51594
Trapanosoma - antigenic variation
(of T. brucei)
- How does Trypanosoma brucei survive in humans? (antigenic variation)
- Immune evasion
- T. brucei is highly susceptible to antibodies
- Lives in the bloodstream
(constantly exposed to Ab's)
- Induces a strong Ab response
- How then, does it survive and thrive in the
same host for periods greater than a year?
- Number of parasite in blood (level of parasitaemia)
over time is not constant - there are peaks and
throughs (time between peaks/troughs = 5-7 days)
- Each wave represents a antigenically
distinct serotype of parasite population
- Ab's generated against the parastie in the first week
will not react with those in the second week and so on
- This change in antigenic profile is called ANTIGENIC VARIATION
- The entire population within the blood at any one point appears to be uniform
- However at a low frequency (about 1 in 1,000,000 cells) there is a distinct difference in cell serotype (SWITCHING)
- However at a low frequency (about 1 in 1,000,000 cells) there is a distinct difference in cell serotype (SWITCHING)
- The entire population within the blood at any one point appears to be uniform
- This change in antigenic profile is called ANTIGENIC VARIATION
- Ab's generated against the parastie in the first week
will not react with those in the second week and so on
- Each wave represents a antigenically
distinct serotype of parasite population
- Number of parasite in blood (level of parasitaemia)
over time is not constant - there are peaks and
throughs (time between peaks/troughs = 5-7 days)
- How then, does it survive and thrive in the
same host for periods greater than a year?
- Lives in the bloodstream
(constantly exposed to Ab's)
- T. brucei is highly susceptible to antibodies
- Immune evasion
- Variable surface glycotprotein (VSG)
- When viewed using electron microscopy the surface of
T. brucei is seen as being very electron dense
- Antisera (Ab's against T. brucei) react strongly to this coat
- When surface proteins of T. brucei
cleaved off using the protease
trypsin then Ab's are unable to bind
- Implying detemrination of the organisms
atigenicity (and therefore antigenic
variance properties) is through this coat
- Implying detemrination of the organisms
atigenicity (and therefore antigenic
variance properties) is through this coat
- What is this coat?
- SDS-PAGE reveals that the coat is mainly
composed of almost a single protein type
(one prominent band on the gel)
- This protein is the variable surface glycoprotein (VSG)
- VSG's areM very immunogenic and their amino
acid sequence varies between parasites of
different parasitaemia peaks (5-7days)
- Structure
- 10 million per cell
- 65kDa glycoprotein (protein
with bound sugar structures)
- Forms about 10% of the cells total protein content
- VSG's form dimers
- Following synthesis; signal sequence (~20aa)-variable domain (~360aa)-conserved domain (~100aa)-hydrophobic sequence (~20aa)
- 10 million per cell
- VSG synthesis
- Transcription and translation
- 20aa signal sequence targets the protein for the ER
- N-terminal signal sequence cleaved within ER lumen
- VSG protein is translated and fed into the ER lumen
- The C-terminal hydrophobic domain binds the VSG to the phospholipid membrane of the ER
- This hydrophobic sequence is then cleaved and the VSG
molecule covalently attached to a glycolipid in the membrane
- The glycolipid is made of;
4 core sugar residues and
phosphatidylinisotol
- The sugar residues are
often branched and
additional residues added
- The sugar residues are
often branched and
additional residues added
- This is called the glycosylphosphatidylinositol (GPI) anchor
- Binds VSGs to the membrane and allows for the tight packing of surface VSGs
- This tight packing prevents immune
complement factors binding (preventing
MAC formation and phagocytosis)
- Also, other essential cell proteins (that cant exhibit
antigenic variance) can 'hide' beneath the VSG canopy
- Also, other essential cell proteins (that cant exhibit
antigenic variance) can 'hide' beneath the VSG canopy
- This tight packing prevents immune
complement factors binding (preventing
MAC formation and phagocytosis)
- Binds VSGs to the membrane and allows for the tight packing of surface VSGs
- Two VSGs then dimerise and are transported (via secretory pathway) to
the cell surface (ER -> golgi -> vesicles -> flagellal pocket -> cell surface)
- The glycolipid is made of;
4 core sugar residues and
phosphatidylinisotol
- This hydrophobic sequence is then cleaved and the VSG
molecule covalently attached to a glycolipid in the membrane
- The C-terminal hydrophobic domain binds the VSG to the phospholipid membrane of the ER
- VSG protein is translated and fed into the ER lumen
- N-terminal signal sequence cleaved within ER lumen
- 20aa signal sequence targets the protein for the ER
- Transcription and translation
- Changing expressn of VSGs (Antigenic variance mechanism)
- VSG genes
- T. brucei entire genome
- 10% (~1,000-1,500 genes) = VSG genes
- Two distinct pools of VSG localisation (within the entire genome)
- 1) subtelomeric (non-telomere DNA, but
towards the end of the chromosome)
- >1,000 VSG genes
- VSGs in large tandem arrays (one VSG after another)
- Mainly found on large
(megabase) chromosomes
- No associated promotors (non are expressed) = VSG store
- >1,000 VSG genes
- 2) telomeric pool
- ~200 VSG genes
- Seen mainly for microchromosomes (kilobases)
- No associated promoters (non are expressed) = VSG store
- Seen mainly for microchromosomes (kilobases)
- ~30-40 VSG genes
- Adjacent to promoters (can be
expressed) = expressin sites (ES)
- Two types of ES
- ~15-20 VSG genes
- Feature (in sequence);
promoter, 70bp repeat
sequence, VSG gene and
telomere
- Metacyclic expression sites (mES)
- In salivary gland of insect (metacyclic phase)
parasites express only one VSG from one mES
- All other VSGs from mES and bES are silenced (allelic exclusion)
- Metacyclic parasites in salivary gland of insect
are now prepared for survival in mammals
- Infection of mammal (human)
- Metacyclic form differentiates into the bloodstream form
- Active mES is now turned off and a single bES is turned on
- Only one VSG gene is expressed at
one time - all other ES's are silenced
- How can the parasite
switch expression
between different VSGs?
- The process of altering the VSG expressn is called "switching"
- *see reglation of VSG switching*
- *see reglation of VSG switching*
- Three mechanisms
- 1) in situ switching
- The active ES is switched of and an
incative ES switched on in its place
- Explains mES and bES
switching between off and on
- Explains mES and bES
switching between off and on
- The active ES is switched of and an
incative ES switched on in its place
- 2) Telomere exchange
- Double standed DNA recombination between
telomeric regions reaults in an inactive VSG
gene being transferred to an active ES
- Explains how VSGs on microchromosomes can be
expressed (from microchromosome VSG store)
- Explains how VSGs on microchromosomes can be
expressed (from microchromosome VSG store)
- Double standed DNA recombination between
telomeric regions reaults in an inactive VSG
gene being transferred to an active ES
- 3) gene conversion
- Single stranded DNA recombination between coding
sequences of an inactive VSG and an active VSG
- Mismatch repair system copies the new
VSG sequence in place of the previous one
- Previously active VSG is lost
- Explains how VSGs from subtelomeric stores can be expressed
- Explains how VSGs from subtelomeric stores can be expressed
- Previously active VSG is lost
- Mismatch repair system copies the new
VSG sequence in place of the previous one
- Single stranded DNA recombination between coding
sequences of an inactive VSG and an active VSG
- 1) in situ switching
- The process of altering the VSG expressn is called "switching"
- Most cells in one populatn express the same VSG
- The immune system selects for antigenically
distinct VSGs -> peaks of parasitaemia
- The immune system selects for antigenically
distinct VSGs -> peaks of parasitaemia
- How can the parasite
switch expression
between different VSGs?
- Only one VSG gene is expressed at
one time - all other ES's are silenced
- Active mES is now turned off and a single bES is turned on
- Metacyclic form differentiates into the bloodstream form
- Infection of mammal (human)
- Metacyclic parasites in salivary gland of insect
are now prepared for survival in mammals
- All other VSGs from mES and bES are silenced (allelic exclusion)
- In salivary gland of insect (metacyclic phase)
parasites express only one VSG from one mES
- Metacyclic expression sites (mES)
- Feature (in sequence);
promoter, 70bp repeat
sequence, VSG gene and
telomere
- ~15-20 VSG genes
- Feature (in sequecne); promoter,
other genes (x7), 70bp reapeat
sequences, VSG gene and telomere
- Boodstream expression sites (bES)
- Other genes include
useful proteins; Fe
transporters,
adenylate cyclase etc.
- Boodstream expression sites (bES)
- Feature (in sequecne); promoter,
other genes (x7), 70bp reapeat
sequences, VSG gene and telomere
- ~15-20 VSG genes
- Two types of ES
- Adjacent to promoters (can be
expressed) = expressin sites (ES)
- ~200 VSG genes
- 1) subtelomeric (non-telomere DNA, but
towards the end of the chromosome)
- Two distinct pools of VSG localisation (within the entire genome)
- 10% (~1,000-1,500 genes) = VSG genes
- T. brucei entire genome
- VSG genes
- Structure
- VSG's areM very immunogenic and their amino
acid sequence varies between parasites of
different parasitaemia peaks (5-7days)
- This protein is the variable surface glycoprotein (VSG)
- SDS-PAGE reveals that the coat is mainly
composed of almost a single protein type
(one prominent band on the gel)
- When surface proteins of T. brucei
cleaved off using the protease
trypsin then Ab's are unable to bind
- Antisera (Ab's against T. brucei) react strongly to this coat
- When viewed using electron microscopy the surface of
T. brucei is seen as being very electron dense
- Regulation of VSG switching
- These are only theories - exact
mechanisms still to be discovered
- In situ switching, telomere exchange and gene
conversion explain how switching may occur
- However, this does not explain why only
one ES is ever active and the rest silenced
- 1) telomeric silencing
- All VSG ES's are located at the telomeres
- Protein complexes may bind to the telomere
that actively supress transcriptional activity
- Proteins that have been shown to repress
transcription at telomere sites in yeast have been
found in T. brucei (more conclusive evidence needed)
- These proteins are; repressor/activator protein 1 (RAP1)
and the silent information regulator (SIR) complex
- These proteins are; repressor/activator protein 1 (RAP1)
and the silent information regulator (SIR) complex
- Proteins that have been shown to repress
transcription at telomere sites in yeast have been
found in T. brucei (more conclusive evidence needed)
- Protein complexes may bind to the telomere
that actively supress transcriptional activity
- All VSG ES's are located at the telomeres
- 2) modified bases
- Trypanosomes contain unusual J-bases
(beta-glycosyl-hydroxy-methyluracil -
modified thymine + glucose)
- J-bases in DNA may act as "read me" or "don't
read me" signals to the transcription machinery
- J-bases inhibitory to transcription
- Found in inactive ES's at
subtelomeric regions
- J-bases inhibitory to transcription
- J-bases in DNA may act as "read me" or "don't
read me" signals to the transcription machinery
- Trypanosomes contain unusual J-bases
(beta-glycosyl-hydroxy-methyluracil -
modified thymine + glucose)
- 3) chromatin modification and structure
- Chromatin = DNA + proteins (histones)
- Modifications to histones and DNA can change
their interaciton and affect gene expression
- Modifications to histones and DNA can change
their interaciton and affect gene expression
- Chromatin = DNA + proteins (histones)
- It is still unclear however how one ES
remains active while all others repressed
- Why one gene is 'selected' and all
others repressed simultaneously
- The ES body
- Seems that gene location within the
nucleus detemrines its expression fate
- In an experiment looking at RNA pol
localisation in T. brucei during the
bloodstream and procyclic (insect) phases
- Navarro and gull (2001)
- Bloodstream form showed two distinct regions of gene
expression (generic euchromatin expression and a second,
smaller region of expression)
- However, in insect form (procyclic) only
the generic region of expression was seen
- This smaller region of expressn
correspnds to the single active ES
- It was also seen that inactive ES's were
kept away from the ES body and there
localisation was perinuclear (around the
nuclear border)
- It is localised wihtin a region of
the nucleus called the ES body
- The ES body can only accomodate one ES
- therefore only one VSG ever expressed
- The ES body can only accomodate one ES
- therefore only one VSG ever expressed
- It was also seen that inactive ES's were
kept away from the ES body and there
localisation was perinuclear (around the
nuclear border)
- This smaller region of expressn
correspnds to the single active ES
- However, in insect form (procyclic) only
the generic region of expression was seen
- Navarro and gull (2001)
- Seems that gene location within the
nucleus detemrines its expression fate
- The ES body
- Why one gene is 'selected' and all
others repressed simultaneously
- 1) telomeric silencing
- However, this does not explain why only
one ES is ever active and the rest silenced
- These are only theories - exact
mechanisms still to be discovered
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