HAT and Immune Invasion - 2a Flashcards Preview

Parasites and Infectious Diseases > HAT and Immune Invasion - 2a > Flashcards

Flashcards in HAT and Immune Invasion - 2a Deck (71):
1

Antigen

 

molecule that triggers production of a specific antibody

2

T. brucei is highly susceptible to

antibodies

  • lives in bloodstream - fully exposed to antibodies response
  • induce STRONG antibody response
  • antibodies are effective at clearing this pathogen

 

3

Immune invasion

  • number of trypanosomes found in blood is NOT constant
  • waves of parasitemia
  • difference between parasitemia peaks 5-7 days

4

Waves of parasitemia

  • each wave represents an antigenically distinct serotype (clone
    • parasites are clonal within peaks

 

  • parasites are antigenically distinct in different waves
  • antibodies produced in the 1st week will not react with parasites generated in the second week

so antibodies don't recognize later peaks

 

correlation - parasite numbers with wave of fever

(wave of host temperature just after parasite peak)

(parasites being killed and releasing contents that are also antigenic - our immune system responds to these lysis products = fever)

5

Change in antigen profile is called

antigenic variation

6

Antigenic variation

(picture)

A image thumb
7

Q image thumb

red parasite increases in number

→ antibodies generated against red

→ red parasite decreases in number

some red parasites change molecule on cell surface that is revealed to the immune system 

→ grow and elicit an immune response

→ decrease in numbers

always looking to stay one step ahead of our immune system

8

Antigenic variation

  • entire population in host appears uniform
  • at low frequency (1 in 1million cells) some cells have a different serotype (SWITCHING)
  • T. brucei has 1500 genes to choose from, plus can recombine those genes

9

The process of switching

antigenic variation

10

Surface of T. brucei cell covered with

electron dense coat

  • protein that covers the surface of the cell and the flagella
  • antisera reacts strongly with surface coat
  • surface coats from different clones are antigenically distinct
    • varies among different parasites and in different ways

 

 

11

Antisera reacts strongly with

surface coat

12

Surface coats from different clones are 

antigenically distinct

13

Trypsin

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

 

14

The electron-dense coat is made of

Variant Surface Glycoproten (VSG)

  • surface coat is made up almost of a single VSG
  • VSG is highly immunogenic
    • electron-dense coat stimulates immune system
  • VSGs from different parasitemia peaks differ in their amino acid sequences
    • only 1 VSG being expressed by 1 parasite in a population at a time
    • different waves have different VSGs

 

15

VSGs are

 

homodimers that are split into 4 regions

  • 10 million per cell
  • ~65 kDa glycoprotein
  • 10% total cell protein

 

16

VSG structure

A image thumb
17

Signal sequence

~20 amino acids

18

Variable domain

~360 amino acids

 

distinguished the different types of VSGs

19

Conserved region

~100 amino acids

20

Hydrophobic sequence

~20 amino acids

21

cDNA sequence indicates VSG has

extension at N- and C- termini

  • in actual protein this sequence is missing

why the difference

  • post-translational modification

 

22

VSG amino terminal 

(picture)

A image thumb
23

VSG amino terminal

  • VSG mRNA translated into protein sequence that's being trafficked into the ER
  • protein into ER
  • the 20 amino acid signal sequence at the amino terminal acts as an ER signal
  • once the ER signal is within the ER lumen it undergoes proteolysis and is clipped off 
  • protein translation carries on until the hydrophobic domain is produced
  • this hydrophobic domain acts as an anchor
  • so the protein is synthesized, most ends up toward ER lumen
  • the hydrophobic terminal at the C terminus of the protein captures and holds the pole of the protein onto the membrane surface
  • the hydrophobic end of the protein interacts with hydrophobic bilayer of the ER
  • that hydrophobic sequence is clipped off but the protein remains bound to the hydrophobic membrane
  • the protein is clipped and the hydrophobic domain is replaced with a glycolipid sugar fat moiety
    • post-translational modification at its C-terminus
  • changed a string of amino acids to a sugar-fat moiety
  • this sugar-fat moiety is referred to as the GPI anchor

24

VSG amino terminal (sum)

  • VSG enters the ER
  • C-terminal
    • hydrophobic
    • binds VSG to the ER membrane
    • cleaved off
    • replaced with a glycolipid (sugar/fat) 
      • covalently linked

 

25

GPI anchor

glycolipid (sugar/fat)

  • consists of core sugar (4) residues + phosphatidylinositol phospholipid
  • sugar component can be branched
  • called glycosylphosphatidylinositol (GPI) anchor

 

way to anchor VSG to membrane without protein/peptide sequence

26

GPI anchor

(picture)

  • blue = phosphatidylinositol phospholipid
  • green = sugar component (can be branched)

 

A image thumb
27

VSG molecules dimerize

  • form a homodimer
  • 2 GPI anchors - 1 from each monomer - anchoring it to the membrane
  • conserved domains come together - lots of cystiene molecules that can form disulfide bonds within the conserved domains
  • protein then transferred via secretory pathway to cell surface

 

28

VSGs covering the cell surface

ER → golgi → vesicles (clathrin coated) → flagella pocket (vesicles fuse via exocytosis) and vesicles form part of the membrane of the flagella pocket, expose VSGs to extracellular environment in flagella pocket, fluid mosaic model in flagella pocket come out of pocket → cover cell surface

 

VSG in flagella pocket, comes out → covers surface

29

VSG on the surface

(picture)

A image thumb
30

Variant surface glycoprotein

functions

GPI anchors VSG to the cell membrane and allows tight packing of VSGs

  • VSGs really close together
  • if you have a protein/peptide sequence, the hindrance effect of the protein/peptide sequence will force the VSGs slightly further apart
  • having a GPI anchor, having glycolipid at the C-terminus takes up less room on the cell membranee so you can pack more VSGs together over the cell and flagella surface
  • forms a canopy over the cell surface and removes antibodies that bind by endocytic proteolysis mechanisms
  • prevents complement getting to the cell surface
    • so no membrane attack complex forms to poke holes in the parasite
  • other surface molecules can hide under the coat so won't generate antibody response
  • VSG coat holds a lot of the immune system away fromt eh cell membrane where they can do damage
  • under this canopy you can hide proteins so hiding from the immune system

 

31

VSG genes

 

  • 10% of T. brucei genome encodes for VSGs
  • 1000-1500 genes
  • 2 distinct genomic localizations within the nuclear genome
    • subtelomeric
    • telomeres

 

32

Subtelomeric VSG genes

  • away from the telomeres
  • >1000 genes (majority of these genes)
  • VSGs with megabase-sized chromosomes (0.9 - 5.7 Mb)
  • in large arrays - genes one after another
  • no adjacent promoter - not expressed (VSG store)
  • so large arrays of genes that the parasite can access if it needs
  • each of the genes is proceeded by 70bp repeats
    • can play a role in getting these subtelomeric genes into other parts of the genome through reocmbination

 

33

Telomeric VSG genes

  • adjacent to the telomere
  • ~ 240 VSG genes

34

2 families of telomeric VSG genes

  • majority (~200) on minichromosomes
    • small chromosomes 50-100kb
    • no adjacent promoters = not expressed (VSG store)
    • regarded as a telomeric store of VSGs
    • so have 2 stores of VSGs - on ein arrays at subtelomeric localization, and this second store associated with telomerse - that the parasite can call on as it needs
  • 30-40 adjacent/near promoters = can be exprssed
    • on the megabase-sized chromosomes
    • EXPRESSION SITES

 

 

35

2 types of expression sites

  • relatively simple expression site (of telomeric VSG genes)

metacyclic expression sites (mES)

  • 15-20 
  • promoter - 70bp repeat sequence - VSG gene + telomere
  • promoter drives expression
  • can be activated and expressed by the infectious metacyclic trypomastigote form of the parasite
  • form of VSG that the parasite expresses in the salivary gland of the tsetse fly as preadaptation to life within the mammalian host

 

bloodstream form expression sites (bsES)

  • more complicated expression site (of telomeric VSG genes)
  • 15-20 
  • promoter - 7al other genes (expresion site associated genes ESAGs) - 70bp repeates - VSG gene + telomere
  • between the promoter and the 70bp repeates has series of ESAGs
    • don't know the function of many
    • some code for adenylate cyclases for signal transduction
    • some code transferrin receptors for eg uptake of iron
    • form active in humans
    • when the parasite is in its long slender bloodstream form that colonizes humans and ungulates, one of these expression sites is active

36

Telomeric VSG genes

on minichromosomes

 

(picture)

A image thumb
37

Telomeric VSG genes

expression sites 

 

(picture)

A image thumb
38

mES
(picture)

A image thumb
39

bsES

A image thumb
40

Expression sites in infection

  1. tsetse fly takes a blood meal (insects metacyclic trypomastigote)
  • metacyclic parasites in insect salivary gland express 1 VSG
  • expression of the VSG is from 1 of the 20 mES
  • all other VSGs (in mES and bsES) are silent (allelic exclusion)
    • 1 simple mES active and all others turned off
  • metacyclic parasite in salivary gland adapted to survive in mammal
    • expression of one of these VSGs on the parasite surface is a preadaption to life in the mammalian host

 

  1. injected metacyclic trypomastigotes transform into bloodstream trypomastigotes
  • infection of mammal
  • metacyclic differentiates into bloodstream form
  • mES is “turned off” and 1 bsES is “turned on”
    • mES expression site is turned off
    • 1 of the 15-20 complicated bsES expression sites is turned on
  • only 1 VSG gene is expressed, all other expression sites are silent (allelic exclusion)

 

  1. trypomastigotes multiply by binary fission in various body fluids eg blood, lymph, spinal fluid
  • during infection
  • only 1 VSG gene is expressed at a time
  • most cells in population express save VSG
  • immune system causes selection of antigenically distinct VSGs
  • (waves of parasites, giving rise to undulating fever)

41

Expression sites in infection 

1. tsetse fly takes a blood meal (injects metacyclic trypomastigote)

  • metacyclic parasites in insect salivary gland express 1 VSG
  • expression of the VSG is from 1 of the 20 mES
  • all other VSGs (in mES and bsES) are silent (allelic exclusion)
    • 1 simple mES active and all others turned off
  • metacyclic parasite in salivary gland adapted to survive in mammal
    • expression of one of these VSGs on the parasite surface is a preadaption to life in the mammalian host

42

Expression sites in infection

2. injected metacyclic trypomastigote transform into bloodstream trypomastigotes

  • infection of mammal
  • metacyclic differentiates into bloodstream form
  • mES is “turned off” and 1 bsES is “turned on”
    • mES expression site is turned off
    • 1 of the 15-20 complicated bsES expression sites is turned on
  • only 1 VSG gene is expressed, all other expression sites are silent (allelic exclusion)

43

Expression sites in infection

3. trypomastigotes multiply by binary fission in various body fluids

eg blood, lymph, spinal fluid

  • during infection
  • only 1 VSG gene is expressed at a time
  • most cells in population express save VSG
  • immune system causes selection of antigenically distinct VSGs
  • (waves of parasites, giving rise to undulating fever)

44

Process of altering VSG

switching

  • during infection
  • only 1 VSG gene is expressed at a time
  • most cells in population express saved VSG
  • immune system causes selection of antigenically distinct VSGs
    • waves of parasites giving rise to undulating fever

45

VSG expression and antigenic variation

how is the parasite able to switch between different VSGs?

3 mechanisms

  • in sit switching
  • telomer exchange
  • gene conversion

 

46

VSG expression and antigenic variation

1. in situ switching

  • aka transcription switching
  • have parasite population, peak of parasitemia
    • how does the parasite that's going to be killed by our immune system switch to a different VSG?
  • the active ES is turned off / inactive ES is turned on
    • one promoter is switched off (that drives VSG221) and activating a promoter on another expression site at a telomere
  • explains how mES and bsES can be turned off/on

47

VSG expression and antigenic variation

in situ switching

(picture)

 

A image thumb
48

VSG expression and antigenic variation

2. telomere exchange

  • double-stranded DNA recombination between telomeric regions results in VSG from inactive site transferring into an active expression site
    • to consider VSGs found at telomeric sites but not at adjacent promoters
    • homologous recombination occurring between the 70bp regions of one of the VSG stores on the minichromosome recombining with telomeric 70bp region in the active VSG
    • active VSG expressing 221 (picture)
    • homlogous recombination at homologous recombination upstream of theVSG gene with the 70bp repeat with VSGB (picture) within one of the minichromosomes
    • so just exchanging telomeres
  • explains how VSGs on minichromosomes can be expresed
    • how do VSG stores get into an active VSG expression site?
    • mechanism that will kick in later onduring chronic infection

49

VSG expression and antigenic variation

telomere exchange 

(picture)

A image thumb
50

VSG expression and antigenic variation

3. Gene conversion

  • single stranded DNA recombination occurs between inactive and active VSGs
    • mismatch repair usually stimulated by DNA damage
    • one of the 2 strands that encodes VSG A (picture) crosses over and invades the DNA sequence of 22
    • both are double-stranded
    • one of the A crosses over and matches up with 221 gene to form heteroduplex
    • the remaining 221 strand is degraded, copied and replaced by the A strand
  • inactive VSG copied OVER previously active VSG
  • previously active VSG is lost
  • explains how subtelomeric sites can be expressed
    • how youget one of the array genes into an active VSG gene

51

VSG expression and antigenic variation

gene conversion

(picture)

A image thumb
52

Explains how mES and bsES can be turned off / on

in situ switching

(aka transcription switching)

53

Explains how VSGs on minichromosomes can be expressed

telomere exchange

  • how do VSG stores get into an active VSG expression site?
  • mechanism that will kick in later on during chronic infection

 

54

Explains how subtelomeric sites can be expressed

  • how you get one of teh array genes into an active VSG gene

 

55

Gene conversion can also generate

mosaics

  • combinations like 221A tog enerate even more variation
    • one of the reasons why the repertoire of VSG genes
    • their ability in 1500
  • then have the ability to alter through making mosaics - one of the reasons why you'll never see a vaccine against T. brucei because the repertoire to stay ahead

56

Regulation of VSG switching

  • in situ switching, telomere exchange, and gene conversion explain how switching may occur
  • does not explain why only 1 gene is active

 

  1. telomeric silencing
  2. modified base
  3. chromatin modification and structure

 

57

Regulation of VSG switching

1. telomeric silencing

  • makes sense of why you have an active gene next to a telomere
  • normally telomeres will recruit other proteins
  • some of these proteins will bind (esp RAP1 in yeast that's repressor/activator protein) to telomeric repeats and acts as a recruiter for other proteins (eg SIR complex)
  • complexes binding and preventing transcription and then translation of mRNA at telomere
  • SIR complex spread into adjacent sequences away from telomeres and cover adjacent genes
  • thee effectively act to block transcription from promoter sequence, turn down and silence genes that are near/adjacent to telomeres
  • can put drug resistance marker adjacent to telomere and can see expression of that reporter going down

58

Regulation of VSG switching

1. telomeric silencing

(picture)

A image thumb
59

Regulation of VSG switching 

2. modified base

  • trypanosomes contain an unusual J-base (β-glucosyl-hydroxy-methyluracil)
    • modified thymidine + glucose tag on its surface
  • J-base takes the place of thymidine within a genome
  • sticking J-base in DNA could as act as a read-me/dont read me signal
  • common in telomeric regions
  • inactive expression sites contain J-base at sub-telomeric regions
  • most likely plays a role in maintaining inactivation of experssion site

 

  • when you look at active VSG sites this modified base appears to be restricted to a series of repeats upstream or at telomeric sites
  • when you look at inactive VSG, this J-base appears to invade the expression site
    • may play a role in maintaining gene expression
    • could play a role in regulatory processes and selecting which VSG is expressed at a given time

 

60

Regulation of VSG switching

2. modified base

(picture)

A image thumb
61

Regulation of VSG switching

3. chromatin modification and structure

  • 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 the nucleosome
  • histones can be modified (methylated or acetylated)
  • modifications can alter how DNA interacts with other molecules ie turn on/off gene expression
  • perhaps some sort of system involving modification of histones plays a role in exposing an active VSG promoter and allowing that to be expressed

62

Regulation of VSG switching

3. Chromatin modification and structure

(picture)

A image thumb
63

Expression site body

  • appears that the active expression site is not within the nucleolus
    • nucleolus within nucleus is the site where most genes are transcribed
  • when you take a bloodstream form parasite and use an antibody against RNA polymerase I (used for transcription of all their genes) you see 2 spots
  • if you take a non-VSG expressing cell (insect form) then you only see one spot

 

  • still unclear how one VSG is active while others are exprssed
  • location!
  • 2 spots in the bloodstream form
  • 1 spot in the procyclic (insect) form

64

Expression site body

bloodstream vs procyclic

(picture)

A image thumb
65

VSG gene localization (FISH)

 

  • the larger of those spots corresponds to the nucleolus
  • when you look to see whwere the active expressoin site is localized (using FISH) the active VSG gene is found very close, if not over the top, of the other spot
  • when you look for other inactive VSG genes - where the gene is actually found in the genome - find that it's away from this second spot
  • when you look at more than all of those VSG genes ands several of the inactive genes - see that they're toward the nuclear envelope

66

VSG gene localization (FISH)

(picture)

A image thumb
67

Active vs Inactive VSG (FISH)

  • smaller spot corresponds to the active ES
  • inactive ES not found within this regoin (perinuclear location)
  • this second active spot (transcriptionally active) is always associated with the active exprssion site
  • that second active spot is the expression site body

68

Active vs Inactive VSG (FISH)

(picture)

A image thumb
69

Expression site body 

(continued)

  • only accommodate small piece of DNA on single chromosome
  • only single ES (VSG) can access ESB at one time
  • there's a competition to get into that active site
  • competition between different VSG genes to get into the expression site body
  • could be what underlies the whole process

 

70

Expression site body

schematic picture

A image thumb
71

Summary

  • only 1 VSG gene out of ~1000-1500 is expressed
  • expression occurs at telomeric expression sites
  • 15-20 mES and 15-20 bsES
    • 40 exprssion sites available
    • half in metacyclic form
    • half in bloodstream form
    • only 1 is active at a time
  • to switch expression, several mechanisms reported
  • several mechanisms play role in maintaining gene expression from 1 site - still unclear how this occurs
  • expression seems to be controlled by physical association of ES with single RNA pol I transcription particle (ESB) per nucleus