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TcdB produced by Clostridium difficile is an important member of the class of large clostridial toxins (LCTs) and is a major virulence factor in pseudomembranous colitis (3). Unfortunately, TcdB, which glucosylates Rho, Rac, and Cdc42 (1), and other LCTs have not been fully utilized or thoroughly studied, since expression of recombinant forms of these toxins in Escherichia coli is difficult. Truncated forms of TcdB have been expressed in E. coli, but they are devoid of receptor binding and translocation activity and thus must be microinjected into target cells and cannot be analyzed in animal models (9). To address these problems, we have utilized a previously described (2, 6) translocation-active, yet nontoxic, form of anthrax toxin to deliver the enzymatic domain of TcdB (TcdB_1-556) to the cytosol of mammalian cells. As described below, a truncated form of anthrax toxin lethal factor (LFn) was genetically fused to the enzymatic domain of TcdB and was used in combination with LF's binary partner, protective antigen (PA), to deliver the glucosylating domain to the cytosol of mammalian cells.

Construction, expression, and purification of LFnTcdB_1-556. lfn was genetically fused to tcdB_1-1668 (the region encoding the enzymatic region of TcdB) by cloning the fragment into the BamHI site of pABII, a derivative of pET15b, which contains the lfn gene with a 3' multiple cloning site, to make the plasmid pLMS200. This genetic fusion resulted in joining the 3' end of lfn at the codon TCC encoding S254, followed by sequences within the multiple cloning site which encoded the linker region and a string of residues (PGGGGGS), with the 5' end of tcdB_1-1668 at the ATG codon encoding M1. Candidate clones were transformed into E. coli BL21(DE3) (Stratagene), and using the pET15b-encoded six-His tag, the fusion protein was expressed and purified according to manufacturer's instructions (Novagen, Madison, Wis.). In the purification, LFnTcdB_1-556 consistently eluted in lower concentrations of imidazole (~60 mM) compared to Ni²⁺ affinity isolation of LFn (elutes in ~250 mM imidazole), suggesting preclusion of the six-His tag by the fusion. Purified LFnTcdB_1-556 migrated within the predicted size range (~94 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and was immunoreactive to both anti-LF and anti-TcdB polyclonal antisera (Fig. 1).

View larger version (60K): [in this window] [in a new window]   FIG. 1.   SDS-PAGE and immunoblot analysis of LFnTcdB1-556. LFnTcdB1-556 was expressed and isolated from E. coli BL-21(DE3). (A) Coomassie-stained SDS-PAGE gel showing LFnTcdB1-556 eluted from a Ni2+ affinity column. Lane 1, prestained molecular weight markers; lane 2, purified LFnTcdB1-556. (B) Immunoblot of LFnTcdB1-556 obtained by using rabbit anti-TcdB antiserum. (C) Lane 1, immunoblot of LFnTcdB1-556 obtained by using rabbit anti-LF antiserum; lane 2, prestained molecular weight markers (applies to both panels B and C).

Analysis of LFnTcdB_1-556 substrate specificity and cytopathic effects (CPEs). To determine the substrate specificity of LFnTcdB_1-556, recombinant Rho, Rac, and Cdc42, as well as cell lysates, were used as targets. Recombinant clones of Rho, Rac, and Cdc42 (a generous gift of Alan Hall) were expressed and purified as glutathione S-transferase (GST) fusions from pGEX2-T according to the manufacturer's instructions (Amersham Pharmacia, Piscataway, N.J.). Chinese hamster ovary (CHO)-K1 (American Type Culture Collection, Manassas, Va.) cell extracts were prepared using a previously described method (5). For the glucosylation assay, CHO cell extracts (2 mg/ml) or GST-RhoA, GST-Rac, and GST-Cdc42 (350-µg/ml concentrations of each) were added to a glucosylation mixture containing 50 mM HEPES, 100 mM KCl, 1 mM MnCl₂, 1 mM MgCl₂, 100 µg of bovine serum albumin/ml, 35 µM [¹⁴C]UDP-glucose (308 Ci/mol; ICN Pharmaceuticals Inc., Irvine, Calif.), and 10 µg of TcdB/ml or 15 µg of LFnTcdB_1-556/ml in a final reaction mixture volume of 20 µl. The reaction mixture was incubated for 2 h at 37°C, resolved by SDS-PAGE on a 15% acrylamide gel, and imaged on a Packard electronic autoradiograph instant imager (Packard Instrument Company, Meriden, Conn.) similarly to previously described methods (4, 5). As shown in Fig. 2, LFnTcdB_1-556 is able to glucosylate recombinant forms of each of the Rho proteins and substrates from cell extracts. The glucosylation profiles for LFnTcdB_1-556 and wild-type TcdB were similar, indicating that the fusion maintained substrate specificity similar to that of TcdB. PA and LFn alone did not glucosylate Rho, Rac, Cdc42, or substrates from cell extracts (data not shown).

View larger version (93K): [in this window] [in a new window]   FIG. 2.   LFnTcdB1-556 (A) and TcdB (B) in vitro glucosylation of Rho proteins and CHO cell lysates. LFnTcdB1-556 and TcdB glucosylation activities were compared using GST fusions of Rho, Rac, and Cdc42 and cell lysates as targets. Lane 1, LFnTcdB1-556 plus GST-Cdc42; lane 2, LFnTcdB1-556 plus GST-Rac; lane 3, LFnTcdB1-556 plus GST-RhoA; lane 4, LFnTcdB1-556 plus CHO cell lysates; lane 5, TcdB plus GST-Cdc42; lane 6, TcdB plus GST-Rac; lane 7, TcdB plus GST-RhoA; lane 8, TcdB plus CHO cell lysates.

View larger version (18K): [in this window] [in a new window]   FIG. 3.   Dose-curve response and time course for LFnTcdB1-556 CPEs. CHO cells were treated with LFnTcdB1-556 and a TcdB control to determine the rate and dose of LFnTcdB1-556 intoxication. (A) Dose-curve comparison for CPE induced by LFnTcdB1-556 plus PA and TcdB. (B) Time course of CPE for LFnTcdB1-556 (100 fmol) plus PA (30 pmol) and TcdB (100 fmol).

View larger version (113K): [in this window] [in a new window]   FIG. 4.   Actin organization in LFnTcdB1-556-treated human fibroblast. Human fibroblasts were treated as follows and were stained with rhodamine phalloidin. (A) Phosphate-buffered saline control; (B) PA plus LFn control; (C) TcdB; (D) LFnTcdB1-556 plus PA.