Trapanosoma - antigenic variation (of T. brucei)

How does Trypanosoma brucei survive in humans? (antigenic variation)
1. Immune evasion
  1. T. brucei is highly susceptible to antibodies
    1. Lives in the bloodstream (constantly exposed to Ab's)
    2. Induces a strong Ab response
      1. 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)
Variable surface glycotprotein (VSG)
1. When viewed using electron microscopy the surface of T. brucei is seen as being very electron dense
  1. Antisera (Ab's against T. brucei) react strongly to this coat
    1. When surface proteins of T. brucei cleaved off using the protease trypsin then Ab's are unable to bind
      1. Implying detemrination of the organisms atigenicity (and therefore antigenic variance properties) is through this coat
    2. What is this coat?
      1. 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.
Regulation of VSG switching
1. These are only theories - exact mechanisms still to be discovered
2. In situ switching, telomere exchange and gene conversion explain how switching may occur
  1. However, this does not explain why only one ES is ever active and the rest silenced
    1. 1) telomeric silencing
      1. 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. 2) modified bases
      1. 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. 3) chromatin modification and structure
      1. Chromatin = DNA + proteins (histones)
        Modifications to histones and DNA can change their interaciton and affect gene expression
    4. It is still unclear however how one ES remains active while all others repressed
      1. 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

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Trapanosoma - antigenic variation (of T. brucei)

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