Anthrax lethal toxin (LT) is a binary intracellular bacterial toxin that specifically targets macrophages and cleaves mitogen-activated map kinase kinases (MAPKKs). However, it is not clear how this inactivation of MAPKKs is linked to the physiology of targeted macrophages, which over-produce IL-1 and TNF. Clearly, analysis of the overall toxic effects of LT is needed. In these studies we propose to use DNA array and proteomic technologies to examine the physiology of LT-targeted macrophages. In the first aim of this study we will generate cDNA arrays from mouse macrophages. These arrays will then be used in the second aim to profile transcriptional differences between LT treated and normal macrophages. Also, in the second specific we will use proteomics to determine which proteins may be targeted and modified in LT treated macrophages. Through a series of important controls, we will dissect these events and determine which are linked to inactivation of MAPKKs, receptor binding, endocytosis and LT cytosolic activity. In the final aim we will use LT mutants to determine if this toxin has effects which may not be linked to proteolysis of MAPKKs. Traditionally, intracellular toxins have been studied at the single target level, and many related cellular events have been ignored. Using DNA arrays and proteomics we will, for the first time, summarized the global effects an intracellular bacterial toxin has on target cell physiology.
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