University of Michigan
Proteomic Profiling of Bacillus Anthracis Infected Macrophages Chen E.I.1*, Prieto J.H.1*, Bergman N.H.2,3, Anderson E.C. 2, Swenson E.E. 2, Hanna P.C. 2, Yates J.R. III1
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The Scripps Research Institute, Department of Cell Biology, La Jolla, CA;
Department of Microbiology & Immunology and 3Bioinformatics Program, University of Michigan Medical School, Ann Arbor, MI; *Authors contributed to the work equally Anthrax is a serious, often fatal disease that has been recently brought to public attention because of its use in bioterrorism and noted presence in several nations’ biowarfare arsenals. The disease is caused by the bacterium Bacillus anthracis, which enters the body as dormant endospores, then uses host macrophages as a sanctuary in which to germinate, begin replication, and produce toxins. Current therapy and vaccine options are suboptimal, and a better understanding of the pathogenesis of B. anthracis— particularly how the bacterium interacts with the host macrophage to establish infection—is essential for the development of new options. The objective of this project is to determine specific B. anthracis and host factors that represent essential elements in anthrax infection. Determination of Host Factors: Using a large-scale proteomic method, Multidimensional Protein Identification Technology (MudPIT), we first define the global protein expression changes of murine macrophage during experimental anthrax and bacterial growth in culture. The experimental infection system uses the B. anthracis 34F2 Sterne strain, a capsule-deficient, toxigenic strain, and the mouse RAW 264.7 macrophage cell line. We focused on the first six hours following infection, as this time frame has been characterized microscopically in previous studies. Protein profiles of mouse RAW 264.7 macrophage cells were identified at 0, 3, and 6 hours post B. anthracis infection and compared with the non-infected macrophage cells harvested at the identical time point (Table 1). Changes of protein expression at 3 and 6 hour time points were uncovered and classified as early response proteins (change initiated at 3 hour) and late response proteins (change initiated at 6 hour) (Figure 1). Determination of B. anthracis Factors: Knowledge deriving from comparing the molecular changes of the invading organism and host organism concurrently as it happens in nature can provide valuable information of the interplay between host-bacteria interactions. However, increased protein complexity and dynamic range in the dual-organism system present a big challenge to
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protein profiling, particularly in the less abundant organism. To overcome this problem, we employed an enrichment step for B. anthracis proteins (differential lysis), and then digested the enriched B. anthracis proteins with two different proteases to increase protein identifications by MudPIT. To enrich for B. anthracis proteins, infected macrophage cells were lysed first and separated into soluble and insoluble fractions (see method). 100ug of each fraction were digested with either trypsin (neutral pH) or pepsin (acidic pH) and then subjected to the MudPIT analysis (see method). Significant increase in B. anthracis protein identification was found by combining the differential lysis and differential digest methods (Table 2). Using this new method, we are able to characterize changes in B. anthracis protein expression during the course of infection.
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