Anthrax toxin entry into polarized epithelial cells

Abstract

We examined the entry of anthrax edema toxin (EdTx) into polarized human T84 epithelial cells using cyclic AMP-regulated Cl- secretion as an index of toxin entry. EdTx is a binary A/B toxin which self assembles at the cell surface from anthrax edema factor and protective antigen (PA). PA binds to cell surface receptors and delivers EF, an adenylate cyclase, to the cytosol. EdTx elicited a strong Cl- secretory response when it was applied to the basolateral surface of T84 cells but no response when it was applied to the apical surface. PA alone had no effect when it was applied to either surface. T84 cells exposed basolaterally bound at least 30-fold-more PA than did T84 cells exposed apically, indicating that the PA receptor is largely or completely restricted to the basolateral membrane of these cells. The PA receptor did not fractionate with detergent-insoluble caveola-like membranes as cholera toxin receptors do. These findings have implications regarding the nature of the PA receptor and confirm the view that EdTx and CT coopt fundamentally different subcellular systems to enter the cell and cause disease.

FIG. 1

EdTx elicits an increase in I_sc from T84 cells. (A) Time courses of CT- and EdTx-induced I_sc. All toxins were applied at time zero. EdTx (10 μg of PA per ml and 0.1 μg of EF per ml) was added to either the apical or basolateral chamber. CT (20 nM) was added to the apical chamber. VIP was added to the control monolayers at the end of the experiment to confirm viability. VIP is a cAMP agonist that is used to show that the cells can respond to increases in cAMP. (B) Dose dependency of EdTx action. The indicated concentrations of PA were added to the basolateral chamber of T84 monolayers in the presence of 0.1 μg of EF per ml. Peak I_sc values (means ± standard deviations at steady state ≈97 min after EdTx application, n = 2) are plotted and fit to Michaelis-Menten kinetics. Apparent ED₅₀, 3 μg/ml.

FIG. 2

The source of the EdTx induced current is Cl⁻ transport. PA (10 μg/ml) and EF (0.1 μg/ml) were added to the basolateral chamber of T84 cells in either HBSS or a gluconate buffer which lacked Cl⁻ (see Materials and Methods). For this and the rest of the time courses, time zero was the time at which toxin was added to the cells. Control monolayers not exposed to EdTx were incubated in gluconate buffer. Cl⁻ was added back to the monolayers in gluconate buffer at 48 min (I_sc) (means ± standard deviations n = 3). VIP was added to control monolayers at the end of the experiment to confirm the viability of the monolayers.

FIG. 3

EdTx-induced I_sc depends on cyclic nucleotide, but not Ca²⁺, as a second messenger. Time courses of I_sc induced by 10 μg of PA per ml and 0.1 μg of EF per ml added basolaterally to T84 monolayers pretreated for 30 min in either 3 mM barium or 100 nM charybdotoxin or not pretreated with an inhibitor. Control monolayers were incubated in HBSS alone.

FIG. 4

PA binds to the basolateral but not the apical membrane of T84 cells. PA (10 μg/ml) was bound to T84 monolayers at 4°C for 1 h, and unbound material was washed away. Cell extracts were analyzed for PA by Western blotting. The anti-PA antibody recognized a nonspecific cellular protein with a size of approximately 55 to 60 kDa (indicated by *), which proved useful as an internal control for protein loading. The molecular size markers are indicated on the outside of each blot and represent molecular sizes of 104, 80, 47, and 33.5 kDa. (A) Western blot of extracts from T84 cells that had been treated with PA apically or basolaterally at 4°C or without treatment. PA recovered from cells treated basolaterally was cleaved to its PA63 form, while the PA standard (not exposed to T84 cell surfaces) was not. (B) Dilutions of protein extracts from T84 cells exposed to PA. Extracts from cells treated basolaterally with PA were diluted in protein sample buffer, as indicated above each lane and Western blotted for PA. Undiluted extracts from cells treated apically with PA were run on the same blot. At least 30-fold-less PA was recovered from cells treated apically with PA than from those treated basolaterally.

FIG. 5

Assembly of PA in T84 cells. PA was bound to T84 cells at 4°C and then incubated at 37°C for the indicated times. A Western blot of cell extracts is shown. Three forms of PA could be detected in the proteins recovered from the basolateral membrane of cells incubated at 37°C: full-length PA (PA83), nicked PA (PA63), and a high-molecular-weight band representing the PA oligomer (indicated by an arrow). No toxin was recovered from the apical membrane.

FIG. 6

Association of the CT-GM1 and PA-receptor complexes with caveola-like membrane domains. Sucrose gradient of extracts from T84 cells were exposed apically to CT B subunits (CTB, 20 nM) or basolaterally to PA (PA83, 20 nM) and analyzed for CT B subunits or PA by Western blotting. Only fractions 10 to 20, which correspond to a linear sucrose gradient from 15 to 32% sucrose (top to bottom), are shown. Less than 1% of the total cellular protein floated into the gradient (fractions 11 and 12, representing 18.2 to 22.8% sucrose). This fraction contains caveolin 1 (40).