Sleeping Sickness and Trypanosomes

1
Sleeping Sickness and Trypanosomes
2
Trypanosomias

• 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

David Bruce, 1855-1931
3
Trypanosome biology
4
The kinetoplast consists of a complex network of
concatenated DNA cirlces
Mensa-Wilmot lecture will tell you what the
circles are for
5
Trypanosome biology
6
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

7
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 involve fusion
  and meiosis (progeny appears largely diploid but
  there are also polyploids)
• Most likely exchange/fusion occurs among detached
  salivary gland epimastigotes

8
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, 14
9
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)

10
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 cause 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/105/6/199
  9.full
• Loss of oxidative phosphorylation locks parasite
  into BS form or the other way around, leaving
  Africa and tse-tse transmission makes the
  mitochondrion dispensible
• (There are trypanosomes infecting many species of
  animals and even plants and every single deer in
  the State of Georgia)

11
Sleeping sickness in man
12
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

13
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.

14
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

15
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

16
Sleeping sickness in man

• In later stages of infection parasites pass the
  blood brain barrier and infect the CNS
• Presence of parasites leads to meningo-encephaliti
  s with progressive neurological involvement,
  which ultimately ends in coma (sleeping sickness)
• Untreated trypanosomiasis is always fatal

17
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

18
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

19
Wild animals are important reservoirs for human
and cattle trypanosomiasis
20
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

21
Why is trypanosomiasis so deadly?

• Infection is characterized by periodic waves of
  parasitemia

22
Why is trypanosomiasis so deadly?

• Infection is characterized by periodic waves of
  parasitemia
• Each wave represents a single antigenically
  distinct clone or serotype

23
Antigenic variation

• The entire trypanosome population seems
  antigenically uniform but at a very low frequency
  divergent (so called switched) serotypes are
  encountered

24
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

25
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/
26
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.

27
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

28
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

29
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 5end, and the VSG mRNA encodes a
  hydrophobic c-terminus absent from the mature
  protein sequence, VSG message is transcribed by
  Pol I

30
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

31
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

32
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
  trans-splicing (details in Mensa-Wilmot lecture)
• This might help control but was shown not to be
  the key to antigenic variation

33
Location in the genome?
34
VSGs are expressed from telomeric polycistronic
expression sites

• Active VSG genes are always at the ends of
  chromosomes (teleomeres)
• 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?

35
Several mechanisms for switching have been
described
36
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

37
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 patterns (Bob Sabatini will
  go over this in detail on Monday)

38
For the next experiment we need a mushroom
Amantia bisporingea, the Destroying Angel
http//www.mushroomexpert.com
39
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
40
African trypansome cellular architecture
Nucleus
Nucleoulus
Kinetoplast
41
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 414759-63
42
How is a single expression site activated?

• The additional spot of PolI is not the nucleolus

Navarro M, Gull K. Nature 414759-63
43
How is a single expression site activated?

• The extranuclear PolI structure is
  transcriptionally active

control
a-amanitin
a-amanitin
Navarro M, Gull K. Nature 414759-63
44
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 414759-63
45
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

46
Lecture ends here

• A few additional slides on diagnosis and
  treatment for those who might be interested

47
Sleeping sickness in man

• Trypansosomiasis is best diagnosed by
  demonstration of parasites in blood smears or
  lymph node exudates
• Parasitemia can be very low at times resulting in
  false negative results
• Several techniques can be use to enrich rare
  parasites in blood sample (centrifugation,
  chromatography)
• Serological assays like this agglutination assay
  are very helpful for epidemiological monitoring

48
Sleeping sickness in man

• At later stages of the disease there might be no
  detectable parasites in the blood
• Lumbar puncture and microscopic identification of
  parasites and detection of elevated protein
  levels in the CSF are needed for diagnosis
• Establishment of CNS infection is important for
  therapy decissions

49
Treatment (haemolymphatic phase)

• Suramine discovered 1921 (rhodesiense)
• Suramine will also kill O. volvolus microfilariae
  which can cause anphylactic shock
• Pentamidine discovered 1941 (gambiense)
• Drug resistance is on the rise

50
Treatment (menigoencephalitic phase)

• Melarsoprol is an organic arsenical developed1949
• It is the only drug effective against late stage
  rhodesiense infection
• Fatality due to severe acute reactive
  encephalopathy can be 1-10
• Drug resistance is a growing problem in
  rhodesiense
• Severe long term neurological (side) effects even
  after successful treatment possible

51
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

52
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

53
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?

54
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

55
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

56
Trans-splicing

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

57
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?

58
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

59
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
60
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

61
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

62
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
63
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)

64
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

65
Treatment (menigoencephalitic phase)

• Difluoromethylornithine (DMFO) is a more recently
  developed drug
• It is less toxic and quite effective
  (resurrection drug) but only effective against
  gambiense
• Target of DMFO and several other trypansome drugs
  is the parasites polyamine metabolism which is
  critical for redox balance
• Production of DMFO and other drugs almost ceased
  due to low commercial potential

66
Expression/Repression works different in insect
and bloodstream stages

• Metacyclic VSG expression sites (20-30) are
  small and only contain the VSG gene
• The promoter seems to be important for expression
  as exchange for a constitutive promoter has 10
  fold effect
• Repression is not very tight and effect
  elongation more than initiation
• Blood stream VSG is expressed out of large
  polycistronic sites, control is extremely tight,
  promoter independent and acts on initiation

67
Expression/Repression works different in insect
and bloodstream stages
68
Antigenic variation
J
(1)
VSG
active
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
J
X
(19)
VSG
inactive
The hyper-modified base J
But is J a chicken or an egg?