Summary of last session

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Summary of last session

• Malaria has a broad range of symptoms most
  prominent are cycles of chills, high fever, and
  sweating
• There are several syndromes of several malaria
  that cause high mortality (P. falciparum), most
  prominently severe anemia and cerebral malaria
• Immuno-toxin versus sequestration as mechanistic
  cause of cerebral malaria
• Sequestration depends on knobs made up of
  parasite derived proteins (PfEMP1 is the most
  prominent ligand)

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Export into the host cell
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Export into the host cell
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Export into the host cell

• Marti et al., Science 306, 1930 -1933 (2004)
  

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Export into the host cell

• Marti et al., Science 306, 1930 -1933 (2004)
  

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Export into the host cell

• Recent work indicates that the Pexel motif is a
  proteolytic cleavage site
• The pexel motif is apparently cleaved in the ER
  and this cleavage is essential for export
• A number of models have been invoked to explain
  how cleavage could help targeting and membrane
  crossing

Boddey et al., Traffic e-pub
7
Oomycetes include plant pathogens causing blight

• Images show soy plant and its pathogen
  Phytophthora sojae
• Oomycetes and their host plants battle with plant
  factors that cause apopotosis of infected tissues
  thus limiting infection and pathogenesis factors
  that counteract those
• Recent work shows that these factors are
  delivered to the cytoplasm of the host cell and
  that this delivery depends on Pexel motif
• Interestingly oligomycete and Plasmodium Pexel
  signals can be interchanged potentially
  suggesting a common mechanism

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Oomycetes include plant pathogens causing blight

• The first 44 aa of a P. sojae pathogenesis factor
  containing a Pexel motif were fused to GFP and
  the protein was expressed recombinantly
• Incubations of roots with purified protein
  results in effective uptake of the protein into
  the cytoplasm of the host cells
• Mutation of the Pexel motif abolishes uptake
• What is your interpretation of this result and
  what (if anything) could this mean for Plasmodium?

Dou et al., Plant Cell 201930-1947
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Var gene -- polymorphism

• Parasites show variation in their antigens as
  well as their cytoadherence phenotype
• Both phenomena correlate with variations in
  PfEMP1/Var

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Var gene -- polymorphism

• Var (and other virulence associated multigene
  families like Rif and stevor) are found
  especially in subtelomeric regions of chromosomes

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Var gene -- polymorphism

• Plasmodium has a relatively limited set of 50
  Var genes
• The genes are however highly polymorphic
• Telomeres have been found clustered both in
  asexual and meiotic stages
• This could permit the opportunity for
  recombination (indeed chimeric genes are
  frequently found)

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Var gene regulation models

• VAR gene expression underlies allelic exclusion,
  that means that out of a number of alleles only a
  single one is expressed at any given time
• Hypotheses as they developed over time
• Location is important (nuclear periphery is
  silenced)
• Telomeric silencing (Sir2) (REP20 is important
  for localization in telomere clusters)
• The VAR promoter is regulated
• A sterile RNA expressed from a promoter in the
  conserved VAR intron is silencing
• Histone modifications are critical for silencing
• Keep in mind that these models are not mutually
  exclusive!

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Var gene regulation models
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VAR promoter DHFR transgenics are drug sensitive,
but can be switched
Open before selection
Closed var promoter
Closed after selection
Open cam promoter
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hDHFR is transcriptionally regulated in all
upsC-DHFR transgenics
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upsC but not cam results in association with
terminal repeats in the nuclear periphery
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upsC-hDHFR activation interferes with binding of
infected RBCs to CD36 and recognition by host Abs
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Summary

• Plasmodium falciparum infection causes severe
  disease and immunity is slow to develop
• Severe malaria is associated with high cytokine
  levels and endothelial adhesion of infected red
  blood cells
• The parasite transports and assembles complexes
  on the surface of the RBS which include a varied
  adhesion protein (PfEMP1)
• The parasites has developed mechanisms to enhance
  variation in PfEMP1 and to express only a single
  gene at the time
• The mechanisms for allelic exclusion seems to
  depend on chromatin remodeling and a conserved
  var gene promoter and a lesser extend the intron

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Theileria -- cell transfomation by intracellular
parasites
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Theileria

• Infects mainly ruminants (cattle, goats, sheep,
  deer)
• Several different species causing both pathogenic
  and benign disease
• Infection in wild animals is mostly asymptomatic

cattle disease
Cape buffalo reservoir
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Distribution of theileriosis
red T. annulata 250 million cattle at
risk orange T. parva 50 million cattle
at risk grey T. buffeli/orientalis/sergenti
relatively benign
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Life cycle of Theileria spec.

• Infects mainly ruminants (cattle, goats, sheep,
  deer, horse)
• Several different species causing both pathogenic
  and benign disease

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Two stages are found in the bovine host Kochs
bodies and piroplasms
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Theileria (sporozoite) invasion differs from
Toxoplasma invasion
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Sporozoites do not enter the host cell apical end
first
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Theileria sporozoites do not form a moving
junction during invasion
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Theileria escapes the vacuole into the cytoplasm

• Escape occurs lt15 min after invasion
• No vacuolar acidification is observed
• Escape coincides with discharge of rhoptries and
  micronemes

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Host cell microtubles are found in close
association with the parasite
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Theileria invasion

• Zipper mechanism of entry into lymphocyte
• Escape from vacuole into cytoplas coincides with
  rhopthry microneme discharge
• Parasites free in the cytoplasm associate with
  host MT

• Animated version

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The Theileria paradox

• Although Theileria replicates in lymphocytes
  these cells seem to proliferate enormously in
  infected animals (most of these proliferating
  lymphocytes are infected) -- this is in contrast
  to other infections like malaria or babesiosis
  where parasite replication is associated with the
  decline of the host cell population causing
  anemia
• Also, the sporozoite (injected by the tick)
  appears to be the only stage capable of invading
  lymphocytes
• How can the parasite spread to new lymphocytes?
• The trick Theileria hijacks and exploits two key
  features of the lymphocytes cell biology cell
  division and growth control

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Divide conquer
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Divide conquer

• Parasites do not egress from (and in the process
  destroy) their host cells and infect new
  lymphocytes but proliferate along with them
• The tight association of parasites with host cell
  microtubules ensures that they are segregated by
  the host cell mitotic spindle between the two
  daughter cells
• A recently divided infected lymphocyte (the arrow
  indicates the cleavage furrow at which
  cytokinesis occurred. Blue (DNA), red (host cell
  centrioles), green (parasite surface membrane),
  HN (host nucleus)

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Theileriosis is a lympho-proliferative disease

• Recall the immunology lecture -- lymphocytes are
  usually arrested and only expand upon antigen
  presentation
• If parasite replication requires host cell
  replication the parasite has to somehow induce
  proliferation of its host cells
• Indeed theileriosis is a lympho-proliferative
  disease
• Swelling and proliferation of the lymph node
  draining the bite site is the first sign of
  disease

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Pathology is mainly due to lymphoproliferation

• Lymphocytes proliferate heavily invading multiple
  organs causing disease similar to a lymphoma
  (cancer of lymphocytes)
• (Top) Infiltration of kidney by Theileria parva
  infected lymphocytes
• (Bottom) Abdominal ulcers due to transformed
  lymphocytes
• Death is in most cases due to infiltration of the
  lung resulting in lung edema (the abnormal build
  up of fluid within the lung)

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Theileria infected cells show characteristics of
transformation

• Theileria infection seems to share many of the
  features seen in the transformation of normal
  cells into cancer cells
• Uncontrolled growth
• Loss of differentiation
• Immortalization (infected cells taken into
  culture will grow indefinitely)
• Growth in the absence of external growth factors
• Enhanced ability to migrate and to infiltrate
  organs
• When cells are cured from parasite infection they
  die (by apoptosis -- this suicide response is
  usually suppressed in cancer cells)

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Transformation by Theileria is completely
reversible

• Theileria infection of transformed cells can be
  cured with BW720c
• Cells cease to proliferate and apoptose
• Optimal serum conditions and IL-2 treatment
  (diamonds) prolong life of cells

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How does Theileria interfere with lymphocyte
growth and cause cancer?
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NF-kB -- a major regulator of lymphocyte growth

• NFkB (nuclear factor, p50 p65) is an important
  and very well studied transcription factor (a
  protein that interacts with the promoter of genes
  and stimulates gene expression)
• It is a major player in the stimulation and
  clonal expansion of lymphcytes (among other
  functions)
• NFkB is bound by IkB (its inhibitor) which
  sequesters it in the cytoplasm and keeps it
  inactive
• Phosphorylation followed by ubiquitinylation and
  degradation of IkB leads to import into the
  nucleus and transcriptional activity
• Theileria interferes with this pathway by causing
  the destruction of IkB

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Theileria induces the degradation of IkBa and b

• NFkB is constitutively activated in transformed
  cells and dependent on the continuos presence of
  parasites
• Theileria seems to induce constitutive
  degradation of IkB a and b

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The IKK complex regulates IkB turnover

• IkB is tagged for destruction by phosphorylation
  through the IKK complex
• In normal lymphocytes this provides a way to
  relay the reception of signals from the surface
  of the cell to gene expression (e.g. stimulate
  clonal expansion)
• Theileria hijacks and activates the IKK signaling
  complex independent of the usually required
  external stimulation

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Hijacking and activation of IKK transforms
infected cells

• Theileria parasites (green) interact with and
  activate IKK (red) of their host lymphocytes
• IKK tags IkB for destruction
• NfKb free of its inhibitor enters the nucleus and
  cells start dividing rapidly

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(No Transcript)
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summary

• Theileria sporozoites invade using a zippering
  mechanism
• The PV is lysed upon rhoptry secretion and the
  parasites resides in the cytoplasm and associates
  with the host cells microtubuli centrosomes
• When the host cell divides the parasite divides
  and segregates alongside using the host cells
  mitotic machinery
• Theileria schizonts transform their hosts
  lyphocytes (induce uncontrolled cancer-like
  growth)
• Transformation is parasite dependent and
  reversible
• Parasites interfere with NFkB growth control by
  activating the IKK signalling pathway