Hypervirulent Clostridium difficile strains, which are associated with increased morbidity and mortality, produce the actin-ADP ribosylating toxin Clostridium difficile transferase (CDT). CDT depolymerizes actin, causes formation of microtubule-based protrusions, and increases pathogen adherence. Here, we show that septins (SEPT) are essential for CDT-induced protrusion formation. SEPT2, -6, -7, and -9 accumulate at predetermined protrusion sites and form collar-like structures at the base of protrusions. The septin inhibitor forchlorfenuron or knockdown of septins inhibits protrusion formation. At protrusion sites, septins colocalize with the GTPase Cdc42 (cell division control protein 42) and its effector Borg (binder of Rho GTPases), which act as up-stream regulators of septin polymerization. Precipitation and surface plasmon resonance studies revealed high-affinity binding of septins to the microtubule plus-end tracking protein EB1, thereby guiding incoming microtubules. The data suggest that CDT usurps conserved regulatory principles involved in microtubule-membrane interaction, depending on septins, Cdc42, Borgs, and restructuring of the actin cytoskeleton.microtubules | septins | actin | bacterial toxin | Clostridium difficile

Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/β3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.

Microtentacles are mostly microtubule-based cell protrusions that are formed by detached tumor cells. Here, we report that the formation of tumor cell microtentacles depends on the presence and dynamics of guanine nucleotide-binding proteins of the septin family, which are part of the cytoskeleton. In matrix-attached breast, lung, prostate and pancreas cancer cells, septins are associated with the cytosolic actin cytoskeleton. Detachment of cells causes redistribution of septins to the membrane, where microtentacle formation occurs. Forchlorfenuron, which inhibits septin functions, blocks microtentacle formation. The small GTPase Cdc42 and its effector proteins Borgs regulate septins and are essential for microtentacle formation. Dominant active and inactive Cdc42 inhibit microtentacle formation indicating that the free cycling of Cdc42 between its active and inactive state is essential for septin regulation and microtentacle formation. Cell attachment and aggregation models suggest that septins play an essential role in the metastatic behavior of tumor cells.