Analyzing a novel mechanism of action of bacterial cAMP producing toxins

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DESCRIPTION (provided by applicant): Our recent discovery that key virulence factors from Bacillus anthracis, edema factor (EF), and Vibrio cholerae, cholera toxin (Ctx), both inhibit protein trafficking to cell-cell junctions is paradigm shifting. EF is a highly active adenylate cyclase and Ctx ADP-ribosylates Gs¿ subunits to constitutively activate host adenylate cyclase. These two cAMP producing toxins reduce the levels and activity of a small GTPase (Rab11) required in the final step of endocytic recycling of cell adhesion molecules (e.g., cadherins) and signaling proteins (e.g., Notch components) to cell-cell junctions, resulting in disruption of the vascular endothelium (EF) or intestinal epithelium (Ctx). This novel effect of EF and Ctx was discovered and genetically dissected in the model system Drosophila melanogaster (fruit fly), and these cell biological mechanisms translate to toxin action in human vascular endothelial cells (EF) and intestinal epithelial cells (Ctx) as well as in vivo in mice (both EF and Ctx). Another key finding, with important practical implications, was that over-expression of Rab11 can reverse the junction disrupting effects of EF and Ctx in vivo in flies and in human cells. In the current revised grant, we propose three integrated aims to elucidate the pathways mediating the barrier disruptive actions of EF and Ctx and to analyze the consequences of this novel cell biological mechanism in disease pathogenesis. In Aim 1 we will examine the pathways by which high sustained levels of cAMP produced by EF/Ctx reduce Rab11 protein levels to derail junctional transport and explore new potential functions of EF and Ctx related to inhibition of exocyst function in immune cells. In Aim 2, we will investigate how inhibition of endocytic recycling promotes leakage across human cell monolayers and in the vasculature during anthrax infection. Since vascular collapse is a frequent cause of death in anthrax, we will also determine whether increasing Rab11 levels or treating with known traffic-promoting agents can reverse the vascular leakage caused by EF. In Aim 3, we will similarly examine the contribution of exocyst inhibition to the massive fluid secretion that is pathognomonic of cholera and whether elevating endocytic recycling via genetic or pharmacological means is protective in vivo. The proposed studies have important translational relevance to treating anthrax since toxins can reach critical lethal levels just as patients begin to seek medical intervention, when antibiotics are no longer effective. Thus, treatments based on restoring endocytic recycling could be used in conjunction with existing anti-toxin therapies (e.g., anti-toxin antibodies, small molecule inhibitors) to neutralize toxins already present in the circulation. An advantage of traffic-promoting agents is that they would intervene at the very last step when vascular integrity collapses and other organ systems fail. Such traffic-promoting compounds might also increase the efficacy of fluid replacements to treat cholera and to treat other barrier disruptive diseases including: ischemia, asthma, dermatitis, IBD, cancer, ciliary diseases, and neurodegenerative disorders.