African Trypanosomes & Sleeping Sickness II

1. African Trypanosomes & Sleeping Sickness II

2. Sleeping Sickness and Trypanosomes I • Life cycle and biology of trypanosomes • Sleeping sickness, differences between gambiense and rhodesiense • Nagana, kachexia and TNF • Drugs used to treat trypanomiasis • Tse tse flies, fly control

3. 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

4. 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?

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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)

13. 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

14. 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

15. 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

16. 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?

17. 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

18. 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

19. 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.

20. 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?

21. 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

22. 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

23. 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

24. 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 (b-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

25. For the next experiment we need a mushroom Amantia bisporingea, the Destroying Angel http://www.mushroomexpert.com

26. VSG is transcribed by Pol I • a-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 a amanitin) • Ribosomal RNA is generally transcribed by Pol1 (which is resistant to the toxin) • VSG transcription is insensitive to a-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? tubulin rRNA VSG Drug

27. African trypansome cellular architecture Nucleus Nucleoulus Kinetoplast

28. 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 Pol I DNA Nature 414:759-63 mammal insect

29. 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

30. How is a single expression site activated? • 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 active VSG inactive VSG Nature 414:759-63

31. Antigenic variation • Only a single VSG gene out of ~1000 is expressed • Expression occurs out of teleomeric expression sites (the tape recorder) • To switch genes on they are transposed into an active expression site by several mechanisms • Expression 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