- Nathan P. Manes‡,
- Heather S. Smallwood‡,
- Haixing Wang‡,
- John Forbes§,
- Philippe Gros§,
- Sergio Uzzau¶,
- Karin D. Rodland‡,
- Fred Heffron∥,
- Richard D. Smith‡ and
- Thomas C. Squier‡
- ‡Pacific Northwest National Laboratory, Richland, Washington 99354, §McGill University, Montreal, Quebec H3G 1Y6, Canada, ¶University of Sassari, 07100 Sassari, Italy and ∥Oregon Health Sciences University, Portland, Oregon 97201
- 1 To whom correspondence should be addressed: Microbiology Group, Pacific Northwest National Laboratory, 902 Battelle Blvd., P. O. Box 999, MSIN: P7-50, Richland, WA 99354. Tel.: 509-376-4834; Fax: 509-372-1632; E-mail: liang.shi{at}pnl.gov.
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.
Footnotes
-
↵2 The abbreviations used are: STM, S. enterica serovar Typhimurium; AMT, accurate mass and time; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; FTICR, Fourier transform ion cyclotron resonance; HBSS, Hanks' buffered saline solution; IHF, integration host factor; LC, liquid chromatography; MG, methylglyoxal; MS, mass spectrometry; Nramp1, natural resistance-associated macrophage protein 1; N+, Nramp1-positive; N-, Nramp1-negative; p.i., post infection; SCV, Salmonella-containing vacuole; SPI, Salmonella pathogenicity island; T3S, type III secretion system; WB, Western blot; NET, normalized elution time; WT, wild type.
-
↵* This work was supported in part by the Laboratory Directed Research and Development Program of the Department of Energy (to L. S.) and by NIAID, National Institutes of Health/Department of Health and Human Services Interagency Agreement Y1-AI-4894-01. Significant portions of this work were carried out at the W. R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the Department of Energy Office of Biological and Environmental Sciences program located at Pacific Northwest National Laboratory. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
-
↵
The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1 and Tables S1-S3.
-
- Received May 15, 2006.
