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African Trypanosomes & Sleeping Sickness II
Sleeping Sickness and Trypanosomes I
Life cycle and biology of trypanosomesSleeping sickness, differences between
gambiense and rhodesienseNagana, kachexia and TNFDrugs used to treat trypanomiasisTse tse flies, fly control
Trypanosomes II
Why is African trypanosomiasis such a deadly disease?
Important pathways discovered along the way to understand this problem: trans splicing and GPI anchors
The nuts & bolts of trypanosme gene expression control
Why is trypanosomiasis so deadly?
Infection is chronic and ultimately fatal if left untreated.
How can the slender trypomastigote form of the parasite survive in the human bloodstream?
Why is trypanosomiasis so deadly?
Trypanosomes are highly susceptible to antibodies and complement
They live fully exposed to antibodies in the blood stream
They induce a very strong antibody response
Still they manage to thrive in the same host for a year or longer, until the host dies
Why is trypanosomiasis so deadly?
The number of parasites found in the blood of humans and animals infected with trypanosomes is not constant, but shows characteristic waves of parasitemia
The window of time between parasitemia peaks is about 5-7 days
Why is trypanosomiasisso deadly?
Infection is characterized by periodic waves of parasitemia
Each wave represents a single antigenically distinct clone or serotype
Antibodies produced in the first week against clone A will not react with clone B
The changing display of different antigens is called antigenic variation
Antigenic variation is an important form of immune evasion
Antigenic variation
The entire population of trypanosomes within an infected animal seems antigenically uniform
But at a very low frequency divergent (so called switched) serotypes are encountered
Antigenic variation
Trypanosomes are covered with a dense surface coat
Variant specific antisera strongly react with this surface coat
Surface coats from different clones are antigenically distinct
Antigenic variation
Trypsin treatment completely removes the surface coat from Trypanosomes (trypsin is a protease, an enzyme that specifically digests proteins)
This treatment also abolishes antibody binding
This suggests that the antigenic determinant on the surface is a protein
Antigenic variation
The surface coat is made up almost entirely by a single protein the Variant Surface Glycoprotein or VSG
This protein is highly immunogenic and distinguishes the clones in successive parasitemia peaks
VSGs from different parasitemia peaks differ in their amino acid sequence
Lessons learned along the way: the GPI anchor
When genes for T. brucei VSGs were sequenced they were shown to encode a c-terminal hydrophopic peptide that could anchor the protein
However when the proteins were sequenced this part was absent -- how is this soluble protein kept in the membrane?
VSG is anchored into the membrane via a glycolipid anchor (glycosyl-phosphatidylinositol or GPI)
Lessons learned along the way: the GPI anchor
Initially thought to be specific for trypanosomes GPI anchors have been shown to be present in all eukaryotic organisms
The GPI anchor is synthesized as a precursor glycolipid in the endoplasmic reticulum by sequential addition of sugar molecules to a phospholipid
The mature precursor contains a terminal ethanolamine phosphate which can form a peptide bond with the c-terminal carboxyl group of the protein
Antigenic variation
GPI anchors allow very dense packing of molecules on the surface of the parasite
VSGs forms a dense coat on the surface of the trypanosome
This coat is equivalent of the coat form by lipophosphoglycan in Leishmania
Antigenic variation
All VSGs are 65 kDA glycoproteins, and are present on the surface as dimers
The outer domain is highly variable and the only conservation detected is the position of cysteines
Other (non-variant) proteins like transferrin receptor or hexose transporter are hidden in the this surface coat
Antigenic variation
6-10% of the total genome of African trypanosomes is coding for VSGs (more than 1000 genes)
Only one is expressed at a given time the other 999 genes are shut down and completely silent (allelic exclusion)
At a low frequency a switch to a different gene occurs, if the host develops antibodies against the previous VSG the new clone is strongly selected
What is the advantage of expressing a single VSG? How is expression controlled? What mechanisms can you think of by which a cell
could control gene expression and protein abundance?
Antigenic variation
mRNA derived from only a single VSG gene can be detected at one time
VSG expression is controlled at the level of transcription initiation
Regulation of promoter activity is used to control gene expression in many organisms
Transcription in trypanosomes is polycistronic
But, only very few promoters have been identified in trypanosomes and they did not seem to control the expression
Also surprisingly transcription in trypanosomes was found to be polycistronic
Polycistronic means that a number of genes are transcribed at the same time into one long messenger RNA
In bacteria this message is translated into protein, in trypanosomes further processing is needed and this processing might confer additional level of control
Transcription in trypanosomes is polycistronic
Individual mature mRNAs are derived from large polycistronic transcripts by a process called trans-splicing
In this process mRNAs for individual genes are cut out of the polycistronic transcript and a short RNA transcribed from a different locus (the splice leader) is attached to it 5’ end
Initially this was thought to be the key to regulation – but it is not.
Antigenic variation
If it is not the promoter or the processing maybe it is the exact location in the genome that predisposes a specific VSG for expression
Where are active and inactive genes in the genome?
How could a location based system switch?
VSGs are expressed from telomeric polycistronic expression sites
Transcription in trypanosome is polycistronic as we have seen
Active VSG genes are allways at the “ends” of chromosomes (telomeres)
Genes are read in (20) expression sites like CDs in CD players but only one CD player appears to be playing at a time
How do you get a new CD in and how are the CD players controlled
Several mechanisms for switching have been discovered
The most common mechanism of VSG switching requires physical transposition of a new VSG gene into the active expression site
Antigenic variation
There are several mechanisms but the most common mechanism of VSG switching requires physical transposition of a new VSG gene into the active expression site
Transposition of VSG genes occurs by intra- or intermolecular recombination
This explains switching but not really why one gene is active and all the others are silent
Antigenic variation
Regulation could be achieved by modification of chromatin (by sticking on a read me or do not read me label)
Indeed active and inactive sites differ in the amount of a special modified base called J (-glucosyl-hydroxy-methyluracil)
But is this the chicken or the egg? Recent work from Dr. Sabatini’s lab here at UGA shows that J is likely not
controlling expression but is important for switching & recombination
For the next experiment we need a mushroom
http://www.mushroomexpert.com
Amantia bisporingea, the Destroying Angel
VSG is transcribed by Pol I -amanitin is a specific and highly
potent RNA polymerase inhibitor Cells have specialized RNA
polymerases to transcribe different genes
In most cells mRNA which encodes proteins is transcribed by the RNA polymerase Pol2 (this enzyme can be inhibited by the toxin amanitin)
Ribosomal RNA is generally transcribed by Pol1 (which is resistant to the toxin)
VSG transcription is insensitive to -amanitin suggesting it is transcribed by the highly processive Pol I (however all other mRNAs for proteins seem to be made using Pol II as everywhere else)
How could this help to explain allelic exclusion?
Drug
rRNA
tubulin
VSG
African trypansome cellular architecture
Nucleoulus
Nucleus
Kinetoplast
How is a single expression site activated?
Location, location, location
PolI is found in two spots in blood stream forms: the nucleolus (where rRNA is made) and a second locus outside of the nucleolus
insect mammal
Pol I
DNA
Nature 414:759-63
How is a single expression site activated?
The additional spot of Pol I is not the nucleolus (Fib in red is a nucleolus marker)
Nature 414:759-63
How is a single expression site activated?
active VSG
inactive VSG
Active, but not inactive VSG expression sites colocalize with the extranuclear Pol I spot. GFP in green shows the position of the respective VSG gene in the nucleus
Nature 414:759-63
Antigenic variation
Only a single VSG gene out of ~1000 is expressedExpression occurs out of teleomeric expression
sites (the tape recorder)To switch genes on they are transposed into an
active expression site by several mechanismsExpression seems to be controlled by physical
association of the expression site with a single POL1 transcription particle per nucleus
There are 1000 CDs, 20 CD players but only one is plugged in