Murine macrophage transcriptional responses to Bacillus anthracis infection and intoxication

Abstract

Interactions between Bacillus anthracis and host macrophages represent critical early events in anthrax pathogenesis, but their details are not clearly understood. Here we report the first genomewide characterization of the transcriptional changes within macrophages infected with B. anthracis and the identification of several hundred host genes that were differentially expressed during this intracellular stage of infection. These loci included both genes that are known to be regulated differentially in response to many other bacterial pathogens and those that appear to be differentially regulated in response to B. anthracis but not other bacterial species that have been tested. These data provide a transcriptional basis for a variety of physiological changes observed during infection, including the induction of apoptosis caused by the infecting bacteria. The expression patterns underlying B. anthracis-induced apoptosis led us to test further the importance of one very highly induced macrophage gene, that for ornithine decarboxylase. Our data show that this enzyme plays an important and previously unrecognized role in suppressing apoptosis in B. anthracis-infected cells. We have also characterized the transcriptional response to anthrax lethal toxin in activated macrophages and found that, following toxin treatment, many of the host inflammatory response pathways are dampened. These data provide insights into B. anthracis pathogenesis as well as potential leads for the development of new diagnostic and therapeutic options.

FIG. 1.

Infection of RAW 264.7 cells by Bacillus anthracis. Murine macrophage-like RAW 264.7 cells are shown untreated (A) and 1 h (B), 3 h (C), and 6 h (D) after inoculation with B. anthracis 34F2 spores at a multiplicity of infection of 10:1.

FIG. 2.

RT-PCR measurement of expression levels for representative genes. A Expression levels for selected genes as measured by DNA microarray. Levels shown reflect induction or repression in cells 3 and 6 h postinfection relative to untreated cells. B RT-PCR analysis of gene expression. RT-PCRs were performed on equal amounts of starting RNA, and control reactions (not shown) demonstrated that there was no contaminating DNA present in the RNA samples.

FIG. 3.

Cell death (necrosis and apoptosis) in B. anthracis infection as measured by enzyme-linked immunosorbent assay. Measurements of necrosis and apoptosis have been normalized in each case to measurements obtained from untreated cells, such that levels of cell death are expressed as the ratio of experimental to untreated cells. Data shown are from representative experiments, and each column is the average of at least three individual measurements. Error bars indicate the standard deviation of each data set.

FIG. 4.

Dampening of immune response genes following treatment with anthrax lethal toxin. Bars indicate the level of induction relative to expression in untreated cells, as measured by DNA microarray. For each gene indicated, induction levels are shown for cells treated with LPS and cells treated with LPS and lethal toxin (LeTx).