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. 2010 Oct 14;467(7317):854-8.
doi: 10.1038/nature09446.

Anthrax Toxins Cooperatively Inhibit Endocytic Recycling by the Rab11/Sec15 Exocyst

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Free PMC article

Anthrax Toxins Cooperatively Inhibit Endocytic Recycling by the Rab11/Sec15 Exocyst

Annabel Guichard et al. Nature. .
Free PMC article

Abstract

Bacillus anthracis is the causative agent of anthrax in humans and other mammals. In lethal systemic anthrax, proliferating bacilli secrete large quantities of the toxins lethal factor (LF) and oedema factor (EF), leading to widespread vascular leakage and shock. Whereas host targets of LF (mitogen-activated protein-kinase kinases) and EF (cAMP-dependent processes) have been implicated in the initial phase of anthrax, less is understood about toxin action during the final stage of infection. Here we use Drosophila melanogaster to identify the Rab11/Sec15 exocyst, which acts at the last step of endocytic recycling, as a novel target of both EF and LF. EF reduces levels of apically localized Rab11 and indirectly blocks vesicle formation by its binding partner and effector Sec15 (Sec15-GFP), whereas LF acts more directly to reduce Sec15-GFP vesicles. Convergent effects of EF and LF on Rab11/Sec15 inhibit expression of and signalling by the Notch ligand Delta and reduce DE-cadherin levels at adherens junctions. In human endothelial cells, the two toxins act in a conserved fashion to block formation of Sec15 vesicles, inhibit Notch signalling, and reduce cadherin expression at adherens junctions. This coordinated disruption of the Rab11/Sec15 exocyst by anthrax toxins may contribute to toxin-dependent barrier disruption and vascular dysfunction during B. anthracis infection.

Figures

Figure 1
Figure 1. LF and EF synergistically inhibit Notch signaling
a–d) Wings (upper panels) and corresponding wg expression in wing imaginal discs (bottom panels) of the following genotypes: a) wild-type (wt), b) wkG4>LF2X (wkG4 refers to the 1348-GAL4 driver), c) wkG4>EF (wing has A/P patterning phenotype superimposed upon thickened veins - arrowhead - and an occasional small notch at the wing margin), d) wkG4>LF2X+EF. e–g) Reticular pattern of Delta (Dl) staining in wing discs with accompanying Z-sections. The stgG4 driver is expressed at higher levels on the dorsal - D - surface than on the ventral - V - surface. e) Wild-type Dl expression has both cell surface (bracket) and vesicle-like intracellular components, and is expressed along the future margin (M) in two parallel lines as well as in vein primordia (which intersect the margin in perpendicular stripes), f) stgG4>LF, g) stgG4>EF. h–o) wings of the following genotypes: h) stgG4>LF, i) stgG4>EF (arrowhead indicates thickened veins), j) stgG4>LF+EF, k) stgG4>Rab11DN, l) stgG4>Rab11wt, m) stgG4>EF+Rab11wt, n) stgG4>EF+Rab11DN, o) stgG4>LF+Rab11DN.
Figure 2
Figure 2. LF and EF inhibit Rab11/Sec15-dependent recycling
a–c) Endogenous Rab11 expression in wing imaginal discs detected by immunofluorescence. Insets are Z-sections from regions of the same discs in these and other panels (brackets indicate the cell surface. a) Wild-type, b) stgG4>EF, c) stgG4>LF disc. d–f) DECad expression detected by immunofluorescence. d) Wild-type (DECad and Dl co-stain in a net-like pattern at points of cell-cell contact - Supplementary Fig. 4a–c), e) stgG4>EF discs, f) stgG4>Rab11DN. g–l) Expression of a UAS-Sec15-GFP construct driven by the stgG4 driver. g) Wild-type - stgG4>Sec15-GFP (large cell surface vesicles of Sec15-GFP co-localize with Rab11 - arrows), h) stgG4>EF+Sec15-GFP, h) stgG4>Rab11DN+Sec15-GFP (vesicular Sec15-GFP expression is similarly dependent on Rab11=Sec4 function in yeast), k) stgG4>EF+ Rab11wt+Sec15-GFP. l) stgG4>LF+Sec15-GFP. Staining differences are quantitated in Supplementary Table 2.
Figure 3
Figure 3. Conserved activity of anthrax toxins in mammals
a–c) Pan-cadherin (pCad) staining (blue) in hBMEC transfected with a human Sec15-GFP construct (green). a) Untreated cells. b) Cells treated with EF toxin (= 3 µg EF + 6 µg PA) for 24 hours. c) Cells treated with LF toxin (= 3 µg LF + 6 µg PA) for 24 hours. Treatment with doses ranging from 0.3 µg to 3 µg gave similar results. d–k) Rescue of Sec15-GFP (green) expression by Rab11-RFP (red) in EF toxin treated hBMEC (0.3 µg EF + 0.6 µg PA): d–g, untreated cells, and h–k, cells treated with 0.3 µg EF + 0.6 µg PA. d,h) Cells transfected with Sec15-GFP alone. e–g, i–k) cells co-transfected with sec15-GFP and wild-type Rab11-RFP. Cells in panels d–k were also stained for pCad (blue). l,m) Expression of pCad (blue) and Dll4 (magenta) in untreated primary human dermal microvascular endothelial cells (1° Dermal MEC = hDMEC) (l), or after treatment with 1 µg EF + 2 µg PA (m). n) Semiquantitative analysis of Hes1 and GapDH RNA expression in hBMEC. First set of lanes: hBMEC infected with B.a. or isogenic mutants: UI = uninfected control; WT = B.a. Sterne bacteria; ΔpX01 = B.a. lacking the pX01 plasmid; ΔLF = B.a. with deletion of LF; ΔEF = B.a. with deletion of EF; ΔLF/ΔEF = B.a. with deletion of both LF and EF. Second set of lanes: effect of the γ-secretase inhibitor DBZ (2 µM) or vehicle control (DMSO) on Hes1 expression in hBMEC. Staining differences are quantitated in Supplementary Table 2. o) Transwell permeability assay of hBMEC cells grown to confluence in a transwell chamber and infected with B.a. WT or isogenic toxin mutants. Leakage across the monolayer was determined 6 hours later by measuring Evans Blue leakage to the bottom chamber at OD 620. Abbreviations for genotypes of bacteria are as in panel (n). Mean and Standard Deviation (represented by error bars) of a representative experiment are shown. p) Vascular effusion in response to subcutaneous infection with B.a. (wt B.a.) or isogenic toxin mutants ΔLF or ΔEF. Effusion was visualized by Evans blue dye leakage (Supplemental Fig. 9d). q) Quantitation of vascular permeability shown in Fig. 3p. ***, p < 0.001. r) Proposed schematic model for the convergent activity of EF and LF on the exocyst. EF reduces Rab11 levels/activity, which indirectly inhibits formation of Sec15 exocyst complexes, while LF acts more directly on Sec15. The combined effect of these two toxins is to reduce cell surface expression of the Notch ligand Dl and cadherins at AJs, and possibly other AJ proteins involved in cell-cell adhesion and barrier maintenance, thereby compromising vascular integrity.

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