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 PKC-alpha 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.

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.

Cytotoxic necrotizing factor type 2 (CNF2) produced by Escherichia coli strains isolated from intestinal and extraintestinal infections is a dermonecrotic toxin of 110 kDa. We cloned the CNF2 gene from a large plasmid carried by an Escherichia coli strain isolated from a lamb with septicemia. Hydropathy analysis of the deduced amino acid sequence revealed a largely hydrophilic protein with two potential hydrophobic transmembrane domains. The N-terminal half of CNF2 showed striking homology (27% identity and 80% conserved residues) to the N-terminal portion of Pasteurella multocida toxin. Methylamine protection experiments and immunofluorescence studies suggested that CNF2 enters the cytosol of the target cell through an acidic compartment and induces the reorganization of actin into stress fibers. Since the formation of stress fibers in eukaryotic cells involves Rho proteins, we radiolabeled these small GTP-binding proteins from CNF2-treated and control cells with a Rho-specific ADP-ribosyltransferase. The [32P]ADP-ribosylated Rho proteins from CNF2-treated cells migrated slightly more slowly in SDS/PAGE than did the labeled proteins from the control cells. This shift in mobility of Rho proteins in SDS/PAGE was also observed when CNF2 and the RhoA protein were coexpressed in E. coli. We propose that Rho proteins are the targets of CNF2 in mammalian cells.

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.