Tomography of flagellar
pocket
Lacomble et al., J Cell Sci. 122:1081-90.
Sleeping Sickness and
Trypanosomes
Trypanosomias
David Bruce, 1855-1931
 Trypanosomes were first
described in frogs 1855
 Griffith Evans identifies T.
evansi as agent of surra (a
horse and camel disease) in
1880
 David Bruce identifies T.
brucei as cause of Nagana
and demonstrates
transmission by Tse-tse
flies
Trypanosome biology
The kinetoplast consists of a complex
network of concatenated DNA cirlces
Mensa-Wilmot lecture will tell you what the circles are for
Trypanosome biology
Tse tse flies
 Parasites are taken up with
the blood meal (stumpy forms
are cell cycle arrested and
‘ready to go’ for the next host
 Transformation into procyclic
trypomastigotes in the midgut
 Migration into the
ectoperitrophic space where
parasites replicate
 Passage into salivary glands,
differentiation into
epimastiogotes which attach to
the epithelium and massively
replicate
 Transformation into infectious
metacyclic trypomastigotes
 Again these are cell cycle
arrested ‘sleepers’
Trypanosmes have sex and likely
it happens in the salivary gland
 Genetic exchange occurs (e.g.
double drug resistance occurs after
coinfection of fly with single
resistant parents)
 Genetic exchange/sex is likely not
obligatory to complete fly
development and population
genetics suggest “modest” sex
 Nobody has seen it, yet it likely
involves fusion and meiosis
(progeny appears largely diploid
but there are also polyploids)
 Most likely this exchange/fusion
occurs among detached salivary
gland epimastigotes
Trypanosmes have sex and likely
it happens in the salivary gland
GFP & RFP only parents early
on in salivary gland
Red, green & yellow progeny
Red, green & yellow progeny
Gibson et al., Parasites & Vectors 2008, 1:4
Insect stages and blood
stream forms are very different
 Different stages express
different sets of surface
proteins
 Insect forms have large
mitochondria with many
cristae
 Insect stages have an aerobic
metabolism and a full
respiratory chain
 Blood stream forms only
engage in glycolysis and
excrete pyruvate and glycerol
 Note that transmission stages
do not replicate and are
arrested in development
(ladies in waiting)
Several species of trypanosomes cause
disease in domestic animals and man
 T. brucei rhodesiense & gambiense cause sleeping
sickness
 T. brucei brucei, T. congolense and T. vivax cause
Nagana in cattle
 T. equiperdum causes sexually transmitted disease in
horses and camels (interestingly, T. equiperdum is a
recent ‘petite’ mutant of T. brucei (loss of mitochondrial
genome or kDNA) Lai et al. 2008, http://www.pnas.org/content/105/6/1999.full
 Loss of oxidative phosphorylation locks parasite into BS
form – or the other way around, leaving Africa and tsetse transmission makes the mitochondrion dispensable
 (There are trypanosomes infecting many species of
animals and even plants and every single deer in the
State of Georgia)
Sleeping sickness in man
Sleeping sickness in man
Trypanosomes multiply
in the tissue around the
initial bite site
This often results in a
characteristic local
inflamation the
trypansomal chancre
From there they enter
the blood and lymphatic
system
Sleeping sickness in man
 Enlargement of the
lymphatic glands
(especially in the
posterior triangle of the
neck) can be an early
sign of the diseasese
(Winterbottom sign, not
as common in
rhodesiense infection).
 Aspiration of swollen
gland often reveals
parasites.
Sleeping sickness in man
 Once parasites enter blood
stream fever sets in (low and
irregular in gambiense and
high and periodic in
rhodesiense
 General toxic symptoms
include headache, facial
oedema, nausea and
vomiting,back and bone pain
 Symptoms at this stage are
rather mild in gambiense but
can be servere in
rhodesiense with often fatal
outcome
Sleeping sickness in man
 The second stadium of
trypansomiasis is
characterized by progressive
anemia and kachexia.
 Both features are primarily
due to extremely high serum
levels of TNFa
 TNFa was isolated both as
factor with tumor necrotic
effect as well as kachexin
inducing wasting in nagana
Sleeping sickness in man
 In later stages of infection
parasites pass the blood
brain barrier and infect the
CNS
 Presence of parasites
leads to meningoencephalitis with
progressive neurological
involvement, which
ultimately ends in coma
(sleeping sickness)
 Untreated trypanosomiasis
is always fatal
Sleeping sickness in man
 The progressive encephalitis
can cause severe dementia
with sometimes aggressive
behavior
 Disease progression especially
CNS invasion is much faster in
rhodesiense
 Gambiense can take a year or
two rhodesiense usually
passes the blood brain barrier
within a month
Nagana is the major impediment
to cattle production in Africa
Almost the entire area
of subsaharan Africa
which is suitable for
cattle is Tsetse
infested
High losses due to
anemia and cachexia
especially in
productive breeds
Wild animals are important reservoirs
for human and cattle trypanosomiasis
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
Why is trypanosomiasis so deadly?
 Infection is
characterized by
periodic waves of
parasitemia
Why is trypanosomiasis so deadly?
 Infection is
characterized by
periodic waves of
parasitemia
 Each wave represents
a single antigenically
distinct clone or
serotype
Antigenic variation
The entire trypanosome
population 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
The surface coat consists of
a single 65 kDa glycoprotein
 A single protein can be
labeled on the surface
of trypanosomes
 Upon parasite lysis this
protein becomes
soluble and can be
purified to homogeneity
fairly easily.
George Cross
http://tryps.rockefeller.edu/
Different antigenic variants have
different surface glycoproteins
 VSGs from different clonal
isolates have the same
molecular weight but vastly
different amino acid
compositions
 Vaccination with a given
VSG protects against
challenge with the
homologous isolate but not
against another variant.
VSGs share a common
structure
 All VSGs are 65 kDA
glycoproteins
 Most contain classical N-linked
glycans and all are anchored via a
GPI glycolipid (cross reacting
determinant)
 Two domains can be cleaved by
trypsin
 The outer domain is highly
variable and the only conservation
detected is the position of
cysteines
 VSG forms dimers
Antigenic variation
 VSG dimers form a densly packed surface coat
 Other (non-variant) proteins like transferrin receptor or
hexose transporter are hidden in this coat
Trypansomes harbor ~1000
different VSG genes
 The genomic organization of
trypanosomes is quite complex with
20 chromosomes and 100 mini
chromosomes
 Great variability of chromosome
size between isolates
 6-10% of the total DNA is coding for
VSGs (~1000 genes)
 Only one is expressed
 3 very peculiar details emerged
from studying the mRNA of VSG: all
trypanosome mRNAs seem to have
the same 5’end, and the VSG
mRNA encodes a hydrophobic cterminus absent from the mature
protein sequence, VSG message is
transcribed by Pol I
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 regulate the expression
of VSG
 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
Transcription is polycistronic
 The 39 first (5’) base pairs of
all trypanosme mRNAs are
identical, this sequence is not
found in the genomic locus of
these genes
 Individual mature mRNAs are
derived from large
polycistronic transcripts and
short SL-RNAs by transsplicing (details in MensaWilmot lecture)
 This might help control – but
was shown not to be the key to
antigenic variation
Location in the genome?
VSGs are expressed from telomeric
polycistronic expression sites
 Active VSG genes are typically at the “ends” of
chromosomes (telomeres)
 They are found in “expression sites”
 Genes are read in (~20) expression sites like tapes
in a tape recorder but only one recorder is playing
at a time
 How do you get a new tape in and how are the
recorders controlled e.g. switched on and off?
Several mechanisms for
switching have been described
Antigenic variation
Transposition of VSG
genes occurs by intraor 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
Indeed active and inactive sites differ in
the amount of a special modified base
called J (b-glucosyl-hydroxy-methyluracil a T variant) and there are newly discovered
differences in histone methylation and
acetylation patterns (Bob Sabatini will go over this
in detail on Monday)
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 antibody detects
two spots in blood
stream forms: the
nucleolus (where
rRNA is made) and a
second locus outside
of the nucleolus
Navarro M, Gull K. Nature 414:759-63
How is a single expression
site activated?
The additional spot of PolI is not the nucleolus
Navarro M, Gull K. Nature 414:759-63
How is a single expression
site activated?
control
The extranuclear
PolI structure is
transcriptionally
active
a-amanitin
a-amanitin
Navarro M, Gull K. Nature 414:759-63
How is a single expression
site activated?
active VSG
inactive VSG
 Labeling of the expression sites using GFP-Lac
 Active, not inactive VSG expression sites colocalize
with the extranuclelarPolI spot
Navarro M, Gull K. Nature 414:759-63
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 promoter independent
Inactive DNA is modified
Expression seems to be controlled by physical
association of ES with a single POL1 transcription
particle per nucleus