B. anthracis translocation across a lung epithelial barrier requires Src activity It was previously shown that B. anthracis could translocate across an A549 cell barrier without apparent disruption of the barrier integrity, suggesting that translocation occurred via an intracellu- lar route [1]. We tested if inhibiting Src activity would reduce bacterial translocation. A549 cells grown on transwell inserts were treated with PP2, SU6656, PP3, or no inhibitor. Spores were added to the apical side of A549 cells and incubated in the presence of the inhibitor. Significantly less bacteria were recovered from the bottom chambers of cells treated with PP2 and SU6656 respectively, compared to the control (Fig. 5, A, ***, p,0.001). PP3, a negative control compound for PP2, did not have any effect on translocation (Fig. 5, A). The inhibitor treatment did not compromise the A549 cell barrier integrity as assessed by FITC- dextran diffusion from the top to the bottom chambers in the inhibitor-treated and the control wells (Fig. 5, B). To rule out the possibility that the inhibitors affected vegetative growth of B. anthracis, which could have resulted in fewer bacteria in the bottom chambers, we examined the effect of the inhibitors on bacterial growth in the tissue culture conditions. No difference was observed (Fig. 5, C). Together, the above results indicate that Src activity is required for B. anthracis translocation across a barrier of lung epithelial cells. Inhibition of Src activity reduces bacterial dissemination in a mouse model of inhalational anthrax In pilot experiments, A/J mice were inoculated with ,1–36106 spores/mouse by intranasal instillation (i.n.). Bacterial counts in the spleen were determined at 24, 48 and 72 hr post inoculation. Few bacteria were found in the spleens at 24 or 48 hr. However, substantial amounts of bacteria were recovered at 72 hr. We also found that 72 hr is the approximate median survival time for mice infected with the indicated spore dose via the i.n. route. To determine if inhibition of Src activity would affect bacterial dissemination in vivo, mice were treated with either SU6656 (7.5 mg/g body weight) [24] or an equivalent volume of solvent by daily i.p. injection as described in Materials and Methods. Mice were inoculated with spores via the i.n. route and bacterial dissemination was examined at 72 hr post inoculation. Signifi- cantly fewer bacteria were recovered from the spleen and blood of Entry of B. anthracis Spores SU6656-treated mice compared to those from the control mice (*, p,0.05) (Fig. 6, A and B). In contrast, there was no significant difference in the lung bacterial counts between the treated and the control groups (Fig. 6, C), suggesting that SU6656 treatment did not affect bacterial survival in the lung. To further examine whether the reduction in bacterial burden could be due to non- specific effects of SU6656, spores were directly injected into the peritoneum (i.p.) or the tail vein (i.v.) of mice. The appropriate spore dose and the median survival time for the i.p. and i.v. routes of infection were determined in pilot experiments. The spleen bacterial burden in i.p. inoculated mice were essentially the same between SU6656-treated and the control mice at 72 hr post inoculation (, median survival time) (Fig. 6, D). For i.v. inoculated mice, blood were collected at 84 hr post inoculation (, median survival time) and dilution plated. No difference was observed in the blood bacterial counts between the treated and the control groups (Fig. 6, E), suggesting that SU6656 did not affect bacterial survival or growth in the blood. The effect of the inhibitor on mouse survival was also tested. The results showed that although SU6656 treatment did not prevent death, it prolonged mouse survival time (Fig. 6, F, p,0.05, compared with the control group). The median survival time was increased by an average of 12 hr with the inhibitor treatment (84 hr for the treatment group vs. 72 hr for the control group, calculated from 4 independent experiments with a total of 37 mice per group). In contrast, in i.p. inoculated mice, there was no difference in the survival curves between the SU6656-treated and the control groups (Fig. 6, G). Together these results strongly suggest that Src activity is important for bacterial dissemination through the respiratory epithelium and virulence in vivo. Discussion The airway and lung epithelium not only functions as a mechanical barrier to inhaled pathogens but also actively participates in host immune responses such as pathogen recognition and production of cytokines, chemokines and anti- microbial peptides. It is thus an important component in the host defense system against microbial pathogens. On the other hand, to establish an inhalational anthrax infection, B. anthracis must breach the respiratory epithelial barrier. Earlier studies suggested that B. anthracis spores potentially could use lung epithelial cells in addition Figure 5. Src activity is required for B. anthracis translocation through an A549 cell barrier. A549 cells grown in transwell inserts were pre- treated with PP2 (10 mM), PP3 (10 mM), and SU6656 (50 mM) or solvent (Ctrl) for 1 hr. Spores were then added to the top chambers as described in Materials and Methods. Media was collected from the bottom chambers after 16 hr of incubation. An aliquot of the media was dilution plated to determine bacterial counts (A). Another aliquot was used to measure fluorescence emission at 520 nm (FITC-dextran) (B). The effect of the inhibitors on bacterial growth was also determined (C) as described in Materials and Methods. The results shown are mean 6 SEM from at least two independent experiments. ***, p,0.001, t test. doi:10.1371/journal.pone.0011665.g005 PLoS ONE | www.plosone.org 7 July 2010 | Volume 5 | Issue 7 | e11665