Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

Abstract

Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from Bacillus anthracis, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of Drosophila and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and in vivo. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and in vivo, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.

Author summary

Recent anthrax outbreaks in Zambia and northern Russia and biodefense preparedness highlight the need for new therapies to counteract fatal late-stage pathologies in patients infected with Bacillus anthracis. Indeed, two toxins secreted by this pathogen—edema toxin (ET) and lethal toxin (LT)—can cause death in face of effective antibiotic treatment. ET, a potent adenylate cyclase, severely impacts host cells and tissues through an overproduction of the ubiquitous second messenger cAMP. Previously, we identified Rab11 as a key host factor inhibited by ET. Blockade of Rab11-dependent endocytic recycling resulted in the disruption of intercellular junctions, likely contributing to life threatening vascular effusion observed in anthrax patients. Here we present a multi-system analysis of the mechanism by which EF inhibits Rab11 and exocyst-dependent trafficking. Epistasis experiments in Drosophila reveal that over-activation of the cAMP effectors PKA and Epac/Rap1 interferes with Rab11-mediated trafficking at two distinct steps. We further describe conserved roles of Epac and the small GTPase Arf6 in ET-mediated disruption of vesicular trafficking and show how chemical inhibition of either pathway greatly alleviates ET-induced edema. Thus, our study defines Epac and Arf6 as promising drug targets for the treatment of infectious diseases and other pathologies involving cAMP overload or related barrier disruption.