ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

Christine Clayton
Molecular Cell Biology of Trypanosomes
Introduction
Salivarian trypanosomes are unicellular parasites that
live in the blood and tissue fluids of mammals. They
cause sleeping sickness in humans and also infect
domestic animals and wildlife, mainly in subSaharan
Africa. The geographical restriction is determined by
the fact that the parasites are usually transmitted from
one mammal to the next by Tsetse flies. They develop
and multiply within the insect midgut and can undergo
a sexual cycle before infection of another mammal
via the insect salivary glands. Salivarian trypanosomes
do not multiply inside cells, unlike the closely
related tropical disease parasites Trypanosoma cruzi
and Leishmania. At present about 300 000 people are
infected with the sleeping sickness trypanosomes Trypanosoma
brucei rhodesiense and Trypanosoma gambiense.
The disease is invariably fatal unless treated,
and very few drugs are available, especially to combat
the late stages of the disease when the parasites invade
the brain. Moreover, most of the available drugs are
either too expensive or unacceptably toxic and drug
resistance is developing.
Trypanosomes and Leishmanias belong to the genus
Kinetoplastida, which branched extremely early in
eucaryotic evolution. As a consequence they exhibit
a number of remarkable deviations from standard
eucaryotic paradigms. In the realm of gene expression,
most transcription is polycistronic, all mRNAs are
trans spliced, there is almost no developmental regulation
of transcription, and the mitochondrial RNAs
are extensively edited. Energy and redox metabolism
also exhibit unique features: for example, the glycolytic
enzymes are compartmentalised in an organelle,
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Figure 1: Electron micrographs of trypanosomes. On the left
are normal trypanosomes with many round glycosomes (Gly).
On the right are cells with a 90% reduction in PEXII (Evelyn
Baumgart, Anatomy Dept.).
the glycosome. At the same time, the parasites show
many conserved features. For example, the glycosome
is a specialised type of peroxisome, and trans splicing
is mechanistically related to cis splicing.
Our work concentrates on Trypanosoma brucei brucei,
a close relative of the sleeping sickness trypanosomes
which is not infective for humans. The parasites grow
in laboratory rodents or in vitro and life-cycle related
differentiation can be mimicked in culture. They are
diploid, carrying about 60 Mb of DNA on 11 chromosome
pairs; a sequencing project is in progress. A full
palette of genetic manipulation methods is available,
including gene replacement by homologous recombination
and inducible gene expression. Conditional
knockouts can be made by placing the gene of interest
under control of a tetracycline-inducible promoter,
then deleting the remaining gene copies via homologous
recombination.
In our research on trypanosomes, we try to keep two
aims in view. Firstly, we concentrate on aspects of
the parasites that uniquely distinguish them from their
mammalian hosts, and may therefore represent suitable
targets for specific anti-parasitic chemotherapy.
Secondly, by examining the similarities and differences
between fundamental processes in trypanosomes,
mammals and yeast we can clearly distinguish
those aspects that are conserved throughout eucaryotic
evolution and therefore likely to be essential from
those that are required for specialised lifestyles.
Control of gene expression
Maciej Drodzd, Antonio Gonzales, Claudia Hartmann,
Henriette Irmer, Luis Quilada
Specialised surface molecules are essential for the
survival and virulence of pathogens. In the case of
trypanosomes, the forms that grow in the mammal
(bloodstream forms) wear a uniform coat of variant
surface glycoprotein (VSG), expressed from a single
gene chosen from a repertoire of up to 1000 different
VSG genes. The VSG expressed can be changed either
through recombination or by transcriptional switches.
This enables the parasites to change their surface coat,
thereby evading the immune response. In the Tsetse
midgut, the VSG is replaced by the EP and GPEET
repetitive, acidic proteins. All these surface proteins
are retained on the plasma membrane by a glycosyl
phosphatidylinositol (GPI) lipid anchor.
Normally, protein-coding genes can only be functionally
expressed by RNA polymerase II. This is because
the mRNAs have to be modified at the 5‘-end by
enzymes that are associated exclusively with RNA
polymerase II. The Kinetoplastids are not subject to
this restriction because the 5‘ spliced leader is capped.
The VSG and the EP/GPEET genes are transcribed
by RNA polymerase I as part of polycistronic tran-
scription units. Nearly all other protein-coding genes
are transcribed by RNA polymerase II, but again transcription
is polycistronic. Although the expression of
many genes has to be strongly regulated to enable
survival in the disparate environments of Tsetse and
mammal, there is no evidence for any developmental
control of RNA polymerase II transcription. Instead,
regulation usually requires sequences which are found
in the 3‘-untranslated regions and control RNA degradation
and/or translation. Polymerase I-mediated transcription
of the VSG genes is, exceptionally, subject to
strong developmental regulation, but the EP/GPEET
gene transcription is only weakly regulated.
To compensate for the lack of transcriptional regulation,
the EP/GPEET mRNAs are extremely unstable
and very poorly translated in bloodstream forms,
so that no protein product is detectable. A uridinerich
26 nt sequence in the 3‘-untranslated region of
EP/GPEET mRNA is essential for this control. We
have examined the mechanism of RNA degradation
in detail. Relatively stable RNAs such as the actin
mRNA appear to be degraded by the standard mechanism
seen in eucaryotes: initial destruction of the
poly(A) tail at the 3‘-end is followed by degradation
of the rest of the mRNA. The EP/GPEET mRNAs
are in contrast rapidly degraded in bloodstream forms
without prior degradation of the poly(A) tail. Detailed
characterisation of the EP RNA secondary structure
indicated that the 26mer is in an exposed, extended
conformation, so it should be accessible to RNA-binding
proteins or specific endonucleases. Searches for
specific 26mer-binding proteins by several methods
have however proved fruitless.
Searches of the available genome sequence have
revealed a number of potential trypanosome proteins
that are very similar to yeast enzymes involved in
mRNA degradation and processing of stable RNAs, or
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