Lecture 2: Immune Evasion by Antigenic Variation

Question 1

True or false: T. brucei infection in man is intracellularly in the bloodstream?

Answer 1

False: extracellularly

Question 2

What causes the peaks and troughs in parasitaemia in T.brucei infection?

Answer 2

QS threshold reached causing differentiation into stumpy form

Elicit immune response that causes parasite destruction and regrowth of parasite population (another wave of parasitaemia starts)

Question 3

What is expressed by parasites in the mammalian bloodstream?

Answer 3

VSGs (variant surface glycoprotein)

Question 4

What is the main function of VSG?

Answer 4

Protect parasite from immune attack because VSG is highly expressed and covers the entire surface of the parasite - high packing density physically obstructs access to plasma membrane by antibodies and prevent MAC formation (prevent complement-mediated killing)

Question 5

True or false: VSGs are antigenically diverse?

Answer 5

True (more than 2000 VSG gene/pseudogenes that produce immunologically distinct VSGs)

Question 6

How many VSG genes are expressed at a time? what is this called?

Answer 6

one

called allelic exclusion

Question 7

Describe the structure of VSG

Answer 7

expressed as homodimers bound to the plasma membrane via C-terminal GPI anchor
VSGs can diffuse freely in the plane of the plasma membrane
- N-terminal signal sequence allows protein to be directed to plasma membrane

Question 8

modelling suggests VSG can adopt two conformations, what are they?

Answer 8

1. tightly packed VSGs elevate VSG above transmembrane proteins to protect the plasma membrane
2. relaxed conformation maintains protective coat at reduced protein density

Question 9

What is the importance of VSGs preventing access of antibodies to plasma membrane proteins?

Answer 9

Antibodies wont be generated against the plasma proteins, antibodies will only be generated against the VSGs

Question 10

What happens to VSG coat in low levels of anti-VSG antibodies?

Answer 10

internalise VSG bound to antibodies (move to posterior end of parasite into the flagellar pocket by endocytosis)
- antibody is degraded by lysosome and the VSG is recycled and returned to the surface

Question 11

What is the name for the directional movement of Ig-VSG complex within the plane of the plasma membrane and what causes this?

Answer 11

Hydrodynamic flow

caused by forward cellular motility of the parasite

Question 12

How is the antibody-VSG complex internalised via the flagellar pocket?

Answer 12

Endocytosis

Question 13

What is resistance to complement dependant on?

Answer 13

The concentration of anti-VSG antibodies

Question 14

True or false: slender forms can remove the VSG-antibody complexes faster then the stumpy forms?

Answer 14

False (stumpy forms can remove the VSG-antibody complexes faster than the slender forms)

Question 15

What is the significance of stumpy forms being able to remove the VSG-antibody complexes faster than the slender forms?

Answer 15

At peak parasitaemia, the host already builds some antibodies against the VSGs but the parasites will be more efficient at degrading the antibodies, allowing them to live longer and increase probability of transmission to tsetse fly.

Question 16

What happens to VSG coat in high levels of anti-VSG antibodies?

Answer 16

complement fixation, agglutination and phagocytosis as Ab-VSG clearance can not protect the parasite and the cells are lysed

Question 17

How can a trypanosome evade complement mediated lysis when anti-VSG antibodies are high in number for a particular VSG?

Answer 17

switch VSG expression

Question 18

What are expression sites?

Answer 18

dedicated sites in the genome located at telomeres from which VSGs are expressed

Question 19

Which polymerase transcribes VSG expression sites? why?

Answer 19

RNA polymerase I

why?
allows for high levels of gene expression(higher rate of transcription than RNA polymerase II)

Question 20

What does it mean that Trypanosomes have polycistronic transcription?

Answer 20

Transcribe all genes on a chromosome into mRNA, which are then processed into individual mature mRNAs by trans-splicing (addition of 5’ splice leader RNA cap) and polyadenylation (3’ PolyA tail)

Question 21

When tagged an inactive expression site, what was observed with tagged RNA Pol I?

Answer 21

RNA Pol I did not co-localise with the inactive expression site (only co-localised with the active expression site)

Question 22

In the insect stages, how is VSG expression repressed?

Answer 22

all VSG expression sites localise to the nuclear envelope and form heterochromatin

Question 23

How are the telomeres involved in repressing expression site transcription (and therefore VSG expression)?

Answer 23

telomeric factors (E.g. RAP1) expert transcriptional control and repress ES transcription (repression is strongest nearer to telomere)

Question 24

What is required for VEX reassembly and VSG-exclusion inheritance?

Answer 24

VEX/CAF-1 interaction

Question 24

What is the result of VEX complex depletion (RNAi knockdown of VEX1/VEX2)?

Answer 24

multi-VSG expression (de-repression of expression sites)

Question 25

How do trypanosomes ensure monogenic expression of VSGs at a time?

Question 26

How are VEX1 and VEX2 involved in ensuring monogenic VSG expression?

Answer 26

VEX1 associates with an SL-RNA compartment (binds SL site)

VEX2 associates with active VSG gene transcription compartment (binds a single Expression site)

VEX1 and VEX2 also interact together to mediate this inter-chromosomal interaction

Question 27

What happens if VEX2 is silenced?

Answer 27

all VSG expression sites can access the splice leader RNA genes and are de-repressed (expression of more than one VSG)

Question 28

What are the four mechanisms that may explain how the trypanosomes switch VSG expression?

Answer 28

1. Transcriptional switch
2. Telomere exchange (recombination changes VSG being expressed in the active expression site)
3. Gene conversion (one VSG gene is exchanged from another part of a chromosome)
4. segmental gene conversion (combine different parts of VSG gene/pseudogenes to into the active expression site to create a new unique VSG)

Question 29

VSG genes and pseudogenes occupy sub-telomeric regions of what three classes of T. brucei chromosome?

Answer 29

mega, intermediate and mini-chromosomes

Question 30

What separates the VSG genes from the expression site-associated genes (ESAGs)? what is the significance of this?

Answer 30

70bp repeats

significance:
thought that may be involved in VSG switching through DSB repair

Question 31

Which VSG switching mechanisms have limited potential for generating diversity? how can this affect the parasite?

Answer 31

Transcriptional switch and telomere exchange

affect parasite because:
- if continue to infect host that has been infected with parasites before, the host likely has antibodies against the VSG

Question 32

Which VSG switching mechanisms have huge potential for generating diversity?

Answer 32

Gene conversion and segmental gene conversion

Question 33

How does T. brucei continually expand the number of usable antigens?

Answer 33

constructing mosaic VSGs through recombination events between individual VSG genes/pseudogenes

Question 34

What is the VSG switching hierarchy during T. brucei infection?

Answer 34

In early infection (first peaks of parasitaemia) = transcriptional switch and telomere exchange events involving ES-associated VSG genes so are not creating random VSGs

Later infection = activation of intact VSG genes from silent chromosomal internal positions (gene conversion)

Late infection = assembly of VSG gene mosaics (segmental gene conversion) - as pressure for VSG switching increases, there may be a higher rate of DSBs that allow mosaic assembly

Question 35

Why might segmental gene conversion occur at late stages of the parasite infection?

Answer 35

Increasing pressure for VSG switching to occur as the infection continues may increase the rate of DSBs which allow mosaic assembly of VSG genes