Research Highlights
October 2006
First Use of Global Proteomics to Quantify Bacterial Proteins Isolated from Host Cells
Systematic investigation of changes can identify proteins important to detecting bacterial pathogens and developing vaccines
Results:Scientists at Pacific Northwest National Laboratory (PNNL) have reported the first systematic investigation using global proteomics to identify changes in the abundance of bacterial proteins associated with macrophage colonization and a new protein that helps the bacterium to survive inside macrophages. Macrophages are large white blood cells, occurring principally in connective tissue and in the bloodstream, that ingest foreign particles and infectious microorganisms. By investigating the changes in Salmonella protein abundances during infection by Salmonella, the researchers identified a novel protein that is required for Salmonella to replicate inside macrophages. These results demonstrate the value of a systematic investigation of bacterial protein changes in identifying proteins of likely importance in detecting bacterial pathogens and developing effective vaccines.
Why it matters: Salmonella enterica serovar Typhimurium (STM) is a facultative intracellular pathogen, meaning that it can grow both inside and outside cells. STM can cause gastroenteritis in humans and a lethal infection in mice that lack a macrophage protein called Nramp1. Because the symptoms resemble human typhoid fever caused by STM, mice infected by STM provide a model system to investigate the development and immunology of typhoid fever in humans.
Methods: To evade a host's resistance mechanisms, STM must alter its proteome following macrophage infection. To identify new colonization and virulence factors that mediate STM production and development, PNNL researchers isolated STM cells from RAW 264.7 (a mouse leukemic cell line) macrophages at various time points following infection. Then they used the PNNL-developed accurate mass and time (AMT) tag proteomic approach to detect the changes in the abundance of STM proteins. The AMT tag approach is critical to identifying key proteins linked to the successful colonization of macrophages. The researchers used the approach to systematically investigate the STM proteins isolated from the macrophages with or without functional Nramp1 at different time-points of infection.
Next steps: Future studies will focus on investigation of 1) the biological functions of newly identified STM protein that is required for colonization of macrophages and 2) Nramp1-affected pathways in STM cells.
Research team: Liang Shi, Josh Adkins, Jim Coleman, Athena Schepmoes, Alice Dohlnakova, Heather Mottaz, Angela Norbeck, Samuel Purvine, Nathan Manes, Heather Smallwood, Haixing Wang, Karin Rodland, Dick Smith, and Tom Squier, all PNNL; John Forbes and Philippe Gros, McGill University, Montreal; Sergio Uzzau, University of Sassari, Italy; and Fred Heffron, Oregon Health Sciences University.
Sponsor: The work is supported by Laboratory Directed Research and Development at PNNL and by the National Institute of Allergy and Infectious Diseases.
![proteomic analysis of the isolated Salmonella](https://webarchive.library.unt.edu/eot2008/20080920044610im_/http://www.pnl.gov/science/images/highlights/biology/20060921.jpg)
This shows the cells of Salmonella isolated from macrophages and the results of proteomic analysis of the isolated Salmonella cells. Investigation of the changes in Salmonella protein abundances during the infection resulted in identification of a novel protein that is required for Salmonella to replicate inside macrophages.
Full Image.
Source: "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." 2006. Journal of Biological Chemistry 281(39):29131-29140. Authors: Shi L, JN Adkins, JR Coleman, AA Schepmoes, A Dohnalkova, HM Mottaz, AD Norbeck, SO Purvine, NP Manes, HS Smallwood, H Wang, J Forbes, P Gros, S Uzzau, KD Rodland, F Heffron, RD Smith, and TC Squier.