Trapanosoma - antigenic variation (of T. brucei)

Trapanosoma - antigenic variation (of T. brucei)

1. 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)

2. 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
       • 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)
               • 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
                             • 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
                             • Two VSGs then dimerise and are transported (via secretory pathway) to the cell surface (ER -> golgi -> vesicles -> flagellal pocket -> cell surface)
               • 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
                         • 2) telomeric pool
                           • ~200 VSG genes
                             • Seen mainly for microchromosomes (kilobases)
                             • No associated promoters (non are expressed) = VSG store
                           • ~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*
                                                       • 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
                                                         • 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)
                                                         • 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
                                                     • Most cells in one populatn express the same VSG
                                                       • The immune system selects for antigenically distinct VSGs -> peaks of parasitaemia
                                 • ~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.

3. 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
       • 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
       • 3) chromatin modification and structure
         • Chromatin = DNA + proteins (histones)
           • Modifications to histones and DNA can change their interaciton and affect gene expression
       • 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