WEEK 2 HAT immune response

Question 1

what is antigenic variation?

Answer 1

modifying the antigens on the cell surface, that is the molecules which trigger production of a specific antibody

Question 2

what is strange about t.brucei and the immune response it elicits in the human?

Answer 2

it is highly susceptible to antibodies
lives in the bloodstream so is fully exposed to the antibody response
it induces a strong antibody response
so how does the parasite thrive in the same host for a chronic infection?

Question 3

how does t.brucei evade the human immune response?

Answer 3

antigenic variation of the cell coat (variant surface glycoprotein)

Question 4

what is the number of trypanosomes found in the blood like?

Answer 4

it is not constant

there are waves of parasitemia

Question 5

how much time is between each wave of parasitemia?

Answer 5

5-7 days

Question 6

each wave in the blood represents what?

Answer 6

antigenically distinct stereotype

Question 7

so what does the antigenically distinct stereotype mean?

Answer 7

antibodies produced in the first week will not react with parasites generated in the second week

Question 8

what is the change in antigenic profile called?

Answer 8

antigenic variation

Question 9

surface of the cell of trypanosoma parasite is covered with what?

Answer 9

electron dense coat or VSG

Question 10

what react strongly with this electron dense coat?

Answer 10

antisera

Question 11

what is evidence that there is indeed an electron dense coat or the variant surface glycoprotein?

Answer 11

trypsin a protease treatment completely removes the surface coat from trypanosomes. the trypsin treatment stops antibody binding and this implies that antigenic variation is caused by a surface protein

Question 12

what is the single protein called that makes up the surface of the coat?

Answer 12

variant surface glycoprotein

Question 13

vsg is highly ______

Answer 13

immunogenic

Question 14

VSGs from different parasitemeia peaks differ in their ___ ___ ___

Answer 14

amino acid sequence

Question 15

what is the variant surface glycoprotein made of?

Answer 15

signal sequence 20 aa
variable domain
conserved region
hydrophobic sequence 20 aa

Question 16

the VSG makes up how much of the total cell protein?

Answer 16

10%

Question 17

GPI ______VSG to cell membrane and allows ____ ____ of VSGs

Answer 17

anchors

tight packing

Question 18

The VSG ____ complement getting to cell surface and enables other surface molecules to ____ underneath the coat, this way they will not generate an ___ ___

Answer 18

prevents
hide
antibody response

Question 19

how many genes are there for VSG what does this mean?

Answer 19

1000-1500 estimate

that it can theoretically have many ways to avoid immune response

Question 20

genomic localisation of VSG genes, what are the 2 distinct pools

Answer 20

subtelomeric

telomeric

Question 21

Explain about subtelomeric (4)

Answer 21

> 1000 genes
within megabased sized chromosomes
large arrays
no adjacent promoter - not expressed - VSG store

Question 22

explain about telomeres

Answer 22

2 families
Minichromosomes no adjacent promotor - not expressed - VSG store

     Expression sites - adjacent promotor - can be expressed

Question 23

What are the 2 types of expression sites?

Answer 23

Metacyclic expression site (mES)

Bloodstream form expression site (bsES)

Question 24

metacyclic parasites in insect salivary gland express how many VSG, consequently……….

Answer 24

one of the mES

all other VSGs (in mES and bsES) are silent (allelic exclusion)

Question 25

What happens when metacyclic form infects mammal?

Answer 25

differentiates into bloodstream form and the mES is turned off and bsES is turned on

Question 26

what is allelic exxlusion in the VSG gene

Answer 26

only 1 VSG gene is expressed all other expression sites are silent

Question 27

the process of altering VSG is called?

Answer 27

switching

Question 28

during infection how many VSG genes are expressed?

Answer 28

only 1 VSG gene is expressed at a time, most cells in the population express the same VSG

Question 29

immune system causes selection of antigenically distinct VSGs which causes what to happen in the host?

Answer 29

waves of parasites giving rise to undulating fever

Question 30

How is the parasite able to switch expression between different VSGs? (3)

Answer 30

in situ switching
telomere exchange
gene conversion

Question 31

Explain in situ switching

Answer 31

the active ES is turned off and the inactive ES is turned on. It explains how mES and bsES can be turned off/on it is the simplest explanation

Question 32

Explain telomere exchangeq

Answer 32

double stranded DNA recombination between telomeric regions results in VSG from inactive site transferring into an active expression site.
Explains how VSGs on minichromosomes can be expressed.
Basically switch telomere

Question 33

Explain gene conversion

Answer 33

single stranded DNA recombination occurs between inactive & active VSGs (mismatch repair usually stimulated by DNA damage)

inactive VSG gene copied OVER previously active VSG

previously active VSG is lost

explains how VSGs at subtelomeric sites can be expressed

Question 34

In situ switching, telomere exchange, and gene conversion explain how switching may occur but does not explain why only 1 gene is active. what 3 processes could?

Answer 34

telomeric silencing
modified base
chromatin modification and structure

Question 35

explain telomeric silencing

Answer 35

The telomere will recruit active proteins and some of these will bind to the telomeric repeats that act as a recruiter to other proteins. You get complexes binding and preventing transcription at the telomere and these have been shown to spread into and away from the telomere into adjacent sequences and can cover adjacent genes and block transcription from the promoter silencing the genes near or adjacent to telom
Observed in t.brucei but not conclusive evidence that it plays a role in antigenic variation (Horn 2009)

Question 36

Explain modified base

Answer 36

trypanosomes contain unusual J base a modified thymidine and glucose. J base could act as a silencer in DNA, inactive expression sites contain Jbase at sub-telomeric regions
most likely plays a role in maintaining inactivation of expression site

Question 37

Explain chromatin modification and structure

Answer 37

chromatin is composed of DNA and protein, allows packing of DNA molecules into cells, main proteins are histones. Histones form structures called nucleosomes, DNA winds around nucleosome
histones can be modified and modifications can alter how DNA interacts with other molecules ie turn on/off gene expression (Horn 2007)

Question 38

Expression occurs where?

Answer 38

telomeric expression sites

Question 39

expression seems to be controlled by ___ ___ of ES with a single ____ POL1 transcription particle (__) per nucleus

Answer 39

physical association
RNA
ESB

Question 40

What happens to the VSG protein as it enters the lumen and ER membrane?

Answer 40

the 20aa signal sequence acts as an ER signal and once that ER signal is in the lumen of the ER itself it undergoes proteolysis and is clipped off. That is why we lose the amino terminal signal sequence.
the hydrophobic domain at the C terminus acts as an anchor, this interacts with the hydrophobic bilayer of the ER.
Proteolysis clips off the hydrophobic bilayer but the protein remains bound to the hydrophobic membrane. The hydrophobic domain is replaced by a glycolipid – undergoes host translation modification at its C terminus.

Question 41

what is the sugar fat molecule called that acts as an anchor?

Answer 41

GPI

glycosylphosphatidylinositol

Question 42

what happens after the GPI has been formed?

Answer 42

what happens next is the monomers of the VSG come together to fotm the homodynamic structure.
systine residues can form disulphide bond between monomers and then you have this variable domain which is right on the outside of the molecule facing the ER lumen.
this molecule is trafficked via the golgi through vesicles eventually fusing through the flagella pocket by exocytosis (see the board drawing he drew) they open up to expose the VSGs as they fuse with the flagella pocket and form some of the membrane and these VSGs are now exposed to the extracelluar matrix, they eventually come out of the flagella pocket and onto the cell surface.

Question 43

what has been said about location and VSG expression and regulation?

Answer 43

It appears that the active expression site is not within the nucleolus.
The nucleolus within the nucleus is the site where most genes are transcribed and when you take a Blood stream form parasite and you use an antibody against RNA polymerase I (these parasites for some reason use RNA polymerase I in the transcription of all of their genes and not RNA polymerase II which is slightly odd) you see two spots. That is just to show they are in the nucleus.

If you take an insect form parasite a non VSG expressing form you only find one spot.

The larger of the 2 spots is the nucleolus, and when you look for where is the active expression site localised to by using insitu fluorescent site it is found very close but not on top of the spot.

Where is the genome carried in the genome it is away from the spot.

What appears to be going on is a second active spot, there is only one, and this transcription reacting spot is always associated with an active transcription site and this second spot is called the EXPRESSION SITE BODY (ESB), mathematical modelling has demonstrated the size of that is only sufficient to express one gene, it is not a very large structure, and so the active expression site is a competition to get into that active site. It is a competition between the different VSG genes to get into the ESB. This is what they think could underline the whole process.

VSG expression and regulation could all be down to location in the nucleus.