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Sleeping Sickness andTrypanosomes I
z Life cycle and biology of trypanosomes
z Sleeping sickness, differences betweengambiense and rhodesiense
z Nagana, kachexia and TNF
z Drugs used to treat typanomiasis
z Tse tse flies
Trypanosomes II
z Antigenic variationz Important pathways
discovered along the wayto understand antigenicvariation: trans splicingand GPI anchors
z Parasites living in theinsect have a differentsurface and metabolism
z The trypansomemitochondrion and RNA-editing
Why is trypanosomiasisso deadly?
z Trypanosomes are highlysusceptible to antibodiesand complement
z They life fully exposed toantibodies in the bloodstream
z They induce a verystrong antibody response
z Still they manage tothrive in the same hostfor a year or longer, untilthe host dies
Why is trypanosomiasisso deadly?
z The number of parasitesfound in the blood ofhumans and lab animalsinfected with trypanosomesis not constant, but showcharactristic waves ofparasitemia
z The difference betweenparasitemia peaks isabout5-7 days
Why is trypanosomiasisso deadly?
z Infection is characterizedby periodic waves ofparasitemia
z Each wave represents asingle antigenically distinctclone or serotype
z E.g. antibodies produce inthe first week against cloneA will not react with clone B
z The changing display ofdifferent antigens is calledantigenic variation
Antigenic variation
z The entire population oftrypanosomes within ananimal seemsantigenically uniform
z But at a very lowfrequency divergent (socalled switched)serotypes areencountered
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Antigenic variation
z Trypanosomes arecovered with a densesurface coat
z Variant specificantisera stronglyreact with this surfacecoat
z Surface coats fromdifferent clones areantigenically distinct
Antigenic variation
z Trypsin treatment completelyremoves the surface coatfrom Trypanosomes (trypsinis a protease, an enzymethat specifically digestsproteins)
z This treatment also abolishesantibody binding
z This suggests that theantigenic determinant on thesurface is made up of protein
Antigenic variation
z The surface coat is made upalmost entirely by a singleprotein the Variant SurfaceGlycoprotein or VSG
z This protein is highlyimmunogenic anddistinguishes the clones insuccessive parasitemia peaks
z VSGs from differentparasitemia peaks differ intheir amino acid sequence
Lessons learned along theway: the GPI anchor
z When cDNAs (mRNAs) of T.brucei VSGs were sequencedthey were shown to encode ac-terminal hydrophopic hichcould anchor the protein
z However when the proteinswere sequence this part wasabsent -- how is this solubleprotein kept in the membrane?
z VSG is anchored into themembrane via a glycolipidanchor (glycosyl-phosphatidylinositol or GPI)
Lessons learned along theway: the GPI anchor
z GPI anchors have beenshown to be present in allorganisms
z The GPI anchor issynthetized as a precursorglycolipid in theendoplasmic reticulum bysequential addition of sugarmolecules to a phospholipid
z The mature precursorcontains a terminalethanolamine phosphatewhich can form a peptidebond with the c-terminalcarboxy group of the protein
Lessons learned along theway: the GPI anchor
z GPI anchor additionoccurs immediately aftertranslation
z The c-terminalhydrophobic portion ofthe protein serves as asignal sequence andcleaved and replacedwith the glycolipid in atrans-peptidation reaction
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Antigenic variation
z GPI anchors allow verydense packing of moleculeson the surface of theparasite
z VSGs forms a dense coaton the surface of thetrypanosome
z This coat is equivalent of thecoat form bylipophosphoglycan inLeishmania
Antigenic variation
z All VSGs are 65 kDAglycoproteins, and arepresent on the surface asdimers
z The outer domain is highlyvariable and the onlyconservation detected isthe position of cysteines
z Other (non-variant)proteins like transferrinreceptor or hexosetransporter are hidden inthe this surface coat
Antigenic variation
z 6-10% of the total genome of african trypanosomesis coding for VSGs (more than 1000 genes)
z Only one is expressed at a given time the other 999genes are shut down and completely silent
z At a low frequency a switch to a different geneoccurs, if the host develops antibodies against theprevious VSG the new clone is strongly selected
z What is the advantage of expressing a single VSG?z How is expression controlledz What mechanism can you think by which a cell
could control which proteins it makes?
Antigenic variation
z mRNA derived from only a single VSG genecan be detected at one time
z VSG expression is controlled at the level oftranscription initiation
z Regulation of promoter activity is used tocontrol gene expression in many organisms
Transcription in trypanosomesis polycistronic
z But, only very few promotershave been identified intrypanosomes and they did notseem to control the expression
z Also surprisingly transcription intrypanosomes was found to bepolycistronic
z Polycistronic means that anumber of genes are transcribeinto one long messenger RNA
z In bacteria this message istranslated into protein, intrypanosomes furtherprocessing is needed and thisprocessing might conferadditional level of control
Transcription in trypanosomesis polycistronic
z The 39 first (5’) base pairs ofall trypanosme mRNAs areidentical, furthermore thissequence is not found in thegenomic locus of these genes
z Individual mature mRNAs arederived from largepolycistronic transcripts by aprocess called trans-splicing
z In this process mRNAs forindividual genes are cut out ofthe polycistronic transcriptand a short RNA transcribedfrom a different locus (thesplice leader) is attached to it5’ end
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Trans-splicing
z The mechanism and enzymes used for transsplicing are very similar to cis-splicing
z Cis splicing is the process that removes theintrons from mRNAs of eukaryotic genes
Antigenic variation
z If it is not the promoter maybe it is the exactlocation in the genome that predisposes aspecific VSG for expression
z But how could that be switched then?
VSGs are expressed from telomericpolycistronic expression sites
z Transcription in trypanosome is polycistronic as wehave seen
z Active VSG genes are allways at the “ends” ofchromosomes (teleomeres)
z Genes are read in (20) expression sites like tapesin a tape recorder but only one recorder is playingat a time
z How do you get a new tape in and how are therecorders controlled
Several mechanisms forswitching have been discovered
Antigenic variation
z Transposition of VSGgenes occurs by intra-or intermolecularrecombination
z This explains switchingbut not really why onegene is active and allthe others are silent
Antigenic variation
z Regulation could be achieved by modification ofchromatin (by sticking on a read me or do not read melabel
z Indeed active and inactive sites differ in the amount ofa special modified base called J (b-glucosyl-hydroxy-methyluracil)
z But is this a chicken or an egg?
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VSG is transcribed by Pol I
z Cells have specialized RNApolymerases to transcribe differentgenes
z In most cells mRNA which encodesproteins is transcribed by thepolymerase Pol2 (this enzyme canbe inhibited by the toxin a amanitin)
z Ribosomal RNA is generallytranscribed by Pol1 (which isresistant to the toxin
z VSG transcription is insensitive toa amanitin suggesting it istranscribed by the highlyprocessive Pol I (however all othermRNAs for proteins seem to bemade by Pol2 as everywhere else)
Drug
rRNAtubulin
VSG
How is a single expressionsite activated?
z Location, location,location
z PolI antibody detectstwo spots in bloodstream forms: thenucleolus (whererRNA is made) and asecond locus outsideof the nucleolus
How is a single expressionsite activated?
z The additional spot of PolI is not the nucleolus
How is a single expressionsite activated?
active VSG
inactive VSG
z Active, not inactive VSG expression sitescolocalize with the extranuclelarPolI spot
Antigenic variation
z Only a single VSG gene out of ~1000 isexpressed
z Expression occurs out of teleomeric expressionsites (the tape recorder)
z To switch genes on they transposed into anactive expression site by several mechanisms
z Expression seems to be controled by physicalassociation of ES with a single POL1transcription particle per nucleus
z There are 1000 tapes, 20 tape recorders butonly one is plugged in