Pathogenic Escherichia coli are responsible for a variety of diseases, including diarrhoea, haemolytic uraemic syndrome, kidney infection, septicaemia, pneumonia and meningitis. Toxins called cytotoxic necrotizing factors (CNFs) are among the virulence factors produced by uropathogenic (CNF1) or enteropathogenic (CNF2) E. coli strains that cause diseases in humans and animals, respectively. CNFs induce an increase in the content of actin stress fibres and focal contacts in cultured cells. Effects of CNFs on the actin cytoskeleton correlated with a decrease in the electrophoretic mobility of the GTP-binding protein Rho and indirect evidence indicates that CNF1 might constitutively activate Rho. Here we show that CNF1 catalyses the deamidation of a glutamine residue at position 63 of Rho, turning it into glutamic acid, which inhibits both intrinsic GTP hydrolysis and that stimulated by its GTPase-activating protein (GAP). Thus, this deamidation of glutamine 63 by CNF1 leads to the constitutive activation of Rho, and induces the reorganization of actin stress fibres. To our knowledge, CNF1 is the first example of a bacterial toxin acting by deamidation of a specific target protein.

CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.

Clostridium botulinum C3 is a recently discovered exoenzyme that ADP‐ribosylates a eukaryotic GTP‐binding protein of the ras superfamily. We show now that the bacterially‐expressed product of the human rhoC gene is ADP‐ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP‐ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

The rho family of GTP-binding proteins regulates actin filament organization. In unpolarized mammalian cells, rho proteins regulate the assembly of actin-containing stress fibers at the cell-matrix interface. Polarized epithelial cells, in contrast, are tall and cylindrical with well developed intercellular tight junctions that permit them to behave as biologic barriers. We report that rho regulates filamentous actin organization preferentially in the apical pole of polarized intestinal epithelial cells and, in so doing, influences the organization and permeability of the associated apical tight junctions. Thus, barrier function, which is an essential characteristic ofcolumnar epithelia, is regulated by rho. and basolateral membranes, high transepithelial resistance to passive ion flow, and a Cl-secretory pathway analogous to that found in native intestinal crypt epithelium. We have also determined the effects of rhoC overexpression in intestinal epithelial cell lines. Our data indicate that rho plays an important regulatory role in determining microfilament organization in the apical pole of columnar epithelial cells and, in so doing, influences the associated tight junction and epithelial permeability. Concurrent with these effects, ZO-1, a tight junction structural element, moves off the membrane, while E-cadherin, the sealing element of the closely related adherens junction, is preserved on the membrane.The rho proteins are members of a large family of small GTP-binding proteins (21 kDa) that are believed to be involved in regulating assembly of the actin cytoskeleton. In unpolarized mammalian cells, rho proteins (rhoA, B, and C) regulate assembly of focal adhesions and basal filamentous (F) actin stress fibers (1). The Clostridium botulinum toxin C3 transferase selectively blocks rho-effector coupling by ADPribosylation of rho on Asn-41 (2-4) and has been a useful tool in examining the biological function of rho proteins. In fibroblasts, C3 transferase induces disassembly of stress fibers that are localized at the base of cells (3, 5). Conversely, microinjection of a constitutively activated form of rho into quiescent serum-starved cells results in the appearance of prominent stress fibers and focal adhesions at the cell-matrix interface (1, 6).Polarized columnar epithelial cells, such as those lining the alimentary tract, airways, and renal tubules, differ markedly from spreading unpolarized cells in which rho biology has largely been studied. For example, intestinal epithelial cells maintain a tall cylindrical form, have apical intercellular tight junctions that serve as barriers to restrict paracellular permeability, and exhibit morphologically defined subdomains of cytoskeletal structure. Stable lateral and basal F-actin filaments form a submembrane cortex thought to assist in maintaining the cylindrical shape of columnar epithelial cells and in anchoring a variety of basolateral membrane proteins. In contrast, the apical pole of polarized epithelial cells consists of a perijunctional tensile ri...

Epsilon-toxin is produced by. Here we present evidence that epsilon-toxin cytotoxic activity is correlated with the formation of a large membrane complex (about 155 kDa) and efflux of intracellular K ؉ without entry of the toxin into the cytosol. Epsilon-toxin induced swelling, blebbing, and lysis of MDCK cells. Iodolabeled epsilon-toxin bound specifically to MDCK cell membranes at 4 and 37°C and was associated with a large complex (about 155 kDa). The binding of epsilon-toxin to the cell surface was corroborated by immunofluorescence staining. The complex formed at 37°C was more stable than that formed at 4°C, since it was not dissociated by 5% sodium dodecyl sulfate and boiling.Epsilon-toxin is produced by Clostridium perfringens types B and D and is responsible for a rapidly fatal enterotoxemia in sheep and other animals which causes heavy economic losses (24). It is synthesized as a relatively inactive prototoxin (296 amino acids) which is converted to a highly active mature protein by proteolytic removal of a basic N-terminal peptide (13 amino acids) (3, 16).Epsilon-toxin is lethal and dermonecrotic. It has been reported to increase intestinal permeability (4), to cause kidney damage (9), to elevate blood pressure (27, 39), and to cause contraction of isolated rat ileum (40). A basic property of epsilon-toxin is that it increases vascular permeability. The toxin binds to vascular endothelial cells and causes severe vascular damage and edema in various organs (brain, heart, lung, and kidney) (5, 9). It was reported that the major pathological changes caused by enterotoxemia appear to occur in the brain (12). Moreover, it was shown that labeled toxin specifically accumulates in the brains of mice after intravenous injection and that the lethal activity of the toxin depends on its specific binding in the brain, probably to a sialoglycoprotein (28,29). Certain amino acids of epsilon-toxin, such as histidine, tryptophan, and aspartic or glutamic acid have been found to be essential for its biological activity (35)(36)(37)(38).Recently, it has been found that epsilon-toxin has 20 and 27% identity with Mtx2 and Mtx3 toxins, respectively, of Bacillus sphaericus (22, 43) and 26.5% identity with the open reading frame c53 of Bacillus thuringiensis. However, the mechanism of action of these mosquitocidal toxins is unknown.The Madin-Darby canine kidney (MDCK) cell line was found to be susceptible to epsilon-toxin. The alteration of cell viability determined by the conversion of 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl) tetrazolium to a water-soluble formazan by the mitochondrial cytochrome systems or by the neutral red assay (an indicator of lysosomal integrity) was correlated with the lethal activity of epsilon-toxin in mice (21, 30). The cytotoxic effects were found to be very rapid (2.5 min) and potentiated by EDTA (21). The morphological effects of epsilon-toxin on cells included a condensation of the nucleus and a progressive swelling of the cells (13). The permeability of polarized MD...