An infectious agent of deception, exposed through proteomics.

Salmonella bacteria, infamous for food poisoning that kills hundreds of thousands of people worldwide, infect by stealth. They slip unnoticed into and multiply inside of macrophages, the very immune system cells that the body relies on to seek and destroy invading microbes.

How do Salmonella bacteria escape detection by macrophages, turning predator cells into prey that are complicit in promoting infection? Until recently, finding out has seemed impossibly complicated, a needle-in-a-haystack proposition involving thousands of proteins, the building blocks that carry out the vital functions of the cells.

But by applying the high-volume sorting and analytical power of proteomics--a detailed survey of microbial proteins present in the 24 hours that follow macrophage infection of a mouse--a team led by Liang Shi, staff scientist at the Department of Energy's Pacific Northwest National Laboratory (PNNL), has turned up a suspect protein.

The discovery of the protein, dubbed STM3117, was described in the September 29 issue of the Journal of Biological Chemistry. By knocking out the gene that codes for STM3117, the researchers subsequently crippled the ability of the microbe to multiply inside macrophages. Shi and colleagues say the protein and two closely related proteins discovered in the study are similar in genetic sequence to peptidoglycan, which is known to make and modify chemicals in the cell walls of the microbe.

Drug and vaccine designers armed with this mouse-model information can target chemicals or immune responses that disrupt peptidoglycan synthesis and other processes linked to Salmonella colonization of macrophages in humans, said Joshua Adkins, a PNNL staff scientist and co-author of Shi's paper, as well as lead author of a related study that was reported in Molecular & Cellular Proteomics in August. Adkins added that a quick identification of these proteins could help physicians assess the virulence of a given strain.

The candidate proteins were winnowed from among 315 possibilities that emerged through a combination of techniques culminating in measurements by Fourier-transform mass spectrometry (FT-MS). A suite of FT-MS instruments customized by co-author and PNNL-based Battelle Fellow Richard D. Smith enabled the team to rapidly separate and identify many proteins at once, even as macrophages were being infected.

Most of the initial candidates were designated "housekeeping" proteins whose numbers relative to those of other proteins remained more or less constant during the course of infection. But 39 proteins shot up in number during bacterial colonization of macrophages, and of those, a handful or so--including STM3117--responded specifically to a macrophage protein associated with resistance to microbial infection. A standard Western blot assay confirmed the increases among that small group of proteins during infection.

The work was funded by PNNL and the National Institutes of Health's National Institute of Allergy and Infectious Diseases. Much of the work was performed at the PNNL-based W.R. Wiley Environmental Molecular Sciences Laboratory.