Anthrax lethal factor and edema factor act on conserved targets in Drosophila

Abstract

Many bacterial toxins act on conserved components of essential host-signaling pathways. One consequence of this conservation is that genetic model organisms such as Drosophila melanogaster can be used for analyzing the mechanism of toxin action. In this study, we characterize the activities of two anthrax virulence factors, lethal factor (LF) and edema factor, in transgenic Drosophila. LF is a zinc metalloprotease that cleaves and inactivates most human mitogen-activated protein kinase (MAPK) kinases (MAPKKs). We found that LF similarly cleaves the Drosophila MAPK kinases Hemipterous (Hep) and Licorne in vitro. Consistent with these observations, expression of LF in Drosophila inhibited the Hep͞c-Jun N-terminal kinase pathway during embryonic dorsal closure and the related process of adult thoracic closure. Epistasis experiments confirmed that LF acts at the level of Hep. We also found that LF inhibits Ras͞MAPK signaling during wing development and that LF acts upstream of MAPK and downstream of Raf, consistent with LF acting at the level of Dsor. In addition, we found that edema factor, a potent adenylate cyclase, inhibits the hh pathway during wing development, consistent with the known role of cAMP-dependent PKA in suppressing the Hedgehog response. These results demonstrate that anthrax toxins function in Drosophila as they do in mammalian cells and open the way to using Drosophila as a multicellular host system for studying the in vivo function of diverse toxins and virulence factors.mitogen-activated protein kinase kinase ͉ c-Jun terminal kinase ͉ downstream of raf1 ͉ development ͉ cAMP-dependent PKA

Bacillus anthracis Edema Toxin Activates Nuclear Glycogen Synthase Kinase 3β

“…The transcription factor cAMP response binding protein (CREB), which is a downstream target of cAMP signaling activated via phosphorylation by protein kinase A (PKA) (31), is activated in macrophages exposed to ET (19,22,27). Conversely, using a Drosophila model, Guichard and colleagues found that EF activates hedgehog signaling in a PKA-dependent manner (14). The possible impact of ET intoxication on mitogen-activated protein kinases is not fully resolved, as Paccani et al also reported that ET can inhibit mitogen-activated protein and stress kinase responses in T lymphocytes (26); however, in a separate study, Comer et al did not detect a similar effect in T lymphocytes isolated from mice treated with ET (5).…”

Bacillus anthracis toxins

“…A zebrafish model of vascular collapse also implicated endothelial cells as the target for LT action [301]. In Drosophila, overexpression of either toxin inhibited Rab11/Sec15 endocytic recycling and the Notch signaling [302,303] that is required for cadherin expression at cell junctions, possibly suggesting increased permeability due to altered cell-to-cell junctions.…”

Fighting anthrax with flies

“…Guichard et al (3) report that there are no receptors onto which PA anthrax toxin can bind encoded in the Drosophila genome. However, it is clear from this study that Drosophila can still be used for studying the effects of the anthrax toxins.…”

“…Most importantly, Guichard et al's study (3) shows that LF and EF function on evolutionarily conserved pathways and provides the platform for secondsite modifier genetic screens. Overexpression of toxins in tissues dispensable for viability of the fly, such as the eye or the wing, create a sensitized genetic background in which mutations that will suppress and͞or enhance the toxins' effect can be readily identified.…”