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.

Interaction of microbial pathogens with host exocytic pathways

“…The approaches used and overall findings with CT were similar to those with ET (Guichard et al, 2010;Guichard et al, 2013;Guichard et al, 2017) Similar to the situation observed with ET, CT was found to affect the barrier function of host cells, in part, through the cAMP effector EPAC (Guichard et al, 2013). Specifically, the effect of CT on the structure of TJs in Caco-2 cells was reversed by treatment with a chemical inhibitor of EPAC.…”

“…The approaches used and overall findings with CT were similar to those with ET (Guichard et al, ; Guichard et al, ; Guichard et al, ). Ectopic expression of CtxA in Drosophila wings caused a Notch phenotype and also inhibited localisation of Delta, Rab11, Sec15, and DE‐cadherin to AJs.…”

A Drosophila Model for Clostridium difficile Toxin CDT Reveals Interactions with Multiple Effector Pathways

“…We therefore wondered whether EF might also block the effects of CDTa. Alone, EF expression causes wing phenotypes consisting of blistered wings with thickened veins and notched margins ( Figure S2A, and Guichard et al, 2017;Guichard et al, 2006). Consistent with its inhibitory activity on Rab11, EF partially suppressed the CDTa wing phenotype, leading to an enlarged and partially restored wing blade ( Figure S2A, quantified in S2B).…”

“…We previously reported that the anthrax toxin Edema Factor (EF), a potent Adenylate Cyclase, inhibits Rab11 activity, leading to junctional defects (Guichard et al, 2017). We therefore wondered whether EF might also block the effects of CDTa.…”

“…To parse these complex interactions in the context of a live organism, we made use of the powerful genetic model Drosophila melanogaster, a widely successful tool for dissecting pathogenic mechanisms and strategies underlying host defense (Bier and Guichard, 2012;Buchon et al, 2013;Capo et al, 2019). For example, previous studies employed transgenic flies to express bacterial effectors such as Pseudomonas ExoS (Avet-Rochex et al, 2007), Helicobacter VacA (Botham et al, 2008), anthrax toxins (EF and LF) (Guichard et al, 2010;Guichard et al, 2017), or Cholera toxin (Ctx) (Blow et al, 2005) (Guichard et al, 2013) to uncover novel aspects of the host cellular response.…”