antigenic variation

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African Sleeping Sicknessand

Antigenic Variation

Lecture 10

Antigenic Variation The entire trypanosome

population seems antigenicallyuniform but at a very lowfrequency divergent (so calledswitched) serotypes areencountered

The switch to a new serotype isnot recognized by the hostantibody population

“Switchers” survive & proliferateleading to a new wave ofparasitemia

Serotype switching continues Antigenic Variation

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Antigenic Variation

T. brucei is covered with a densesurface coat

Variant specific antisera stronglyreact with surface coat

Surface coats from different clonesare antigenically distinct

Antigenic Variation

Trypsin (or other protease)treatment completely removes thesurface coat from T. brucei

This treatment also abolishesantibody binding

This suggested that the antigenicdeterminant on the surface ismade of protein

No protease treatment

+ protease treatment

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Surface coat consists of a single glycoprotein

65 kDa glycoprotein C-terminus anchored in the membrane

(GPI-anchor) Only epitopes in the N-terminal 1/3 are

exposed Constant and variable regions VSG forms dimers

VSGs from different clonal variantshave same molecular weight, butdifferent amino acid composition

Different VSG share only 16% aminoacid similarity, but yet adopt a nearlyidentical tertiary structure!

Variant Surface Glycoprotein

• Single VSG type uniformlycovers surface of parasite (107

copies)• VSG forms 12-15 nm electron

dense surface coat• VSG dimers form a densly

packed surface coat

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Variant Surface Glycoprotein

Different VSG share only 16%similarity, but yet adopt anearly identical tertiarystructure!

Variableregion

Constantregion

T. brucei life cycle

non-dividingfuel=glucose

VSG coatmito=“low”

Dividing formfuel=glucose

VSG coatmito=“off”

non-dividingfuel=?

mVSG coatmito=?

Dividing formfuel=amino acids

Procyclin coatmito=“on”

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T. brucei has ~ 1000 different VSG genes

Great variability of chromosome sizeamong isolates

11 diploid megabase chromosomes,intermediate size, and about 100minichromosomes - all classes containVSG genes

6-10% of the total DNA codes for VSGs(~1000 genes)

Only a single VSG is expressed ata time!

At a low frequency a switch to a differentgene occurs, the host developedantibodies against the previous VSG sothe new clonal cell line is stronglyselected.

Genome organization11 Megabase chromosomes (1-6 Mbp)

1-7 Intermediate chromosomes (200-700 kbp)~100 Minichromosomes (50-150 kbp)

VSG Antigenic Variation

What is the advantage to expressing a single VSG?

What mechanisms can you think of that could controlgene expression and protein abundance?

How is VSG expression controlled?

VSGswitch

Immune destruction

by host Proliferation

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Genomic Location of VSGs

The VSG Expression Site

Long polycistronic transcript Approximately 20-40 Bloodstream expression sites (BES) in the genome Active VSG genes are always at the “ends” of the chromosomes

(telomeres)

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VSG in Minichromosomes

VSG genes atminichromosometelmomers

Switching via telomereconversion or reciprocaltelomere exchange

Mechanisms of Switching

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Creation of Mosaic VSGs

VSG switching

Transposition of VSGgenes occurs by intra-or intermolecularrecombination

This explains switchingbut not really why onegene is active and allthe others are silent

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Expression Sites

VSG

VSG

active

inactiveJJJJJJJJ JJ JJJJJJ JJ

JJJJJJJJJ

JJJX

The hyper-modified Base J

But is J a chicken or an egg?

Regulation could be achieved by modification of chromatin

β-glucosyl-hydroxy-methyluracil a T variant

Base J

Expression Site Body (ESB) How is a single expression site

activated? LOCATION!

Differential localization ofRNA polymerase I rRNA transcription in other

eukaryotes by RNA Pol I usually RNA Pol II transcribes

proteins coding sequences

Localizes to nucleolus in PFand BSF

Extranulcleolar in BSF BloodstreamProcyclic

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Expression Site Body (ESB)

Procyclic

Bloodstream

Red: anti-fibrillarin - nucleolus marker Green: anti-RNA Pol I

The additional spot of RNA Pol I localization is NOT the nucleolus

Expression Site Body (ESB)

Active, not inactive VSG expression sites co-localize with theextranuclear Pol I spot.

GFP shows the position of the respective VSG genes in the nucleus

Active221ES

Inactive121ES

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Transcriptional analysis of expression sites

Transcription of ES sites duringdevelopment

Initiation occurs in several sites, butis abortive

Only in an active ES site is RNAelongation productive

Hypothesis: there is a limitedsupply of factors (transcription)connecting Pol I polymerase toelongation/processing machinery

Hypothesis: these factors arelocated in the ESB

Antigenic Variation Key Points

General features of Antigenic Variation (non-viral)Requires a family of variant sruface antigen genesRequires a mechanism to express only one gene at a timeRequires a mechanism to switch genes

Trypanosomes - ~2000 VGS genes (variant surface glycoprotein) Expression occurs out of telomeric expression sites (ES) (tapes/tape

recorder or CDs/ CD player) Expression seems promoter independent To switch genes on, they are transposed into an active expression site by

several mechanisms Expression seems to be controlled by a physical association of ES with a

single RNA Pol I transcription particle (location) per nucleus

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Subnuclear location of Pol I

Navarro Model

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Taking a Tryp(anosome) Across theBlood-Brain Barrier - Part 1

Data acquiredfrom animalmodels - experimental infections with T. brucei brucei

Laminin α5

Endothelium

Major structural elements

Review by Masocha et al 2007 Phys & Behav 92:110-114


Laminin α4 Laminin α5

Trypanosome

Masocha et al 2004 J. Clin Invest 114:689-694

Endothelialmembrane

Parenchymalmembrane

Laminin

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In vitro BBB Model


Laminin α4 Laminin α5

Grab et al 2004 J. Parasitol 90:970-979.

Data acquiredfrom in vitro BBBtissue culture models:T. brucei gambiensi

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Grab et al 2004 J. Parasitol 90:970-979.

antigenic variation

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