| The lethal factor (LF) component of Bacillus anthracis lethal toxin (LeTx) cleaves mitogen activated protein kinase kinases (MAPKKs) in a variety of different cell-types, yet not all cells are susceptible to the toxin. Previous studies revealed that this toxin rapidly kills macrophages from specific genetic backgrounds whereas most other cell types are resistant. The reason for this selective killing is unclear, but suggests other factors may also be involved in LeTx intoxication. In the current study, DNA membrane arrays were used to identify broad changes in macrophage physiology after treatment with LeTx. Expression of genes, regulated by MAPKK activity did not change significantly, yet a series of genes under glycogen synthase kinase-3-beta (GSK-3beta) regulation changed expression following LeTx treatment. Correlating with these transcriptional changes, GSK-3beta was found to be below detectable levels in toxin-treated cells, and, an inhibitor of GSK-3beta, LiCl, sensitized resistant IC-21 macrophages to LeTx. In addition, zebrafish embryos treated with LeTx showed signs of delayed pigmentation and cardiac hypertrophy; both processes are subject to regulation by GSK-3beta. A putative compensatory response to loss of GSK-3beta was indicated by differential expression of three motor proteins following toxin treatment, and kiflc, a motor protein involved in sensitivity to LeTx, increased expression in toxin-sensitive cells yet decreased in resistant cells following toxin treatment. Differential expression of microtubule associating proteins and a decrease in the level of cellular tubulin were detected in LeTx-treated cells, both of which can result from loss of GSK-3beta activity. In addition to examining the cellular impact of LeTx on macrophages, studies were performed in order to identify additional factors that govern LeTx sensitivity among different cell-types. Specifically, comparisons were made regarding the rate of toxin entry among macrophage and non-macrophage lines. These studies revealed differences in the rate of toxin entry among the cell lines tested, which, in turn, could contribute to the differences in susceptibility of these lines. Together, the data presented in this thesis provide new information on LeTx's overall influence on macrophage physiology, and suggest that loss of GSK-3beta as well as changes in kinesin motor proteins and microtubule stability contributes to cytotoxicity. |
