Toxin B from Clostridium difficile is a monoglucosylating toxin that targets substrates within the cytosol of mammalian cells. In this study, we investigated the impact of acidic pH on cytosolic entry and structural changes within toxin B. Bafilomycin A1 was used to block endosomal acidification and subsequent toxin B translocation. Cytopathic effects could be completely blocked by addition of bafilomycin A1 up to 20 min following toxin treatment. Furthermore, providing a low extracellular pH could circumvent the effect of bafilomycin A1 and other lysosomotropic agents. Acid pH-induced structural changes were monitored by using the fluorescent probe 2-(p-toluidinyl) naphthalene-6-sulfonic acid, sodium salt (TNS), inherent tryptophan fluorescence, and relative susceptibility to a specific protease. As the toxin was exposed to lower pH there was an increase in TNS fluorescence, suggesting the exposure of hydrophobic domains by toxin B. The change in hydrophobicity appeared to be reversible, since returning the pH to neutrality abrogated TNS fluorescence. Furthermore, tryptophan fluorescence was quenched at the acidic pH, indicating that domains may have been moving into more aqueous environments. Toxin B also demonstrated variable susceptibility to Staphylococcus aureus V8 protease at neutral and acidic pH, further suggesting pH-induced structural changes in this protein.The large clostridial toxins are a unique class of virulence factors produced by at least three pathogenic clostridial species. Clostridium difficile produces toxins A and B, Clostridium novyi produces alpha-toxin, and Clostridium sordellii produces lethal toxin and hemorrhagic toxin. These toxins not only are unique because of their exceptionally large size (ranging from 260 to 308 kDa) but also demonstrate a novel enzymatic activity (1, 3, 4). Each of these toxins targets members of the Ras superantigen of GTPases by acting as a glycosyltransferase. Toxin A, toxin B, lethal toxin, and hemorrhagic toxin all use UDP-glucose as a cosubstrate, whereas alpha-toxin uses UDPGlcNAc to modify targets.The mechanism of action for C. difficile toxins A and B is of particular interest since this organism causes pseudomembranous colitis, a serious human disease usually occurring in hospitalized patients undergoing antibiotic therapy (2). C. difficile toxin A acts as an enterotoxin and is considered to be the major contributor to the intestinal damage caused by C. difficile. Toxin B is an effective cytotoxin that demonstrates less cell tropism than toxin A and is responsible for systemic intoxication (1).While there has been significant progress in identifying the enzymatic action of these toxins, little is known about how these proteins translocate to the cytosol of target cells. By definition, intracellular bacterial toxins must cross the target cell membrane in order to enter the interior of the target cell. The most common means of accomplishing this appears to be via a three-step process: (i) receptor binding, (ii) receptortriggered endocytosis, and (iii)...

SummaryClostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxication of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase-dependent and caspase-independent apoptosis. The apoptotic pathways became evident when caspase-3-processed-vimentin was detected in TcdBtreated HeLa cells. Caspase-3 activation was subsequently confirmed in TcdB-intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB-induced cell death, but did not alter the time-course of cytopathic effects. A similar effect was also observed in MCF-7 cells, which are deficient in caspase-3 activity. The time-course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB 1-556 ). Unlike TcdB treated cells, intoxication with TcdB 1-556 or expression of TcdB 1-556 in a transfected cell line, did not stimulate caspase-3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB 1-556 treated cells did not demonstrate caspase-3 activation these cells were apoptotic as determined by differential annexin-V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase-independent apoptosis as a result of substrate inactivation and may evoke caspasedependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.

TcdB from Clostridium difficile glucosylates small GTPases (Rho, Rac, and Cdc42) and is an important virulence factor in the human disease pseudomembranous colitis. In these experiments, in-frame genetic fusions between the genes for the 255 amino-terminal residues of anthrax toxin lethal factor (LFn) and the TcdB 1-556 coding region were constructed, expressed, and purified from Escherichia coli. LFnTcdB 1-556 was enzymatically active and glucosylated recombinant RhoA, Rac, Cdc42, and substrates from cell extracts. LFnTcdB 1-556 plus anthrax toxin protective antigen intoxicated cultured mammalian cells and caused actin reorganization and mouse lethality, all similar to those caused by wild-type TcdB.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 3Ј multiple cloning site, to make the plasmid pLMS200. This genetic fusion resulted in joining the 3Ј 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 5Ј 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 (ϳ60 mM) compared to Ni 2ϩ affinity isolation of LFn (elutes in ϳ250 mM imidazole), suggesting preclusion of the six-His tag by the fusion. Purified LFnTcdB 1-556 migrated within the predicted size range (ϳ94 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and was immunoreactive to both anti-LF...

Clostridium sordellii lethal toxin (TcsL) is a large clostridial toxin (LCT) that glucosylates Ras, Rac, and Ral. TcsL differs from other LCTs because it modifies Ras, which does not cycle from cytosol to membrane. By using a suite of inhibitors, steps in cell entry by TcsL were dissected, and entry appears to be dependent on endosomal acidification. However, in contrast to TcdB, TcsL was substantially slower in its time course of entry. TcsL cytopathic effects (CPE) were blocked by bafilomycin A1 and neutralized by antiserum up to 2 h following treatment of cells with the toxin. The slow time course of intoxication and relatively high cytopathic dose were alleviated by exposing TcsL to acid pH, resulting in a time course similar to that of TcdB. The optimal pH range for activation was 4.0 to 5.0, which increased the rate of intoxication over 5-fold, lowered the minimal intoxicating dose by over 100-fold, and allowed complete substrate modification within 2 h, as shown by differential glucosylation. Fluorescence analysis of TcsL with 2-(p-toluidinyl) naphthalene-6-sulfonic acid as a probe suggested the acid pH stimulated a hydrophobic transition in the protein, a likely prelude to membrane insertion. Finally, acid entry by TcsL caused TcdB-like morphological changes in CHO cells, which suggestings that acid activation may impact substrate recognition profiles for TcsL.

The enzymatic moieties of anthrax toxin enter the cytosol of mammalian cells via a pore in the endosomal membrane formed by the protective antigen (PA) moiety. Pore formation involves an acidic pH-induced conformational rearrangement of a heptameric precursor (the prepore), in which the seven 2␤2-2␤3 loops interact to generate a 14-strand transmembrane ␤-barrel. To investigate this model in vivo, we labeled PA with the fluorophore 7-nitrobenz-2-oxa-1,3-diazole (NBD) at cysteine residues introduced into the 2␤2-2␤3 loop. Each labeled PA was bound to CHO cells, and NBD fluorescence was monitored over time in stirred cell suspensions or by confocal microscopy. A strong increase was observed with NBD at positions 305, 307, 309, and 311, sites where side chains are predicted to face the bilayer, and little change was seen at residues 304, 306, 308, 310, and 312, sites where side chains are predicted to face the pore lumen. The increase at position 305 was inhibited by membrane-restricted quenchers, low temperature, or various reagents known to affect toxin action. Of the 24 NBD attachment sites examined, all but three gave results qualitatively consistent with the ␤-barrel model. Besides supporting the ␤-barrel model of membrane insertion, our results describe the time course of insertion and identify PA residues where NBD gives a strong signal upon membrane insertion in vivo.The pathogenesis of Bacillus anthracis depends on two major virulence factors: an antiphagocytic poly(D-glutamic acid) capsule and a toxin that is believed to be responsible for the major symptoms of anthrax (8, 12). Anthrax toxin belongs to the binary bacterial toxins, a class of intracellularly acting toxins in which the enzymatic (A) and receptor-binding (B) proteins are released from the bacteria as separate nontoxic proteins. The A and B proteins then combine to form toxic noncovalent complexes on receptor-bearing mammalian cells. Anthrax toxin has two separate enzymatic A moieties: edema factor (EF; 89 kDa), a calmodulin-and Ca 2ϩ -dependent adenylate cyclase (13,29), and lethal factor (LF; 90 kDa), a Zn 2ϩ -dependent protease that cleaves most mitogen-activated protein kinase kinases (9, 30). EF and LF bind competitively to an activated form of protective antigen (PA; 83 kDa), the receptor-binding and pore-forming B moiety of anthrax toxin. PA then delivers EF and LF to the cytosol, where they modify their substrates.The interaction of PA, LF, and EF occurs after PA binds to either of two receptors at the cell surface (7, 24) and is proteolytically activated (20). A cellular protease of the furin family cleaves receptor-bound PA, removing an N-terminal 20-kDa piece (PA 20 ) and leaving a 63-kDa piece (PA 63 ) bound to the receptor. Once freed from PA 20 , PA 63 spontaneously selfassociates to form a ring-shaped heptamer (also called the prepore) (17). PA 63 is capable of permeabilizing cells to Rb ϩ and Na ϩ under acidic conditions (16) and can form pores (channels) in planar phospholipid bilayers even in the absence of receptor (5). Oli...