Targeted Protein Degradation by Salmonella under Phagosome-mimicking Culture Conditions Investigated Using Comparative Peptidomics

doi:10.1074/mcp.M600282-MCP200

Targeted Protein Degradation by Salmonella under Phagosome-mimicking Culture Conditions Investigated Using Comparative Peptidomics*S

From the ^‡Fundamental Science Division, ^¶Environmental Molecular Science Laboratory, Pacific Northwest National Laboratory, Richland, Washington 99352 and ^§Department of Molecular Microbiology and Immunology, Oregon Health and Sciences University, Portland, Oregon 97239

‖To whom correspondence and reprint requests should be addressed: Dept. of Molecular Microbiology and Immunology, OHSU Mail Drop L220, 3181 S. W. Sam Jackson Park Rd., Portland, OR 97239. E-mail: heffronf{at}ohsu.edu

Abstract

The pathogen Salmonella enterica is known to cause both food poisoning and typhoid fever. Because of the emergence of antibiotic-resistant isolates and the threat of bioterrorism (e.g. contamination of the food supply), there is a growing need to study this bacterium. In this investigation, comparative peptidomics was used to study S. enterica serovar Typhimurium cultured in either a rich medium or in an acidic, low magnesium, and minimal nutrient medium designed to roughly mimic the macrophage phagosomal environment (within which Salmonella are known to survive). Native peptides from cleared cell lysates were enriched by using isopropanol extraction and analyzed by using both LC-MS/MS and LC-FTICR-MS. We identified and quantified 5,163 peptides originating from 682 proteins, and the data clearly indicated that compared with Salmonella cultured in the rich medium, cells cultured in the phagosome-mimicking medium had dramatically higher abundances of a wide variety of protein degradation products, especially from ribosomal proteins. Salmonella from the same cultures were also analyzed using traditional, bottom-up proteomic methods, and when the peptidomics and proteomics data were analyzed together, two clusters of proteins targeted for proteolysis were tentatively identified. Possible roles of targeted proteolysis by phagocytosed Salmonella are discussed.

Footnotes

Published, MCP Papers in Press, January 16, 2007, DOI 10.1074/mcp.M600282-MCP200
↵¹ The abbreviations used are: Log, logarithmic; Stat, stationary; MgM, acidic, magnesium-depleted minimal medium; LB, Luria-Bertani; SCX, strong cation exchange; SPE, solid phase extraction; %FP_est., estimated percentage of false positive peptide identifications; AMT, accurate mass and elution time; NET, normalized elution time; PolyP, polyphosphate.
↵* Portions of this work were supported by the National Institute of Allergy and Infectious Diseases, (National Institutes of Health (NIH)/Department of Health and Human Services through interagency agreement Y1-AI-4894-01) and the NIH National Center for Research Resources (RR18522). Additional supporting results and protocols are available at the National Institute of Allergy and Infectious Diseases funded Administrative Resource for Biodefense Proteomics Research Centers under the PNNL data section (www.proteomicsresearch.org). 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.
↵S The on-line version of this article (available at http://www.mcponline.org) contains supplemental material.
- Received August 1, 2006.
- Revision received January 8, 2007.