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Holger Barth is turning toxins into protein shuttles

Holger Barth works with a special kind of Trojan horses. The toxicologist from Ulm is investigating bacterial toxins. These proteins manage, in a similar way to the ancient Greeks before them, to open the barricaded portal of the cells with a trick, whereupon they start wreaking destruction.

Holger Barth began focusing on the investigation of the C2 toxin in the mid-1990s while he was doing his doctorate and, back then, could not have predicted that this toxin would one day become “his” toxin, from which he would extract many secrets. At the time, the Institute for Experimental and Clinical Pharmacology and Toxicology led by the renowned toxicologist Klaus Aktories, had just relocated to Freiburg. Barth, whose contract with the DKFZ had just ended, was given the task of finding out how the C2 toxin, which is produced by Clostridium botulinum, is taken up by the cell.

Nature is an excellent model for industry

Holger Barth has been professor at the Institute for Pharmacology and Toxicology at the University Hospital of Ulm for almost three years. (Photo: Pytlik, BioRegionUlm)
Barth, who did his doctoral thesis on the regulation of the cell cycle, soon realised that he enjoyed toxicology. “I realised that these toxins had huge potential,” said Barth. These proteins are highly specialised and they use clever ways to enter the cells by using the cells’ own protein transport pathways. This way, they are not degraded by the cell machinery and are able to enter the cell without being damaged. Barth is fascinated by another feature of the toxins. The toxins specifically modify a substrate in the cell and do in principle the same as the industry: produce selective inhibitors or activators.

“His toxin”, the C2 toxin, had long time been in the mighty shadow of C1, another toxin produced by Clostridium botulinum. Many other toxins have been discovered, but C2 still fascinates Barth the most. After he had investigated the effect and structure of this toxin, he is now looking into the pharmacological aspects, i.e. application.

Passenger and transporter

C2 is a member of the group of binary actin-ADP ribosylating toxins that destroy the actin filaments of the cytoskeleton. The best-known toxin of this group, which consists of only a few species, is anthrax. C2 was discovered in 1980 by Japanese researchers and Barth’s supervisor, Professor Aktories, later clarified the toxin’s molecular structure. Only recently, Barth and Aktories succeeded in deciphering the crystal structure of C2.
The effect of C2 toxin on cultured mammalian cells. The photo shows control cells, without C2 toxin.
The effect of C2 toxin on cultured mammalian cells. The photo shows control cells, without C2 toxin.
After three hours, C2 has almost completely destroyed the cytoskeleton. (Photos: Dr. Sascha Pust, AG Barth)
After three hours, C2 has almost completely destroyed the cytoskeleton. (Photos: Dr. Sascha Pust, Barth research group)
The toxin group has a specific binary character. The C2 toxin consists of two individual proteins (the binding/translocation component C2II and the enzyme component C2I). To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. The individual proteins are non-toxic, something that is also positive for laboratory work.

C2I is the actual toxin that destroys the cytoskeleton, but it requires C2II as transporter. Holger Barth clarified the interesting mechanism behind this. For a long time, little had been known about the transporter. Upon proteolytic activation (C2II then becomes C2IIa), C2IIa rapidly forms ring-shaped heptamers that bind to carbohydrate structures on the cell surface. C2I assembles with the C2IIa heptamers and the complete complex is taken up by receptor-mediated endocytosis. The binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface is necessary for C2I to bind on the cell.

A proton pump prepares unfolding

The attachment of three enzymes leads to endocytosis. From a certain pH value in the endosomal compartment, the C2IIa heptamers insert into the endosomal membrane and form pores. The acid environment is caused by protons that are pumped from the cytosol into the inside of the endosomes. The enzymes (C2I) are unfolded when a specific acidity has been reached. So-called chaperons then fold C2I up again and lock the enzyme.

Beware when the helpers are released

Barth discovered these cellular helpers at the same time as some colleagues in the USA. In the meantime, the Ulm researchers have discovered additional helpers that contribute to protein folding. Barth refers to these helpers as a virtual “machine”, which helps the “foreign” protein to cross the cell membrane. This is a complex process, because the foreign protein must initially become water-soluble, then fat-soluble and finally once again water-soluble.

Back in the 1980s, Japanese researchers found out that C2 is toxic in very small concentrations. Investigations by Barth’s team confirmed these findings. C2 led to apoptosis (programmed cell death) in common cell types (e.g. epithelial cells, fibroblasts). The Ulm researchers also found out that, while the cytoskeleton was destroyed within a few hours, the cultured cells survived for as long as 24 hours.

Fusion proteins use Trojan property

Besides the biochemical and cell biological characterisation of toxins and their effect on mammalian cells, Barth’s team is also investigating the use of recombinant proteins. The researchers used the nontoxic C2 translocation component as a shuttle to transport proteins that would not otherwise be able to enter the cells. In 2007, Barth’s team succeeded in discovering the cellular mechanism of a new Salmonella toxin, which could be used to investigate the long-term reactions of SpvB-intoxicated cells. SpvB, a Salmonella enterica virulence factor, also attacks actin (just like the Clostridium toxin C2) and is transported by intracellular Salmonella directly into the cytosol of host cells.

Barth and his colleagues also found out that mammalian cells have a natural defence mechanism against this Salmonella toxin and degrade the toxin in the cytosol. This is different from the C2I protein of Clostridium botulinum of which the tiniest amounts are fatal for the cell. The researchers are now hoping to use this transport system to introduce DNA repair enzymes into the cells. According to Barth, this approach appears to be very interesting for damaged DNA in tumour cells, like for example p53, a protein that functions as tumour suppressor.

Piggy backed into the cell

The members of Barth’s team (the photo shows almost the entire crew) have a very trusting relationship (Photo: Pytlik, BioRegionUlm)
The members of Barth’s team (the photo shows almost the entire crew) have a very trusting relationship (Photo: Pytlik, BioRegionUlm)
Barth’s team has used the shuttle qualities of C2 for another artificial fusion toxin (C2IN-C3). The researchers used this fusion toxin to transport the natural enzyme C3 into the cell. According to Barth, this is of great importance because C3 is currently the only RHO inhibitor known. RHO is a central molecular switch and is involved in many cellular reactions. “Our recombinant fusion toxin is an effective C3 transporter in all eukaryotic cell types tested,” said Barth who assumes that the specific binding of C3 might also have a therapeutic potential.

Transporter principle is research capital

Barth sees C2’s transporter principle as his major research capital. At present, one of Barth’s colleagues is investigating the possibility of coupling a universal adapter to C2. If they succeed, then the researchers will no longer be required to work on the genetic level and be hampered by patent restrictions. The principle of using C2 as a transporter for bulky proteins does not always work and has to be tested in each individual case. According to Barth, this depends on whether the enzyme can be folded back. The size of the blind protein passenger does not appear to be as important as previously believed.

Barth can also envisage the pharmaceutical application of these highly specific toxins. For example, one of the group’s fusion proteins was sold to a company from Tübingen thanks to its neuroregenerative potential. Does it seem unrealistic to commercially exploit a fusion protein in Ulm? The toxicologist is very well aware that many small steps will have to be taken before this becomes reality. And he is also aware of the fact that the enthusiasm is often slightly dampened by the huge amount of work required.

Positive memories and enthusiasm

When Barth speaks about the lucky circumstances of his academic career it becomes clear what drives his team forward. Barth’s gratitude for those that supported him and enabled him to take part in their visions and to honour the freedom of academic research is contagious and Barth is full of praise for the trusting relationship of the group he works with. If he had one wish then Barth would like to have enough money to continue financing the posts of his team when their contracts come to an end.

wp, 10.03.2008 © BIOPRO Baden-Württemberg GmbH

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