A Comprehensive Subcellular Proteomic Survey of Salmonella Grown under Phagosome-Mimicking versus Standard Laboratory Conditions

Roslyn N Brown,Fred Heffron,James A Sanford,Richard D Smith,Brooke L Deatherage,Joshua N Adkins,Jea H Park,Boyd L Champion

doi:10.1155/2012/123076

Abstract

Towards developing a systems-level pathobiological understanding of Salmonella enterica, we performed a subcellular proteomic analysis of this pathogen grown under standard laboratory and phagosome-mimicking conditions in vitro. Analysis of proteins from cytoplasmic, inner membrane, periplasmic, and outer membrane fractions yielded coverage of 25% of the theoretical proteome. Confident subcellular location could be assigned to over 1000 proteins, with good agreement between experimentally observed location and predicted/known protein properties. Comparison of protein location under the different environmental conditions provided insight into dynamic protein localization and possible moonlighting (multiple function) activities. Notable examples of dynamic localization were the response regulators of two-component regulatory systems (e.g., ArcB and PhoQ). The DNA-binding protein Dps that is generally regarded as cytoplasmic was significantly enriched in the outer membrane for all growth conditions examined, suggestive of moonlighting activities. These observations imply the existence of unknown transport mechanisms and novel functions for a subset of Salmonella proteins. Overall, this work provides a catalog of experimentally verified subcellular protein locations for Salmonella and a framework for further investigations using computational modeling.

Highlights

The pursuit of a systems-level understanding of bacterial physiology requires knowledge about the identity, function, and relative abundance of proteins, and insight into the subcellular localization of these proteins
Growth of Salmonella in defined, acidic media with low concentrations of phosphate and magnesium induces expression of Salmonella pathogenicity island 2 (SPI-2) genes that are required for intracellular survival and replication [15,16,17,18,19]. mLPM has been shown to induce expression and secretion of SPI2-related virulence factors [14] and was used in this study to mimic the environment of a macrophage phagosome
We have previously investigated the proteome response of Salmonella to phagosome-mimicking in vitro conditions [19, 26]; the use of subcellular fractionation presented an opportunity for obtaining better proteome coverage, especially of proteins that are typically underrepresented in global proteomic strategies, in addition to highlighting the subcellular location of proteins of interest

Summary

Introduction

The pursuit of a systems-level understanding of bacterial physiology requires knowledge about the identity, function, and relative abundance of proteins, and insight into the subcellular localization of these proteins. There is a growing appreciation for the presence of bacterial “moonlighting proteins,” that is, those proteins that have a secondary function depending on subcellular location [1,2,3]. Verified localization provides a foundation for describing proteins that are “hypothetical,” uncharacterized, or that contain domains of unknown function. With the increasing use of systems biology approaches, including genome-scale models of metabolism [4] and regulation to study microbial functions, experimentally founded protein localization on a global scale is necessary to produce more accurate model constraints

Methods

Results

Conclusion