Toxins A and B of Clostridium difficile are UDP-glucose glucosyltransferases that exert their cellular toxicity primarily through their abilities to monoglucosylate, and thereby inactivate, Rho family small GTPases. Toxin A also hydrolyzes UDP-glucose, although this activity is not well characterized. In this study, we measured the kinetics of UDP-glucose hydrolysis by toxins A and B and found significant differences in the catalytic activities of these two structurally homologous toxins. The toxins displayed similar Michaelis constants (K m ) for UDP-glucose, but the maximal velocity (V max ) of toxin B was ϳ5-fold greater than that of toxin A. Toxins A and B exert their enzymatic actions intracellularly, and, interestingly, we found that each toxin absolutely required K ؉ for optimal hydrolase activity; Na ؉ was inactive. The toxins also required certain divalent cations for activity and exhibited a significantly greater V max and lower K m in the presence of Mn 2؉ as compared with Mg 2؉ . We conclude that C. difficile toxins A and B are cation-dependent UDP-glucose hydrolases that differ significantly in their catalytic activities, a finding that may have important implications in understanding their different cytotoxic effects.

The catalytic domain of Bordetella pertussis adenylate cyclase toxin (ACT) translocates directly across the plasma membrane of mammalian cells to induce toxicity by the production of cAMP. Here, we use electrophysiology to examine the translocation of toxin into polarized epithelial cells that model the mucosal surfaces of the host. We find that both polarized T84 cell monolayers and human airway epithelial cultures respond to nanomolar concentrations of ACT when applied to basolateral membranes, with little or no response to toxin applied apically. The induction of toxicity is rapid and fully explained by increases in intracellular cAMP, consistent with toxin translocation directly across the basolateral membrane. Intoxication of T84 cells occurs in the absence of CD11b/CD18 or evidence of another specific membrane receptor, and it is not dependent on post-translational acylation of the toxin or on host cell membrane potential, both previously reported to be required for toxin action. Thus, elements of the basolateral membrane render epithelial cells highly sensitive to the entry of ACT in the absence of a specific receptor for toxin binding.The adenylate cyclase toxin (ACT), 3 a multifunctional, single polypeptide toxin, is expressed by six of the eight members of the genus Bordetella and was discovered by detection of adenylate cyclase enzymatic activity in a commercial pertussis vaccine (1). ACT derives its cytotoxic effects from delivery of its 400-amino acid adenylate cyclase enzymatic domain into the cell cytoplasm, resulting in the unregulated, calmodulindependent conversion of ATP into cAMP (2-4). Translocation, the process by which the catalytic domain is delivered across the cytoplasmic membrane, is unaffected by cytochalasin D or ammonium chloride and is dependent on the interaction of the ϳ1000-amino acid cell-binding domain with the cell membrane (5).This cell-binding domain is homologous to the members of the RTX (repeat-in-toxin) family of pore-forming bacterial toxins, such as Escherichia coli hemolysin, HlyA, and several leukotoxins from organisms such as Pasteurella hemolytica and Actinobacillus actinomycetemcomitans (6, 7). The RTX region of ACT oligomerizes in the cell membrane independently of translocation, forming cation-selective pores and causing hemolysis of erythrocytes and nonapoptotic death of nucleated cells (8 -14). Membrane interaction and pore formation by ACT can occur in artificial lipid bilayers and liposomes and are influenced by lipid and glycolipid composition (11,(15)(16)(17)(18). Although ACT intoxicates a broad range of cells and is able to associate with artificial lipid membranes containing no proteins, the ␤ 2 -integrin, CD11b/CD18 (Mac-1), which is expressed on phagocytic leukocytes, has been shown to increase the potency of ACT by an order of magnitude and has been considered a receptor for ACT (19). Accordingly, most of the studies on the functional, cytotoxic effects of ACT have focused on CD11b/CD18-positive cells, beginning with the initial observation that the...

The effects of purified toxin A in vitro on the shape and function of polymorphonuclear leukocytes (PMNL) were examined. Toxin A induced changes in adherent PMNL shape from a compact spherical or pyramidal shape to a thin and rope-like shape. This change in shape was accompanied by rearrangement of the F-actin cytoskeleton into aggregates. Toxin A-treated PMNL exhibited increased adherence and expressed less L-selectin and more Mac-1, compared with untreated PMNL. In contrast to these proinflammatory actions, toxin A impaired both directed and non-directed PMNL migration in response to N-formylmethionylleucylphenylalanine. In addition, toxin A decreased the oxidative activity of adherent PMNL stimulated by recombinant human tumor necrosis factor-alpha. These effects could be explained by toxin A-induced glucosylation of the signaling small-size guanine 5'-triphosphate-binding proteins of the Rho family in human PMNL.

Cyclic AMP-(cAMP) and calcium-dependent agonists stimulate chloride secretion through the coordinated activation of distinct apical and basolateral membrane channels and ion transporters in mucosal epithelial cells. Defects in the regulation of Cl -transport across mucosal surfaces occur with cystic fibrosis and V. cholerae infection and can be life threatening. Here we report that secramine B, a small molecule that inhibits activation of the Rho GTPase Cdc42, reduced cAMPstimulated chloride secretion in the human intestinal cell line T84. Secramine B interfered with a cAMP-gated and Ba 2+ -sensitive K + channel, presumably KCNQ1/KCNE3. This channel is required to maintain the membrane potential that sustains chloride secretion. In contrast, secramine B did not affect the Ca 2+ -mediated chloride secretion pathway, which requires a separate K + channel activity from that of cAMP. Pirl1, another small molecule structurally unrelated to secramine B that also inhibits Cdc42 activation in vitro, similarly inhibited cAMPdependent but not Ca 2+ -dependent chloride secretion. These results suggest that Rho GTPases may be involved in the regulation of the chloride secretory response and identify secramine B an inhibitor of cAMP-dependent K + conductance in intestinal epithelial cells.

Clostridium difficile is a major cause of antibiotic-associated diarrhea. While treatment regimens for C. difficile have been available for decades, they remain less than optimal due to the frequent recurrences that occur after therapy is completed. Moreover, the morbidity and expense associated with C. difficile have underscored the need for more effective preventive measures than are currently available. In this review, we outline the current recommendations for treatment and prevention of C. difficile infection and, highlight some promising new approaches that may help to control this common nosocomial pathogen in the future.