Figure 6.

5′-Nucleotidase activity of AdsA enhances B. anthracis survival in blood. (A, top) Mutanolysin extracts from B. anthracis strain Sterne (WT) or its isogenic adsA variant were incubated with radiolabeled [¹⁴C]AMP. The generation of adenosine was measured by TLC. (bottom) Quantification of relative abundance of adenosine. Data are the mean of three independent analyses; error bars indicate SEM. (B) Mutanolysin extracts were analyzed by immunoblotting with antibodies directed against B. anthracis AdsA or BasC (anti-BasC), a control protein not involved in adenosine production. Bars, 5 µm. (C) Fluorescence microscopy images of wild-type B. anthracis Sterne and its isogenic adsA mutant stained with antiserum against B. anthracis AdsA (top) or nonreactive serum (NRS) and Cy3-labeled secondary antibodies (red), as well as Hoechst staining of nucleic acids (blue). Data are representative of two independent analyses. (D) Radiolabeled [¹⁴C]AMP was incubated with 2 µM of purified B. anthracis AdsA in the presence of 5 mM of the indicated metal cations, and generation of [¹⁴C]adenosine ([¹⁴C]Ado) was measured by TLC and a PhosphorImager. Data are representative of three independent analyses. (E) Survival of wild-type and adsA B. anthracis Sterne in rat blood over time, measured as CFUs on agar plates. Data are the mean of two independent analyses; error bars indicate SEM.