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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