Many bacterial toxins destroy the shape and function of their host cells – some of them degrade the cytoskeleton, others have a tendency to inhibit the elasticity of the cytoskeleton. Prof. Dr. Gudula Schmidt and her team at the University of Freiburg are investigating the effect of the substance CNF-1 and have already discovered the mechanism that is initiated by this toxin.
The bacterium Escherichia coli has many facets. Most of the strains used by scientists for their experiments are harmless to humans, as are those that colonise the microflora of the human intestinal tract. However, there are some bacteria that can lead to intestinal and urinary tract infections. These bacteria produce the toxin cytotoxic necrotising factor 1 (CNF-1), a protein that attacks epithelial cells that cover and line tissue. However, the protein has also been shown to have an effect on immune cells. Visible consequences of the toxin are parts of the skin that die off or disorders of the immune system. "How does the toxin act on the molecular level? And can its activity be switched off?" These are questions Prof. Dr. Gudula Schmidt in the Department I of the Institute of Experimental and Clinical Pharmacology and Toxicology at the University of Freiburg is trying to answer. Over the last few years she has gained insights that provide her with a fairly good understanding of the underlying mechanisms.
The key to everything is the cytoskeleton of the target cells. “The cytoskeleton is a scaffold of protein fibres in the interior of cells that are continuously rearranged,” said Schmidt who was born in Bochum in 1967. The cytoskeleton gives the cells stability at the same time as enabling them to change their shape and move around. This latter ability is important for macrophages of the immune system, which patrol the organs in search of intruders. The fibres, of which the cytoskeleton is composed, consist of numerous monomers of the protein actin. The individual components attach to or dissociate from each other, enabling the cytoskeleton to expand or retract, thereby maintaining an elasticity that enables it to adapt to given situations. The process of dissociation is mediated by proteins of the Rho-GTPase family. Rho-GTPase proteins are located on the cell membrane or in the cell where they react to external signals. If, for example, a macrophage detects an intruder, the Rho-GTPases induce signalling cascades in the cell that in turn induce the rearrangement of the actin skeleton of the cell, enabling the immune cell to move towards its target.
“CNF-1 targets a specific Rho-GTPase which expands the actin skeleton,” said Schmidt. Around thirteen years ago, the biologist clarified this mechanism during her postdoctoral period at the Freiburg Institute of Experimental and Clinical Pharmacology and Toxicology. In parallel with another group of scientists, Schmidt was the first to understand how a bacterial toxin functions, in that it leads to the expansion rather than to the retraction of the cytoskeleton, which is more common for many other bacterial toxins. She began her studies from scratch, with only the knowledge that the toxin had some kind of effect on the cytoskeleton. “But we were also lucky,” said Schmidt. “During my doctorate at the Max Planck Institute of Molecular Physiology in Dortmund, I worked with Ras-GTPases that are permanently active in tumour cells due to specific mutations, and which are also responsible for some typical tumour cell characteristics. I found out that CNF-1 had a similar effect on Rho-GTPAses as the mutations of Ras. In addition, the mutations occurred at identical amino acids.”
“What exactly happens in a cell during intoxication? Schmidt found out that CNF-1 was bound by a still unknown receptor located on the surface of the host cell and is endocytosed by way of membrane vesicles. The acid environment inside these vesicles leads to the unfolding of the protein, which subsequently forms a pore in the membrane through which it enters the cell. When CNF-1 meets a Rho-GTPase molecule, it binds to specific amino acids and is then able to exert its action, i.e. prevents a specific reaction inside the Rho-GTPase molecule. Rho-GTPases are active when bound to energy-rich GTP molecules and inactive when GTP is hydrolysed to GDP. The hydrolysis of GTP is key in this mechanism: CNF-1 blocks this process, which in turn keeps Rho in a permanently active state. Therefore, new components are continuously added to the cytoskeleton and the fibres in the cell interior remain rigid.
Under the microscope, an intoxicated cell can be seen to flatten – just like a punctured football. CNF-1-affected cells no longer divide, because cell division is normally mediated by the actin skeleton. However, the cells’ nuclei continue to divide, which leads to a large number of chromosome sets. “A kingdom with many kings will eventually collapse,” said Schmidt explaining the mechanism which leads to the death of the cells after a few days. In future, Schmidt and her team hope to find a receptor on the target cells of CNF-1 which mediates the endocytosis of the bacterial toxin. She is also interested in elucidating the effects of other CNF types, since CNF-1 is not the only toxin of its kind. In a new project, for which the researchers have just applied for funding, Schmidt and a number of other research groups from Freiburg are aiming to find out whether and how bacterial toxins can be used to block the migration of tumours. “The clarification of the functional mechanisms of bacterial toxins is like detective work,” said Schmidt. “We are gradually collecting more and more evidence and are getting closer to solving the “crime”. This research is what has kept Schmidt at the Freiburg-based Institute of Toxicology where she is now a professor (apl. Prof.).
Further information: Prof. Dr. Gudula Schmidt Institute of Experimental and ClinicalPharmacology and ToxikologyAlbertstr. 2579104 FreiburgTel: +49 (0)761/ 203 5316Fax: +49 (0)761/ 203 5311E-mail: Gudula.Schmidt(at)pharmakol.uni-freiburg.de