African Trypanosomes

1
African Trypanosomes Sleeping Sickness II
2
Sleeping Sickness and Trypanosomes I

• Life cycle and biology of trypanosomes
• Sleeping sickness, differences between gambiense
  and rhodesiense
• Nagana, kachexia and TNF
• Drugs used to treat trypanomiasis
• Tse tse flies

3
Trypanosomes II

• Why is African trypanosomiasis such a deadly
  disease?
• Important pathways discovered along the way to
  understand this problem trans splicing and GPI
  anchors
• The nuts bolts of trypanosme gene expression
  control

4
Why is trypanosomiasis so deadly?

• What is surprizing about the life of the slender
  trypomastigote form of the parasite in the human
  bloodstream?

5
Why is trypanosomiasis so deadly?

• Trypanosomes are highly susceptible to antibodies
  and complement
• They live fully exposed to antibodies in the
  blood stream
• They induce a very strong antibody response
• Still they manage to thrive in the same host for
  a year or longer, until the host dies

6
Why is trypanosomiasis so deadly?

• The number of parasites found in the blood of
  humans and animals infected with trypanosomes is
  not constant, but shows characteristic waves of
  parasitemia
• The window of time between parasitemia peaks is
  about 5-7 days

7
Why is trypanosomiasisso deadly?

• Infection is characterized by periodic waves of
  parasitemia
• Each wave represents a single antigenically
  distinct clone or serotype
• Antibodies produced in the first week against
  clone A will not react with clone B
• The changing display of different antigens is
  called antigenic variation
• Antigenic variation is an important form of
  immune evasion

8
Antigenic variation

• The entire population of trypanosomes within an
  infected animal seems antigenically uniform
• But at a very low frequency divergent (so called
  switched) serotypes are encountered

9
Antigenic variation

• Trypanosomes are covered with a dense surface
  coat
• Variant specific antisera strongly react with
  this surface coat
• Surface coats from different clones are
  antigenically distinct

10
Antigenic variation

• Trypsin treatment completely removes the surface
  coat from Trypanosomes (trypsin is a protease, an
  enzyme that specifically digests proteins)
• This treatment also abolishes antibody binding
• This suggests that the antigenic determinant on
  the surface is a protein

11
Antigenic variation

• The surface coat is made up almost entirely by a
  single protein the Variant Surface Glycoprotein
  or VSG
• This protein is highly immunogenic and
  distinguishes the clones in successive
  parasitemia peaks
• VSGs from different parasitemia peaks differ in
  their amino acid sequence

12
Lessons learned along the way the GPI anchor

• When cDNAs (a DNA copies of messenger RNAs) of T.
  brucei VSGs were sequenced they were shown to
  encode a c-terminal hydrophopic region which
  could anchor the protein
• However when the proteins were sequenced this
  part was absent -- how is this soluble protein
  kept in the membrane?
• VSG is anchored into the membrane via a
  glycolipid anchor (glycosyl-phosphatidylinositol
  or GPI)

13
Lessons learned along the way the GPI anchor

• Initially thought to be specific for trypanosomes
  GPI anchors have been shown to be present in all
  eukaryotic organisms
• The GPI anchor is synthesized as a precursor
  glycolipid in the endoplasmic reticulum by
  sequential addition of sugar molecules to a
  phospholipid
• The mature precursor contains a terminal
  ethanolamine phosphate which can form a peptide
  bond with the c-terminal carboxyl group of the
  protein

14
Lessons learned along the way the GPI anchor

• GPI anchor addition occurs immediately after
  translation
• The c-terminal hydrophobic portion of the protein
  serves as a signal sequence and cleaved and
  replaced with the glycolipid in a
  trans-peptidation reaction

15
Antigenic variation

• GPI anchors allow very dense packing of molecules
  on the surface of the parasite
• VSGs forms a dense coat on the surface of the
  trypanosome
• This coat is equivalent of the coat form by
  lipophosphoglycan in Leishmania

16
Antigenic variation

• All VSGs are 65 kDA glycoproteins, and are
  present on the surface as dimers
• The outer domain is highly variable and the only
  conservation detected is the position of
  cysteines
• Other (non-variant) proteins like transferrin
  receptor or hexose transporter are hidden in the
  this surface coat

17
Antigenic variation

• 6-10 of the total genome of African trypanosomes
  is coding for VSGs (more than 1000 genes)
• Only one is expressed at a given time the other
  999 genes are shut down and completely silent
• At a low frequency a switch to a different gene
  occurs, if the host develops antibodies against
  the previous VSG the new clone is strongly
  selected
• What is the advantage of expressing a single VSG?
• How is expression controlled?
• What mechanisms can you think of by which a cell
  could control gene expression and protein
  abundance?

18
Antigenic variation

• mRNA derived from only a single VSG gene can be
  detected at one time
• VSG expression is controlled at the level of
  transcription initiation
• Regulation of promoter activity is used to
  control gene expression in many organisms

19
Transcription in trypanosomes is polycistronic

• But, only very few promoters have been identified
  in trypanosomes and they did not seem to control
  the expression
• Also surprisingly transcription in trypanosomes
  was found to be polycistronic
• Polycistronic means that a number of genes are
  transcribed at the same time into one long
  messenger RNA
• In bacteria this message is translated into
  protein, in trypanosomes further processing is
  needed and this processing might confer
  additional level of control

20
Transcription in trypanosomes is polycistronic

• The 39 first (5) base pairs of all trypanosme
  mRNAs are identical, furthermore this sequence is
  not found in the genomic locus of these genes
• Individual mature mRNAs are derived from large
  polycistronic transcripts by a process called
  trans-splicing
• In this process mRNAs for individual genes are
  cut out of the polycistronic transcript and a
  short RNA transcribed from a different locus (the
  splice leader) is attached to it 5 end

21
Trans-splicing

• The mechanism and enzymes used for trans-splicing
  are very similar to cis-splicing
• Cis splicing is the process that removes the
  introns from mRNAs of eukaryotic genes
• Splicing is accomplished by a complex of small
  nuclear proteins and RNAs - the spliceosome

22
Trans-splicing

• Trans-splicing (cutting and joining two different
  RNAs) is very similar to cis-splicing (cutting
  and joining within the same RNA)

23
Antigenic variation

• If it is not the promoter maybe it is the exact
  location in the genome that predisposes a
  specific VSG for expression
• But how could that be switched then?

24
VSGs are expressed from telomeric polycistronic
expression sites

• Transcription in trypanosome is polycistronic as
  we have seen
• Active VSG genes are allways at the ends of
  chromosomes (telomeres)
• Genes are read in (20) expression sites like CDs
  in CD players but only one CD player appears to
  be playing at a time
• How do you get a new CD in and how are the CD
  players controlled

25
Several mechanisms for switching have been
discovered
The most common mechanism of VSG switching
requires physical transposition of a new VSG
gene into the active expression site
26
Antigenic variation

• Transposition of VSG genes occurs by intra- or
  intermolecular recombination
• This explains switching but not really why one
  gene is active and all the others are silent

27
Antigenic variation

• Regulation could be achieved by modification of
  chromatin (by sticking on a read me or do not
  read me label)
• Indeed active and inactive sites differ in the
  amount of a special modified base called J
  (b-glucosyl-hydroxy-methyluracil)
• But is this the chicken or the egg?
• Recent work from Dr. Sabatinis lab here at UGA
  shows that J is likely not controlling expression
  but is important for switching recombination

28
For the next experiment we need a mushroom
Amantia bisporingea, the Destroying Angel
http//www.mushroomexpert.com
29
VSG is transcribed by Pol I

• a-amanitin is a specific and highly potent RNA
  polymerase inhibitor
• Cells have specialized RNA polymerases to
  transcribe different genes
• In most cells mRNA which encodes proteins is
  transcribed by the RNA polymerase Pol2 (this
  enzyme can be inhibited by the toxin a amanitin)
• Ribosomal RNA is generally transcribed by Pol1
  (which is resistant to the toxin)
• VSG transcription is insensitive to a-amanitin
  suggesting it is transcribed by the highly
  processive Pol I (however all other mRNAs for
  proteins seem to be made using Pol II as
  everywhere else)
• How could this help to explain allelic exclusion?

tubulin
rRNA
VSG
Drug
30
African trypansome cellular architecture
Nucleus
Nucleoulus
Kinetoplast
31
How is a single expression site activated?

• Location, location, location
• PolI antibody detects two spots in blood stream
  forms the nucleolus (where rRNA is made) and a
  second locus outside of the nucleolus

Pol I
DNA
Nature 414759-63
mammal
insect
32
How is a single expression site activated?
Nature 414759-63

• The additional spot of Pol I is not the nucleolus
  (Fib in red is a nucleolus marker)

33
How is a single expression site activated?
active VSG
inactive VSG
Nature 414759-63

• Active, but not inactive VSG expression sites
  colocalize with the extranuclear Pol I spot. GFP
  in green shows the position of the respective VSG
  gene in the nucleus

34
Antigenic variation

• Only a single VSG gene out of 1000 is expressed
• Expression occurs out of teleomeric expression
  sites (the tape recorder)
• To switch genes on they are transposed into an
  active expression site by several mechanisms
• Expression seems to be controlled by physical
  association of the expression site with a single
  POL1 transcription particle per nucleus
• There are 1000 CDs, 20 CD players but only one is
  plugged in