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August 2009

Cell Scavengers Look to Take Bite Out of Infectious Disease

PNNL researchers use global proteomics to study macrophage response against Salmonella enterica

Peptide abundances of all identified macrophage and S. Typhimurium (STM) proteins. A total of 1121 proteins are shown, which include 1006 macrophage and 115 STM proteins. Peptide abundances for all identified macrophage and STM proteins in the non-infection (NI) control and at different time points of infection are indicated by colors that range from black (peptide abundance = 0%) to red (peptide abundance ≥0.2%). Enlarge Image

Results: Researchers from Pacific Northwest National Laboratory applied a novel global proteomic approach to better understand the extent to which macrophages respond to infection by a form of Salmonella.  Recognized as the first global proteomic analyses of time course responses of mouse macrophages to S. enterica serotype Typhimurium (S. Typhimurium) infection, the study may lead to new strategies for diagnosing, treating, and vaccinating against infectious disease. 

Macrophages, from the Greek for "eating cells," are white blood cells that act as cellular scavengers by ingesting dying and invading bacterial cells, but they recognize and refuse to eat their own kind.  The macrophages swallow the bacterial cells and then release powerful anti-bacterials that prevent bacteria from further dividing and infecting host cells. S. Typhimurium, which can be transmitted by ingesting contaminated food or water, is a pathogen that grows in the gastrointestinal tract of many animal species.  

By infecting susceptible mouse macrophages with S. Typhimurium and using global proteomics to analyze the proteins at various time points following infection, researchers identified 1,006 macrophage proteins.  The peptide abundances of 244 macrophage proteins, or 24 percent of the total macrophages identified, changed significantly after infection.  The functions of the Salmonella-affected macrophage proteins were diverse, including production of antibacterial nitric oxide, production of prostaglandin H2, and regulation of intracellular traffic.  The diversity in functions demonstrated a global macrophage response to Salmonella infection. 

The researchers used Western Blot analysis to confirm the proteomic results. The analysis also revealed that Salmonella infection increases mitochondrial abundance of the enzyme superoxide dismutase and decreases the abundance of the sorting nexin protein SNX6, most likely through the bacterial virulence factor SopB.  Superoxide dismutase acts like a natural antioxidant, repairing cells and reducing the damage caused by superoxide.  Sorting nexin proteins help cells move proteins to the correct location within the endomembrane system.

Why it matters: Macrophages are critical in autoimmune diseases such as rheumatoid arthritis, diabetes, and multiple sclerosis.  They also play a role in HIV infection and can stimulate cancer cells.  Researchers gain valuable information about the strengths and weaknesses of our immune system using mouse cell models such as used here.  This study leads to a better understanding of macrophage responses to infection, provides insight into disease development, and suggests targets for therapeutic intervention.

Salmonella-infected macrophages are regularly used to show the molecular mechanisms essential for the interaction between macrophage and intracellular pathogens.  Macrophages are important in controlling Salmonella-mediated systemic infection in susceptible mice.  They also help control morbidity and mortality in unvaccinated infected mice, while helping eliminate S. Typhimurium in vaccinated mice.

Methods: Using a liquid chromatography-mass spectrometry proteomic approach, the researchers analyzed the time course responses of the cell lysate of mouse RAW 264.7 macrophages after infection with S. Typhimurium.  They studied samples collected at four different time points following infection (0, 2, 4, and 24 hours.

What's Next: Future work will focus on understanding the roles of Salmonella-affected macrophage proteins that could lead to improved host-based therapeutics to intracellular pathogens.

Acknowledgments: This work was supported by the Laboratory Directed Research and Development program at PNNL and the National Institute of Allergy and Infectious Diseases (interagency agreements Y1-AI-4894-01 and Y1-AI-8401-01). Significant portions of this work were performed using EMSL, a national scientific user facility sponsored by the U.S. Department of Energy's Office of Biological and Environmental Research, located at PNNL. The research team includes Joshua Adkins, Saiful Chowdhury, Therese R.W. Clauss, Jason McDermott, Heather M. Mottaz-Brewer, Angela Norbeck, Liang Shi, Heather Smallwood, and Richard D. Smith of PNNL, and Fred Heffron and Hyunjin Yoon of Oregon Health Sciences University.

Reference: Shi L, SM Chowdhury, HS Smallwood, H Yoon, HM Mottaz-Brewer, AD Norbeck, JE McDermott, TRW Clauss, F Heffron, RD Smith, and JN Adkins. 2009. "Proteomic Investigation of the Time Responses of RAW 264.7 Macrophages to Infection with Salmonella enterica." Infection and Immunity 77(8):3227-3233

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