Lecture 9 and 10
African Sleeping Sickness
and
Antigenic Variation
Learning Objectives
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Understand African Sleeping sickness symptoms.
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Understand disease transmission & epidemiology.
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Learn some unique features about trypanosomes
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Define antigenic variation and how it contributes
to the disease.
Define molecules and mechanisms involved in the
process of antigenic variation.
Infectious Diseases
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Emerging Infectious Diseases
New diseases (mainly viral agents)
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Re-emerging Infectious Diseases
Tuberculosis, Poliomyelitis
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Neglected Diseases (the big 3)
AIDS, Malaria, Tuberculosis
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Most (“The Great”) Neglected Diseases
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Tropical Parasitic diseases
Numbers of people infected with
parasitic diseases
Disease with HIGH morbidity
and QL losses:
Disease with HIGH mortality:
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Malaria - 489 M
Sleeping Sickness 0.5 M
Chagas disease - 18 M
Visceral Leishmaniasis - 4 M
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Calculated World Pop:
10/15/09:
6.94 Billion
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Schistosomiasis - 200 M
Onchocerciasis - 18 M
Filiariasis - 650 M
Ascariasis - 1.4 B
Hookworm diseases - 1.3 B
Cutaneous leishmaniasis 8M
Food and waterborne
protozoan - 1.5 B
Varies depending on calculator used
Trypanosomatidae Characteristics
Trypanosoma cruzi - Chagas Disease
• Early diverging eukaryotes
• Flagellated parasitic protists
Leishmania species - Leishmaniasis
• Vector borne pathogens
• Complex life cycles
Trypanosoma brucei - African Sleeping Sickness
• Trypanosoma brucei
Model parasite
Two culture forms
Divergent eukaryotic branch with
demonstrated RNA interference
Cell Motility
p
Left-handed helix
Attached to cell body
1 µm
a
Trypanon - auger cell
Trypanosoma
Trypanosoma brucei Life Cycle
Note: position
of kDNA
Non-invasive
HAT - re-emerging disease
DDT
HAT was nearly eliminated in 1960s: now a re-emerging,
uncontrolled Neglected Tropical Disease
Trypanosomiasis - Public Health
• >60 million people at risk (↑)
only 3-4 million screened
re-emerging and uncontrolled
•
~300,000 people infected/yr
ONE parasite can cause infection!
fatal if left untreated
•
Few drugs for treatment
serious side effects (toxic)
increase of drug resistance
Barrett et al. (2007) Br J Pharm 152:1155.
• No Vaccine available!
Available Registered Drugs
Drug
Year of
1st Use
Stage
Target
Toxicity
Suramin
1922
Early
Unknown
Highly
toxic
Toxic
Pentamidine
1940
Early
Unknown;binds DNA
accumulates in mito
Melarsoprol
1949
Late
Unknown;
Complexes thiols
Highly
toxic
Eflornithine
1981
Late
Ornithine
decarboxylase
Less
toxic
“Resurrection drug”
14 day IV treatment
at 6 hr intervals
DB289 - only new drug for Sleeping sickness that made it to advanced clinical trials!
trials discontinued due to unforeseen toxicity issues
nifurtimox-eflornithine combination therapy (NECT)
Urgent need for new drug treatments
African Trypanosomiasis History
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In 1895 David Bruce discovered in
South Africa that trypanosomes are
transmitted by the tsetse fly and
cause nagana in livestock. Several
years later, in Uganda, he discovered
that trypanosomes cause sleeping
sickness in humans.
Sir David Bruce
1855-1931
Trypanosoma brucei brucei
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Definitive Host
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Non-pathogenic to humans
Causes Nagana - very similar
to human disease
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Zebu - sensitive
Ruminants - antelope, livestock
(cattle, sheep goats)
1890’s British colonial farmers
were losing there European bred
cattle to a wasting disease
native cattle were more tolerant
“in low or depressed spirits” (Zulu)
Treatment - drugs are
expensive and used to treat
human sleeping sickness
N’Dama - tolerant
The fly who would be king
In the early 1890s the British colonial
farmers of Zululand were faced with
the decimation of their European
breeds of cattle by a wasting disease
called nagana, a word meaning in Zulu
“in low or depressed spirits.”
Some native cattle were unaffected.
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Almost the entire area of sub-Saharan Africa
which is suitable for cattle is Tsetse infested
High losses due to anemia and cachexia
especially in productive breeds
Losses in meat, milk production, tractive
power - estimated $ 5 billion.
Distribution of Disease and Vector
Glossina subgroups
Riverine/forest
Forest
There are a number of
species within each subgroup
36 sub-Saharan countries
are considered endemic
for one or the other form
of the disease.
Savannah
Trypanosoma brucei complex
T. b. brucei
game animals/livestock
(nagana)
T. b. rhodesiense
E. African trypanosomiasis
T. b. gambiense
W. and Central African
sleeping sickness
Morphologically indinstinguishable species
All are transmitted via the bite of the Tsetse fly
• Trypanosome lytic factor (TLF) found in human sera
• component of HDL (high density lipoprotein) fraction
• human parasites resistant to TLF
• resistance associated with decreased uptake of HDL
TLF-mediated Lysis
1992 Parasitology Today
• In susceptible T. brucei (brucei) , TLF is taken up by
receptor-mediated endocytosis, targeted to the lysosome where
it causes lysosomal breakdown and autodigestion of the cell.
• Resistant T. brucei (gambiense, rhodesiense ) bind TLF, but do
not endocytose it.
Trypanosoma brucei complex
Comparison of T. brucei subspecies
tse-tse vector
ecology
transmission
cycle
non-human
reservoir
epidemiology
disease
progression
parasitemia
asymptomatic
carriers
East
West
rhodesiense
gambiense
G. morsitans
dry bush or
woodland
G. palpalis
rainforest,riverine,
lakes
animal-fly-human,
ungulate-fly-human
human-fly-human***
wild animals
domestic animals
high
endemic, some
epidemics
slow (~1 yr) acute
chronic
low
rare
common
sporadic, safaris
rapid, often fatal
Reservoirs and Ecology
Wild animal reservoirs
Wildebeest
Warthog
Bush buck
Host range of Trypanosoma brucei
Spotted hyena
Spotted
hyena
Eland
Eland
Warthog
Warthog
Waterbuck
Waterbuck
Lion
Lion
Bushbuck
Bushbuck
Wild dog
Wild
dog
Giraffe
Giraffe
Impala
Impala
Buffalo
Buffalo
Zebra
Zebra
Hippo
Hippo
Coke’s Hartebeest
Coke’s
Hartebeest
Reedbuck
Reedbuck
Cattle
Cattle
African Sleeping Sickness in Man
Tsetse fly bite
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All 23 species of Glossina
are potential vectors
Both male and females
take blood meals
Metacyclic trypomastigotes
in saliva enter bite wound
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~40,000 parasites/bite
Experimentally infected
animal with a single parasite
Parasite replication at bite
site
Acute Symptoms
• 1-2 week asymptomatic
incubation period
• sometimes a local inflammation
• 'trypanosomal chancre'
• parasite replication at bite site
• Can be confused with a simple
boil
• invasion of blood characterized by
irregular fever and headache
• T. rhodesiense can develop into
full infection quickly
• T. gambiense is progresses more
slowly to serious disease
Lymphatic stage
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Disease progression often involves
invasion of lymphatics
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Winterbottom’s sign
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Swelling of cervical lymph nodes
Rash
Itching
Edema
Continued febrile attacks
Weight loss (wasting)
weakness
Winterbottom’s sign
A Human Trypanosome Infection
Classic progression: Ross and Thomson, 1910
Cyclical pattern of fever accompanied by increase in parasitemia
Antigenic Variation
Late Stage HAT
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parasites crossing blood-brain barrier result in CNS involvement
and nervous impairment
•
•
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•
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described as meningoencephalitis
increased apathy and fatigue
confusion and somnolence
motor changes including tics, slurred speech, incoordination
convulsions, coma
progression to CNS involvement is rapid (weeks) in rhodesiense
and slow (6-12 months) in gambiense
Death
How do Tryps cause disease?
Trypanosome
Immunobiology:
field still in its
infancy
Host makes
IgM and IgG
Molecular
Mimicry
Taking a Tryp(anosome) Across the
Blood-Brain Barrier - Part 1
Review by Masocha et al 2007 Phys & Behav 92:110-114
Endothelium
Major structural elements
Data acquired
from animal
models experimental
infections with
T. brucei brucei
Laminin α5
Taking a Tryp(anosome) Across the
Blood-Brain Barrier - Part 1
Masocha et al 2004 J. Clin Invest 114:689-694
Endothelial
membrane
Laminin
Trypanosome
Parenchymal
membrane
Laminin α4
Laminin α5
In vitro BBB Model
Taking a Tryp(anosome) Across the
Blood-Brain Barrier - Part 2
Grab et al 2004 J. Parasitol 90:970-979.
Data acquired
from in vitro BBB
tissue culture models:
T. brucei gambiensi
Laminin α4
Laminin α5
Taking a Tryp(anosome) Across the
Blood-Brain Barrier - Part 2
Grab et al 2004 J. Parasitol 90:970-979.
Why is Sleeping Sickness so Deadly?
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T. brucei is highly susceptible to
antibodies and complement
They live fully exposed to
antibodies in the bloodstream, in
constant contact with host immune
response
They induce a very strong antibody
response
Still they manage to survive and
thrive (replicate) in the host for
more than a year.
Why is Sleeping Sickness so Deadly?
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Infection is
characterized by
periodic waves of
parasitemia
Each wave
represents a single
antigenically distinct
clone or serotype
Antigenic Variation
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The entire trypanosome
population seems antigenically
uniform but at a very low
frequency divergent (so called
switched) serotypes are
encountered
The switch to a new serotype is
not recognized by the host
antibody population
“Switchers” survive & proliferate
leading to a new wave of
parasitemia
Serotype switching continues
Antigenic Variation
Antigenic Variation
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T. brucei is covered with a dense
surface coat
Variant specific antisera strongly
react with surface coat
Surface coats from different clones
are antigenically distinct
Antigenic Variation
No protease treatment
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Trypsin (or other protease)
treatment completely removes the
surface coat from T. brucei
This treatment also abolishes
antibody binding
This suggested that the antigenic
determinant on the surface is
made of protein
+ protease treatment
Surface coat consists of a single glycoprotein
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65 kDa glycoprotein
C-terminus anchored in the membrane
(GPI-anchor)
Only epitopes in the N-terminal 1/3 are
exposed
Constant and variable regions
VSG forms dimers
VSGs from different clonal variants
have same molecular weight, but
different amino acid composition
Different VSG share only 16% amino
acid similarity, but yet adopt a nearly
identical tertiary structure!
Variant Surface Glycoprotein
• Single VSG type uniformly
covers surface of parasite (10 7
copies)
• VSG forms 12-15 nm electron
dense surface coat
• VSG dimers form a densly
packed surface coat
Variant Surface Glycoprotein
Variable
region
Constant
region
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Different VSG share only 16%
similarity, but yet adopt a
nearly identical tertiary
structure!
Roles of VSG
T. brucei life cycle
non-dividing
fuel=?
mVSG coat
mito=?
Dividing form
fuel=amino acids
Procyclin coat
mito=“on”
Dividing form
fuel=glucose
VSG coat
mito=“off”
non-dividing
fuel=glucose
VSG coat
mito=“low”
T. brucei has ~ 1000 different VSG genes
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Great variability of chromosome size
among isolates
11 diploid megabase chromosomes,
intermediate size, and about 100
minichromosomes - all classes contain
VSG genes
6-10% of the total DNA codes for VSGs
(~1000 genes)
Only a single VSG is expressed at
a time!
At a low frequency a switch to a different
gene occurs, the host developed
antibodies against the previous VSG so
the new clonal cell line is strongly
selected.
Genome organization
11 Megabase chromosomes (1-6 Mbp)
1-7 Intermediate chromosomes (200-700 kbp)
~100 Minichromosomes (50-150 kbp)
VSG Antigenic Variation
VSG
switch
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Immune
destruction
by host
Proliferation
What is the advantage to expressing a single VSG?
What mechanisms can you think of that could control
gene expression and protein abundance?
How is VSG expression controlled?
Genomic Location of VSGs
The VSG Expression Site
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Long polycistronic transcript
Approximately 20 Bloodstream expression sites (BES) in the
genome
Active VSG genes are always at the “ends” of the chromosomes
(telomeres)
VSG in Minichromosomes
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VSG genes at
minichromosome
telmomers
Switching via telomere
conversion or reciprocal
telomere exchange
Mechanisms of Switching
Creation of Mosaic VSGs
VSG switching
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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
Expression Sites
Regulation could be achieved
by modification of chromatin
JJJJJ
JJJJ
active
JJJJJ
X
VSG
J J J J JJJJ JJJJ JJJJ
inactive
VSG
The hyper-modified Base J
β-glucosyl-hydroxy-methyluracil
a T variant
Base J
But is J a chicken or an egg?
Expression Site Body (ESB)
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How is a single expression site
activated? LOCATION!
Differential localization of
RNA polymerase I
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rRNA transcription in other
eukaryotes by RNA Pol I
usually RNA Pol II transcribes
proteins coding sequences
Localizes to nucleolus in PF
and BSF
Extranulcleolar in BSF
Procyclic
Bloodstream
Expression Site Body (ESB)
Procyclic
Bloodstream
Red: anti-fibrillarin - nucleolus marker
Green: anti-RNA Pol I
The additional spot of RNA Pol I localization is NOT the nucleolus
Expression Site Body (ESB)
Active
221ES
Inactive
121ES
Active, not inactive VSG expression sites co-localize with the
extranuclear Pol I spot.
GFP shows the position of the respective VSG genes in the nucleus
Transcriptional analysis of expression sites
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Transcription of ES sites during
development
Initiation occurs in several sites, but
is abortive
Only in an active ES site is RNA
elongation productive
Hypothesis: there is a limited
supply of factors (transcription)
connecting Pol I polymerase to
elongation/processing machinery
Hypothesis: these factors are
located in the ESB
Another
example of the
differential
expression
Antigenic Variation Summary
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Only a single VSG gene out of ~1000 is expressed
Expression occurs out of telomeric expression sites
(tapes/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 a physical
association of ES with a single RNA Pol I
transcription particle (location) per nucleus