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.

The intracellular signaling involved in the mechanism of action of zonula occludens toxin (ZOT) was studied using several in vitro and ex vivo models. ZOT showed a selective effect among various cell lines tested, suggesting that it may interact with a specific receptor, whose surface expression on various cells differs. When tested in IEC6 cell monolayers, ZOT-containing supernatants induced a redistribution of the F-actin cytoskeleton. Similar results were obtained with rabbit ileal mucosa, where the reorganization of F-actin paralleled the increase in tissue permeability. In endothelial cells, the cytoskeletal rearrangement involved a decrease of the soluble G-actin pool (-27%) and a reciprocal increase in the filamentous F-actin pool (+22%). This actin polymerization was time-and dose-dependent, and was reversible. Pretreatment with a specific protein kinase C inhibitor, CGP41251, completely abolished the ZOT effects on both tissue permeability and actin polymerization.In IEC6 cells ZOT induced a peak increment of the PKCct isoform after 3 min incubation. Taken together, these results suggest that ZOT activates a complex intracellular cascade of events that regulate tight junction permeability, probably mimicking the effect of physiologic modulator(s) of epithelial barrier function. (J. Clin. Invest. 1995. 96:710-720.)

Cytotoxic necrotizing factor type 1 (CNF1) from strains of pathogenic Escherichia coli induces in human epithelial HEp-2 cells, a profound reorganization of the actin cytoskeleton into prominent stress fibres and membrane ruffles. We report here that this process is associated with induction of phagocytic-like activity. CNF1-treated cells acquired the ability to ingest latex beads as well as non-invasive bacteria such as Listeria innocua, which were taken as a model system. Uptake of bacteria was similar to pathogen-induced phagocytosis, since L. innocua transformed with DNA coding for the pore-forming toxin listeriolysin O behaved, with respect to intracellular growth, like the invasive, pathogenic species L. monocytogenes. Our results raise the possibility that, in vivo, pathogenic CNF1-producing E. coli may invade epithelia by this novel induced phagocytic-like mechanism.

Structural changes in the gut microbial community have been shown to accompany the progressive development of colorectal cancer. In this review we discuss recent hypotheses on the mechanisms involved in the bacteria-mediated carcinogenesis, as well as the triggering factors favoring the shift of the gut microbiota from a mutualistic to a pro-carcinogenic configuration. The possible role of inflammation, bacterial toxins and toxic microbiota metabolites in colorectal cancer onset is specifically discussed. On the other hand, the strategic role of inflammation as the keystone factor in driving microbiota to become carcinogenic is suggested. As a common outcome of different environmental and endogenous triggers, such as diet, aging, pathogen infection or genetic predisposition, inflammation can compromise the microbiota-host mutualism, forcing the increase of pathobionts at the expense of health-promoting groups, and allowing the microbiota to acquire an overall pro-inflammatory configuration. Consolidating inflammation in the gut, and favoring the bloom of toxigenic bacterial drivers, these changes in the gut microbial ecosystem have been suggested as pivotal in promoting carcinogenesis. In this context, it will become of primary importance to implement dietary or probiotics-based interventions aimed at preserving the microbiota-host mutualism along aging, counteracting deviations that favor a pro-carcinogenic microbiota asset.

The mechanism whereby the morphology and connectivity of the dendritic tree is regulated depends on an actin dynamics that, in turn, is controlled by Rho GTPases, a family of small GTP-binding proteins encompassing Rho, Rac, and Cdc42 subfamilies. Cytotoxic necrotizing factor 1 (CNF1), a protein toxin from Escherichia coli, constitutively activates Rho GTPases, thus leading to remodeling of the actin cytoskeleton in intact cells. Here, we show that the modulation of cerebral RhoA and Rac1 activity induced by CNF1 in mice leads to (i) rearrangement of cerebral actin cytoskeleton, (ii) enhanced neurotransmission and synaptic plasticity, and (iii) improved learning and memory in various behavioral tasks. The effects persist for weeks and are not observed in mice treated with a recombinant CNF1, in which the enzymatic activity was abolished by substituting serine to cysteine at position 866. The results suggest that learning ability can be improved through pharmacological manipulation of neural connectivity.cytotoxic necrotizing factor 1 ͉ brain ͉ bacterial toxins ͉ dendritic spines ͉ drug therapy