Lecture 2 Flashcards
(22 cards)
T. brucei infection in man
- Lives extra-cellularly in mammalian bloodstream
- Elicits host immune response
- Peaks and throughs of parasitaemia represent repeated cycles of immune destruction and regrowth
Variant surface glycoproteins
- Abundant surface protein (7-10% of cellular protein)
- Electron dense, homogenous surface coat
- No known enzymatic/receptor function - parasite protective
- Over 2000 VSG genes/pseudogenes which are immunologically distinct (<25% identity)
Structure of VSGs
Homodimer of two 50-60kDa subunits
Held on plasma membrane by GPI anchor
- Large N-terminal domain of 250-400 resiudes
- 1-2 smaller C-terminal domains (20-40 residues) connected by flexible linkers
- A, B, or C N-terminals and 6 types of C-terminal domains based on sequence and cysteine residues
- 5.7x10^6 VSG dimers
function of VSGs
- Shields antibody binding to proteins e.g. hexose transporter and lipids
- Prevents membrane attack complex formation
Two conformations of VSGs
- Tightly packed VSGs above transmembrane proteins - 40A gaps, 155A height
- Relaxed conformation maintains protective coat at reduced protein density - 60A gaps, 140A height
VSG antibody response
Trypanosomes internalise VSG-Ig complex, degrade antibodies and return VSG to surface
Occurs via flagellar pocket (5% surface) but pool of VSGs recycled in 12 minutes
VSG-IgG turns over even quicker
Removal of antibodies from surface
Movement of immune complexes depends on cellular motility
Hydrodynamic flow acting on swimming trypanosomes causes directional movement of IgG-VSG with plasma membrane plane
- Antibody acts as molecular sail (sweeps Ab-VSG complex back to flagellar pocket
- Uptake occurs via endocytosis
What does increased IgG concentration cause regarding intact cells
Increased IgG creates more resistance to complement mediated lysis
Short vs stumpy trypanosomes with regards to VSG-antibody removal
Short stumpy removes VSG-antibody complex faster than long slender
Short stumpy have greater resistance to antibody-mediated killing
Stumpy forms predominate at peak parasitaemia and extend period which infection is transmitted to tsetse flies
How long do trypanosomes multipy in blood for?
Until antibody response results in lysis if recognised variants
Switched VSG variants have selective growth advantage
VSG genes in bloodstream form
VSG genes expressed from expression sites at telomeres
Expression sites transcribed by RNA polymerase I (512-fold higher rate of VSG mRNA production compared to Pol II transcribed gene)
Polycistronic transcription - individual mRNAs generated by processing polycistronic transcript
Bloodstream expression sites contain expression site associated genes that encode proteins like transferrin receptors
ESAG repertoire differs between different ESs.
Trypanosome mRNA processing by mono cistron formation
Mono-cistrons formed from polycistronic primary transcript after addition of:
- Spliced-leader RNA - 39 nucleotides at 5’ end of mRNA
- PolyA tail added to 3’ end
chromosomes in trypanosomes
One VSG expressed at a time
Types of chromosomes in trypanosomes:
- megabase (11 diploid pairs, 1-6Mbp)
- intermediate (1-5 chromosomes, 200-900kb)
- Minichromosomes (100 chromosomes, 30-150kb)
How many expression site bodies are in BSF T. brucei
1
Nuclear lamina pathway
Nuclear lamina creates repressive environment
VSG ESs localise to nuclear envelope in insects
Knockdown of RAP1 silences VSGs
Chromatin regulatory factors repress transcription near promoter
VEX1, VEX2 and CAF-1
VEX1 - Enriched in ESB but lacks orthologs in other cells - Binds telomeric DNA and VEX2
VEX2 - Ortholog of nonsense-mediated-decay helicase, UPF1. In humans, UPF1 ortholog influences mRNA biogenesis
CAF-1 - Histone chaperone - loads H3-H4 dimers onto DNA during replication
VEX/CAF-1 interaction - required for VEX reassembly and VSG exclusion inheritance
What occurs when VEX complex depletes
multi-VSG expression
VEX1/2 double RNAi knockdown is cytocidal
Also causes de-repression of VSG-ESs
mRNA maturation via trans-splicing
SL-RNA transcribed from two arrays of >100 SL RNA genes
VSG forms stable inter-chromosomal interaction with SL-array for mRNA splicing/VSG monogenic expression
VEX2 - associates with active VSG transcription compartment
VEX1 - associates with SL-RNA compartment
VEX2 sustains exclusive interaction between single active VSG ES and SL RNA genes – if VEX2 expression silenced all VSG ESs can access SL-array and are de-repressed
Cell configures chromatin architecture to facilitate interaction between SL RNA genes and active VSG ES - ensures sufficient SL RNA is available and not limiting for VSG mRNA expression (VSG = 10% total cellular protein)
VSG expression factory
Chromosomal context of VSG genes
VSG genes/pseudogenes occupy megabase, intermediate and minichromosomes
VSG genes separated from ESAGs by 70bp repeats
Adjacent to telomere repeats
Repetitive sequencing important for VSG switching
VSG switching hierarchy
In situ switches and telomere conversion events involving expression site-associated VSGs
VSG genes activation from silent chromosomal internal positions
Assembly of VSG gene mosaics
Explain an overview of antigenic variation
1, VSG promotes T. brucei survival in bloodstream by protection/immune evasion
- 1 VSG type expressed at any given time
- Anti-VSG antibodies lead to parasite elimination - trypanosome can clear low Ab levels
- Switching - Old VSG coat replaced by new one to protect from immune destruction
- VSG pseudogene recombination provides almost limitless antigenic repertoire
