Entry of B. anthracis Spores Figure 7. A working model of the mechanism of B. anthracis spore internalization by epithelial cells. B. anthracis spores interact with an unknown cell surface receptor. The signal is transduced via a pathway including c-Src, class IA PI3K, and the Rho-family GTPase Cdc42. Eventually F- actin polymerization is activated locally around spore attachment sites, leading to spore internalization. doi:10.1371/journal.pone.0011665.g007 The results also provided evidence indicating that the signaling pathway elucidated here is specifically required for spore entry into epithelial cells but not macrophages. This is not surprising considering that the cellular receptors mediating spore entry into these two types of cells are almost certain to be distinct. Oliva et al. reported that integrin aMb2 (Mac-1) was the receptor mediating phagocytosis of wild type spores by macrophages [26]. Since Mac- 1 is not expressed in epithelial cells, a different receptor should be required for spore uptake by epithelial cells. Studies to identify the epithelial receptor are currently underway in our laboratory. The results from the transwell assays indicate that inhibition of Src-mediated spore entry dramatically reduced B. anthracis dissemination through epithelial cells. This led us to investigate if inhibition of Src would have a similar effect on dissemination in vivo. Although knockout mice are commonly used to examine the importance of a host factor in pathogenesis, c-src2/2 mice show a number of abnormalities such as a small body size, a partial absence of bone marrow, abnormal decidualization and in some genetic background post-natal lethality (Jackson Laboratories http://jaxmice.jax.org). Therefore they were not chosen for this study due to the consideration that these abnormalities may complicate the interpretation of results. Instead, we used the SFK inhibitor SU6656. This inhibitor only inhibits four members of the SFK family, Src, Fyn, Yes and Lyn and thus is far more selective than PP1 or PP2, the other two commonly used SFK inhibitors [27]. The results showed that SU6656 treatment at the indicated dosage significantly reduced bacterial dissemination from the lung to the spleen and blood, suggesting that Src activity is important for bacterial dissemination in vivo. The observation that SU6656 did not significantly affect bacterial counts in the lungs of i.n. inoculated mice, in the spleen of i.p. inoculated mice, or in the blood of i.v. inoculated mice suggests that under the experimental conditions used in these studies SU6656 treatment does not affect the ability of the host to clear the pathogen, the ability of phagocytes to the pathogen to lymphoid tissues, or pathgoen growth in vivo. Thus the reduction of bacterial counts in the spleen and blood of SU6656-treated, i.n. inoculated mice is likely due to a disruption in the dissemination process from the lung to other organs rather than due to non-specific effects of the inhibitor. The SU6656 treatment did not completely abolish dissemination. This is likely due to incomplete inhibition of the kinase activity by the inhibitor and/or dissemination via other routes that do not require this signaling pathway. The effect of SU6656 treatment on mouse survival is consistent with its effect on dissemination. At the dose used, SU6656 significantly improved the survival of i.n. inoculated mice by prolonging the median survival time, whereas it did not affect the survival of i.p. inoculated mice. Death was not prevented since dissemination was not completely blocked. Together these results provide evidence suggesting Src-dependent spore entry into epithelial cells is important for dissemination and virulence in vivo. Combined with the previous finding that lung epithelial cells rather than macrophages or neutrophils are responsible for bacterial lysate-induced host resistance to B. anthracis [11], the data highlights the need for more studies to understand the molecular details of spore-epithelium interactions and their impact on bacterial pathogenesis and host defense. It should be noted that lung epithelial cells may not be the only cell type affected by SU6656 treatment in vivo. Despite all the control animal experiments, the possibility that the effect of SU6656 treatment on dissemination and survival is due to altered activities in other cell types cannot be excluded. Further tests in mouse models lacking the specific epithelial receptor will permit more targeted examination of the complex interplays between the pathogen and the lung epithelium. That disruption of SFK activities can impair dissemination of microbial pathogens was previously described for Streptococcus pneumoniae, although in a somewhat different context. S. pneumoniae was shown to migrate across respiratory epithelial cells by hijacking the host transcytosis pathway for transporting polymeric immunoglobulin receptor (pIgR) across polarized epithelial cell [28]. This transcytosis pathway is mediated by the SFK member c- Yes. In c-yes2/2 mice, dissemination of S. pneumoniae from the respiratory system to the blood was delayed compared to that in the wild-type mice [28]. Our results from the SFK inhibitor PLoS ONE | www.plosone.org 9 July 2010 | Volume 5 | Issue 7 | e11665