In Cultured Cells

Toxin-mediated glucosylation of Rho explains the cytoskeletal disruption seen in cells treated with either of the C. difficile toxins. Somewhat differing effects of both toxins have been reported on the cytoskeletal organization in Hep-2 cells (Fiorentini etal., 1989).

Cytoskeletal changes and a surface blebbing induced by ToxB cytoskeletal disruption were described in a variety of cell types, as determined by immuno-chemistry and electron microscopy (Malorni et ai, 1990). ToxA has been reported to cause a nuclear polarization (Fiorentini et ai, 1990), a multinucleation in the human leukemic T cell line Jurkat (Fiorentini et al., 1992), as well as a surface blebbing with apoptosis-like cell death independent of calcium in rat intestinal cells (Fiorentini etal., 1993). All these effects are likely consequences of a disruption of the actin cyto-skeleton. The subsequent cell rounding and inhibition of cell proliferation are logical general consequences of an irreversible progressive loss of functional Rho. Indeed a transient overexpression of Rho pro teins can confer some resistance to the effects of both ToxA and B (Giry etal., 1995; Just etal., 1994a). Hyperphosphorylation of certain cellular proteins, as observed in ToxB-treated astrocytes (Ciesielski-Treska ef a/., 1991) and McCoy cells (Schue et al., 1994), might also occur after inactivation of Rho proteins and cytoskeletal disruption, although the signaling processes are not known.

Of special interest with respect to the roles of the C. difficile toxins effects on tight junctions in disease are reports of effects on cultured intestinal cells. The T-84

cell line derived from human colon carcinoma, and cultured in vitro as a polarized epithelium, forms the tight junctions essential for epithelial barrier function. Both ToxA and B caused the disintegration of these tight junctions, measured as a loss of barrier function correlated with disruption of actin filaments (Hecht et al., 1988, 1992). This event occurred even at low toxin concentrations which did not cause rounding of the cells. Indeed, recent work with C. botulinum exoenzyme C3 demonstrated that Rho regulates the tight junctions and perijunctional actin organization in T-84 cells (Nusrat et al., 1995). This is consistent with the notion that the C. difficile toxins may disrupt epithelial barrier function due to glucosylation, and thereby inactivation, of Rho.

The functions of the three subtypes of Rho proteins may vary in cells from different tissues, as well as in normal versus tumor cells (Coso et al., 1995; Nishiyama etal., 1994; Olson etal., 1995). If we assume that the C. difficile toxins attack Rac and Cdc42 as well as Rho in vivo, it is not surprising that the long-term consequences of toxin treatment differ between target cell types, as exemplified by the diversity of secondary effects described for both toxins. Thus the inhibition of macro-molecular synthesis caused byToxB in normal human fibroblasts (Florin and Thelestam, 1981) is expected, because cell rounding and the concomitant loss of cell surface fibronectin (Ahlgren et al., 1983) in anchorage-dependent cell lines will inhibit DNA-synthesis (Ben-Ze'ev et al., 1980). By contrast, ToxB did not affect DNA synthesis in transformed B cell lines, whereas it inhibited cytokinesis which depends directly on a functioning actin cytoskeleton (Shoshan et al., 1990). Finally, both toxins were reported to affect human monocytes causing cytokine release (Daubener etal, 1988; Flegel etal., 1991; Siffert etal., 1993), and ToxA was found to stimulate intracellular calcium release and a chemotactic response in human granulocytes (Pothoulakis et al., 1988). Whether these effects are due to glucosylation of Rho proteins or depend on some other separate effects of the toxins remains to be seen.

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