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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
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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 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.
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After three hours, C2 has almost completely destroyed the cytoskeleton. (Photos: Dr. Sascha Pust, Barth research
group)
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 receptormediated 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.
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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)
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
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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
Article
01-Mar-2008
BioRegionUlm
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