Polylysine-Mediated Translocation of the Diphtheria Toxin Catalytic Domain through the Anthrax Protective Antigen Pore

Anthrax toxin is a tripartite system consisting of two catalytic moieties, lethal factor (LF) and edema factor (EF),1,2 and a receptor binding/pore forming moiety, protective antigen (PA; MW = 83 kDa).3−6 These three individually nontoxic proteins combine to elicit many of the disease manifestations caused by Bacillus anthracis. After release from the bacteria, PA binds to its cellular receptors7−12 and is cleaved by cell-surface furin to a 63 kDa form (PA63).13,14 PA63 self-assembles to form a heptameric4,5,10 or octameric prepore,15 which then binds the enzymatic LF and EF moieties, yielding a series of complexes at the cell surface.15−18 These complexes are endocytosed,19,20 and exposure to acidic conditions of the endosomal compartment causes the PA prepore to undergo a conformational change to the pore state.3,21,22 The pore, inserted into the endosomal membrane, translocates the LF and EF moieties to the cytoplasm,23−25 where they modify their respective intracellular targets to the benefit of the bacterium. The anthrax toxin system has been studied extensively to learn how a proteinaceous toxin pore is able to translocate a protein across a phospholipid bilayer. Certain heterologous proteins may be potentiated to undergo PA-dependent translocation by fusion with the PA binding26 N-terminal domain of LF (LFn, residues 1–263) or the corresponding domain of EF. Thus, for example, fusing LFn to the N-terminus allows heterologous proteins and peptides (e.g., the catalytic domain of Pseudomonas exotoxin A,27 diphtheria toxin,28 ricin29 or shiga toxin,30 and the cytotoxic T lymphocyte epitope from Listeria monocytogenes(31)) to be delivered to the cytosol via PA. The ability of LFn to potentiate proteins for PA-dependent translocation stems from its ability to bind to the mouth of the PA pore and orient its disordered, highly charged leader into the lumen of the pore.32 N- to C-terminal translocation occurs in vitro in planar bilayers in the presence of a transmembrane pH gradient corresponding to that between the acidic lumen of the endosome and the neutral cytosol,33 and a charge state-dependent Brownian ratchet mechanism has been proposed.34 In cellular assays of PA-dependent translocation, we have often used DTA, the catalytic domain of diphtheria toxin, as a heterologous effector protein, as its delivery to the cytosol may be readily detected by measuring the inhibition of protein synthesis. Many years ago, we observed that DTA with a hexa-His tag at the N-terminus undergoes significant PA-dependent entry into cells, and we then showed that short N-terminal tracts of Lys or Arg, as well as His, also fostered translocation of DTA via the PA pore.35 Consistent with this finding, it has been reported that an N-terminal His6 tag can promote PA-dependent entry of an active domain of the C2 toxin and epidermal cell differentiation inhibitor of Staphylococcus aureus.36 In the study presented here, we fused tracts of up to 15 Lys residues to the N- or C-terminus of DTA and examined their ability to promote translocation of DTA into cells. These studies, together with experiments performed in parallel in planar bilayers, suggest that the PA pore is able to translocate appropriately tagged proteins across membranes in both N to C and C to N direction, although translocation is less efficient in the C to N direction.