Bacterial protein toxins: current and potential clinical use.

Natural toxins are the product of a long-term evolution, and act on essential mechanisms in the most crucial and vital processes of living organisms. They can attack components of the protein synthesis machinery, actin polymerization, signal transduction pathways, intracellular trafficking of vesicles as well as immune and inflammatory responses. For this reason, toxins have increasingly being used as valuable tools for analysis of cellular physiology, and in the recent years, some of them are used medicinally for the treatment of human diseases. This review is devoted to protein toxins of bacterial origin, specifically those toxins that are currently used in therapy or those under study for their potential clinical applications. Bacterial protein toxins are all characterized by a specific mechanism of action that involves the central molecular pathways in the eukaryotic cell. Knowledge of their properties could be used for medical purposes.

A multinucleating Escherichia coli cytotoxin perturbs cell cycle in cultured epithelial cells.

Pathogenic Escherichia coli strains produce a number of virulence-associated factors, among which cytotoxic necrotizing factor 1 (CNF1). CNF1 is a chromosomally encoded toxin that permanently activates the small GTP-binding proteins of the Rho family (Rho, Rac and Cdc42) by catalizing their deamidation at a specific glutamine residue. This activation modulates a high number of cellular functions, including the reorganization of the actin cytoskeleton, the promotion of cell spreading and the multinucleation. Indeed, accumulating evidence indicates that, in addition to the well-characterized Ras GTPases, also Rho family proteins are crucial in different points of cell cycle regulation. Here, we report that CNF1 induces a block of the cell cycle at the G(2)/M transition in epithelial cell line HEp-2, and up-regulates cyclin B1 and p53 proteins confining them in the cytoplasm region. The ability of CNF1 to perturb cell cycle progression could play a role in E. coli pathogenicity.

Multinucleation and pro-inflammatory cytokine release promoted by fibrous fluoro-edenite in lung epithelial A549 cells.

An unusual cluster of malignant mesothelioma was evidenced in Biancavilla, a Sicily village where no inhabitant had been significantly and professionally exposed to asbestos. Mineralogical and environmental studies led to the identification of a new prismatic amphibole, named fluoro-edenite. We previously reported, by using the human lung epithelial A549 cells, that prismatic fluoro-edenite was unable to induce changes that could be somehow related to cellular transformation, and this was in accordance with studies carried out in vivo. More recently, a fibrous amphibole with a composition very similar to that of prismatic fluoro-edenite, was identified in Biancavilla. This fibrous fluoro-edenite was shown to induce mesothelioma in rats. In keeping with this effect in vivo, in the present work we observed multinucleation and spreading, common features of transformed cells, as well as pro-inflammatory cytokine release in A549 cells. Such cell changes occurred without interfering with the passage of the resulting multinucleated cells through the cell cycle and without condemning cells to death. Hence, in lung epithelial cells, fibrous fluoro-edenite behaved similarly to the unrelated asbestos type crocidolite, whose connection with severe inflammation and cancer of the lung is renowned.

Cytotoxic necrotizing factor 1 hinders skeletal muscle differentiation in vitro by perturbing the activation/deactivation balance of Rho GTPases.

The current knowledge assigns a crucial role to the Rho GTPases family (Rho, Rac, Cdc42) in the complex transductive pathway leading to skeletal muscle cell differentiation. Their exact function in myogenesis, however, remains largely undefined. The protein toxin CNF1 was herein employed as a tool to activate Rho, Rac and Cdc42 in the myogenic cell line C2C12. We demonstrated that CNF1 impaired myogenesis by affecting the muscle regulatory factors MyoD and myogenin and the structural protein MHC expressions. This was principally driven by Rac/Cdc42 activation whereas Rho apparently controlled only the fusion process. More importantly, we proved that a controlled balance between Rho and Rac/Cdc42 activation/deactivation state was crucial for the correct execution of the differentiation program, thus providing a novel view for the role of Rho GTPases in muscle cell differentiation. Also, the use of Rho hijacking toxins can represent a new strategy to pharmacologically influence the differentiative process.

Hijacking Rho GTPases by protein toxins and apoptosis: molecular strategies of pathogenic bacteria.

Certain bacterial toxins and type-III-translocated virulence factors have a peculiar property: they exert part of their actions by modulating Rho GTPases. These toxins target the actin cytoskeleton of host cells and reorganize it to their own advantage, either to facilitate macropinocytosis, which is required for invasive bacteria to enter cells, or to block pathogen sequestration by macrophages. In addition, by acting on Rho GTPases, bacteria may also interfere with the fate of host cells, favoring survival or death depending on their needs. Rho GTPases control the activation of NF-kappaB, which is involved in the expression of antiapoptotic proteins and mediates immunological responses as well. Here, we give a perspective on how NF-kappaB may participate in linking Rho-acting toxins and apoptosis.

Epithelial cells and expression of the phagocytic marker CD68: scavenging of apoptotic bodies following Rho activation.

Macropinocytosis is a ruffling-driven process which drives the ingestion of large particles by both macrophages and epithelial cells. In this context, we have previously described a Rho-activating bacterial toxin from E. coli, the cytotoxic necrotizing factor 1 (CNF1), which allows epithelial cells to macropinocytose not only latex beads and bacteria, but also apoptotic cells in a fashion similar to that of professional phagocytes. We herein report that (i) epithelial cells express the typical phagocytic marker CD68, (ii) Rho activation by CNF1 varies the intracellular localization of CD68, which appears to be co-distributed, as in macrophages, with the homologous lysosomal protein Lamp-1. Together with the capability of digesting apoptotic cells following their internalization, our findings indicate that Rho-activated epithelial cells behave in most respects as professional phagocytes.

Activation of rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells.

Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced "switching on" of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages.