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Applied and Environmental Microbiology, August 2008, p. 5201-5210, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.02890-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

High-Throughput, Single-Cell Analysis of Macrophage Interactions with Fluorescently Labeled Bacillus anthracis Spores{triangledown}

Bojana Stojkovic,1,§ Eric M. Torres,1,§ Angela M. Prouty,1 Hetal K. Patel,1 Lefan Zhuang,1 Theresa M. Koehler,3 Jimmy D. Ballard,4 and Steven R. Blanke1,2*

Department of Microbiology,1 Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801,2 Department of Microbiology and Molecular Genetics, University of Texas—Houston Medical School, Houston, Texas 77030,3 Department of Microbiology and Immunology, The University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 731044

Received 21 December 2007/ Accepted 29 May 2008

The engulfment of Bacillus anthracis spores by macrophages is an important step in the pathogenesis of inhalational anthrax. However, from a quantitative standpoint, the magnitude to which macrophages interact with and engulf spores remains poorly understood, in part due to inherent limitations associated with commonly used assays. To analyze phagocytosis of spores by RAW264.7 macrophage-like cells in a high-throughput, nonsubjective manner, we labeled B. anthracis Sterne 7702 spores prior to infection with an Alexa Fluor 488 amine-reactive dye in a manner that did not alter their germination, growth kinetics, and heat resistance. Using flow cytometry, large numbers of cells exposed to labeled spores were screened to concurrently discriminate infected from uninfected cells and surface-associated from internalized spores. These experiments revealed that spore uptake was not uniform, but instead, highly heterogeneous and characterized by subpopulations of infected and uninfected cells, as well as considerable variation in the number of spores associated with individual cells. Flow cytometry analysis of infections demonstrated that spore uptake was independent of the presence or absence of fetal bovine serum, a germinant that, while routinely used in vitro, complicates the interpretation of the outcome of infections. Two commonly used macrophage cell lines, RAW264.7 and J774A.1 cells, were compared, revealing significant disparity between these two models in the rates of phagocytosis of labeled spores. These studies provide the experimental framework for investigating mechanisms of spore phagocytosis, as well as quantitatively evaluating strategies for interfering with macrophage binding and uptake of spores.

* Corresponding author. Mailing address: Department of Microbiology, B103 CLSL, University of Illinois, 601 South Goodwin, Urbana, IL 61801. Phone: (217) 244-2412. Fax: (217) 244-6697. E-mail: sblanke{at}life.uiuc.edu

{triangledown} Published ahead of print on 13 June 2008.

§ B.S. and E.M.T. contributed equally to this work.

Applied and Environmental Microbiology, August 2008, p. 5201-5210, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.02890-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Gut, I. M., Prouty, A. M., Ballard, J. D., van der Donk, W. A., Blanke, S. R. (2008). Inhibition of Bacillus anthracis Spore Outgrowth by Nisin. Antimicrob. Agents Chemother. 52: 4281-4288 [Abstract] [Full Text]  


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