Transcriptional profiling of Bacillus anthracis during infection of host macrophages

Abstract

The interaction between Bacillus anthracis and the mammalian phagocyte is one of the central stages in the progression of inhalational anthrax, and it is commonly believed that the host cell plays a key role in facilitating germination and dissemination of inhaled B. anthracis spores. Given this, a detailed definition of the survival strategies used by B. anthracis within the phagocyte is critical for our understanding of anthrax. In this study, we report the first genome-wide analysis of B. anthracis gene expression during infection of host phagocytes. We developed a technique for specific isolation of bacterial RNA from within infected murine macrophages, and we used custom B. anthracis microarrays to characterize the expression patterns occurring within intracellular bacteria throughout infection of the host phagocyte. We found that B. anthracis adapts very quickly to the intracellular environment, and our analyses identified metabolic pathways that appear to be important to the bacterium during intracellular growth, as well as individual genes that show significant induction in vivo. We used quantitative reverse transcription-PCR to verify that the expression trends that we observed by microarray analysis were valid, and we chose one gene (GBAA1941, encoding a putative transcriptional regulator) for further characterization. A deletion strain missing this gene showed no phenotype in vitro but was significantly attenuated in a mouse model of inhalational anthrax, suggesting that the microarray data described here provide not only the first comprehensive view of how B. anthracis survives within the host cell but also a number of promising leads for further research in anthrax.

FIG. 1.

Gel electrophoresis of RNA samples isolated from B. anthracis-infected RAW 264.7 cells. Lane A contained a typical sample of total RNA isolated from an infected culture using a simple extraction procedure, and only the eukaryotic 28S and 18S rRNA species are visible (arrows indicate eukaryotic [28S and 18S] and prokaryotic [23S and 16S] rRNA bands). Lane B contained a sample prepared using the differential lysis-based protocol described in the text, and both eukaryotic and prokaryotic rRNA bands are visible. Lane C contained RNA prepared like that in lane B and further purified using the MicrobEnrich kit (Ambien) to remove eukaryotic RNA. All lanes contained equivalent amounts of RNA (roughly 5 μg).

FIG. 2.

Sample distance matrix showing the levels of relatedness (as measured by Pearson correlation) between samples isolated from B. anthracis growing within murine macrophages at 1, 2, 3, 4, 5, and 6 h postinfection. Samples are organized in the same order in both rows and columns, and the square at the intersection of a given row and a given column is shaded according to the relatedness between the samples (the diagonal is blank, since it represents each sample's comparison to itself). Black indicates a very high degree of similarity between two samples, and progressively lighter shades of gray indicate lower levels of relatedness. White squares indicate a Pearson correlation of <0.6.

FIG. 3.

Survival of mice infected with B. anthracis parent (Sterne 34F₂) and mutant (ΔGBAA1941) strains. DBA/2 mice were inoculated via intratracheal injection of 1.5 × 10⁴ spores of either the parent or mutant strain. The total initial group sizes were 27 mice (parent) and 26 mice (mutant), and the percentage of each group surviving is shown relative to time. A log rank test showed that the difference between the two survival curves is statistically significant, with a P value of 0.0003.