Transcript ppt - Striepen Lab

African Trypanosomes &
Sleeping Sickness II
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, fly control
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
Why is trypanosomiasis
so deadly?
 Infection is chronic and
ultimately fatal if left
untreated.
 How can the slender
trypomastigote form of
the parasite survive in the
human bloodstream?
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
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
Why is trypanosomiasis
so 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
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
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
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
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
Lessons learned along the
way: the GPI anchor
 When genes for T. brucei
VSGs were sequenced they
were shown to encode a cterminal hydrophopic peptide
that 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 (glycosylphosphatidylinositol or GPI)
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
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
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
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 (allelic
exclusion)
 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?
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
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
Transcription in trypanosomes
is polycistronic
 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
 Initially this was thought to be
the key to regulation – but it is
not.
Antigenic variation
If it is not the promoter or the processing maybe
it is the exact location in the genome that
predisposes a specific VSG for expression
Where are active and inactive genes in the
genome?
How could a location based system switch?
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
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
Antigenic variation
 There are several mechanisms
but the most common mechanism
of VSG switching requires
physical transposition of a new
VSG gene into the active
expression site
 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
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. Sabatini’s lab here at UGA shows that J is
likely not controlling expression but is important for switching &
recombination
For the next experiment we
need a mushroom
Amantia bisporingea, the
Destroying Angel
http://www.mushroomexpert.com
VSG is transcribed by Pol I
tubulin
rRNA
Drug
VSG
 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 aamanitin 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?
African trypansome cellular
architecture
Nucleus
Nucleoulus
Kinetoplast
How is a single expression
site activated?
Location, location,
location
PolI is found in two
spots in blood stream
forms: the nucleolus
(where rRNA is
made) and a second
locus outside of the
nucleolus
Pol I
DNA
Nature 414:759-63
insect
mammal
How is a single expression
site activated?
Nature 414:759-63
The additional spot of Pol I is not the
nucleolus (Fib in red is a nucleolus marker)
How is a single expression
site activated?
active VSG
Nature 414:759-63
inactive VSG
 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
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