Parasite Genomics II

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Parasite Genomics II

• Antigenic variation in Trypanosomes
• VSG structure
• Switching mechanisms
• Antigenic variation and adhesion in malaria
• PfEMP and VAR genes
• Adhesion and switching

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Metacyclic VSGs Limited
Blood stream VSGs 1000s
PARP 2 forms
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Parasitemia in Sleeping Sickness
Modified from Ross and Thomson, 1910.
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Antigenically distinct clones appear at regular
intervals.Each one expresses a new VSG.
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Trypanosome antigenic variation

• More than 1000 VSG genes throughout genome.
• Only a single VSG expressed at any one time.
• Expressed from blood stream expression site (BES)
• Evidence for some order of antigen expression.
• Metacyclic VSGs revert to a few common types
• Switching occurs in the absence of selection, but
  selection leads to dominance of new clones.

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VSG structure
Acyl tails are remodeled after synthesis Potential
target for inhibition
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Despite highly divergent sequences in the
variable region, the tertiary structure of
the VSGs remains highly conserved.
VSG molecules pack on the cell surface to make a
uniform coat
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Trypanosomes change their coats by multiple
mechanisms. Most require DNA rearrangements. The
genome contains 20 telomeric expression sites
and 1000 VSG genes, Only 1 is active at a
time (BES in the review) How does this happen?
RAD51 dependent homologous recombination
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Control of VSG expression
Open chromatin
Single active expression site (BES) Promoter is
pol I-like, constitutively active Elongation
controls expression
Vanhamme et al., Trends Parasitol. 2001
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Multiple other genes are at the expression site,
including receptors.
20 different ESAG clusters express slightly
different isoforms of transferrin Optimal binding
of transferrin from different mammalian hosts.
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Insect surface coat
Tsetse fly
In the insect, the trypanosome switches the coat
to express PARP Functions in defense and adhesion?
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Metacyclic VSGs
Blood stream VSGs
PARP
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Summary Trypanosome Antigenic Variation

• Surface antigens vary with life cycle stage
• Procyclin in insect stages
• Metacyclic VSG (single gene) following initial
  vertebrate infection
• Blood stream VSGs from one of 20 telomeric sites
  (polycistronic)
• Antigenic variation involves in situ switching,
  telomere exchange or gene conversion, single
  protein coat per cell
• Many internal copies, 20 BSEs, only one is active
• Abundant pseudogenes may increase antigenic
  repertoire
• Multiple BSEs transcribed, only active one is
  elongated and forms a stable transcript
• Transcribed by Pol I in a unique site in the
  nucleus

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Global Partnership
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Plasmodium Life Cycle
Constant Ag
Variable Ags
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Malaria

• Plasmodium falciparum
• Reaches high parasitemia levels/obstructs blood
  vessels
• Cyclic fever, anemia, diarrhea--later renal
  failure, coma, death
• High level resistance to chloroquine

1-3 million death / yr most under age 5
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Partial immunity to malaria infection
Immunity develops over time. Children most
susceptible, decreased symptoms with age. Some
evidence for antibody-mediated protection.
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Knobby phenotypes

• Infected RBCs develop knobs
• Protrusions contain adhesins involved in
  attachment to epithelial/endothelial cells
• Avoidance of clearance in the spleen

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Cerebral malaria

• Sequestration of infected RBCs in capillaries of
  the brain
• Reduced blood flow, coma, death
• Mediated by specific adhesins and recognition of
  endothelial cell receptors

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Antigenic variation and adhesion

• Mediated by gene / protein family called
• VAR (variant antigen genes)
• PfEMP1 (erythrocyte membrane protein 1)
• Single variant expressed by a infected cell
• Rapid switching rate
• Populations are always mixed
• Genome sequence reveals 50 related genes
• Telomeric or internal

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Rate of switching 2 Early in cell cycle, many
vars Single var in mature cell Selection may be
positive (receptor adhesion) negative
(antibody)
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Selecting for monomorphic variants
Mixed variants in culture
Selected variants
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Export of malaria proteins
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(No Transcript)
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Adhesion of infected RBCs
Placental
Cerebral
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VAR gene family organization
Var genes show evidence of recombination Mitotic
rearrangements? Meiotic crossing over?
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Mechanisms of switching
Silencing involves interaction between
promoter and intron sequences
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VAR gene expression

• Model of nuclear organization
• VARs organized in groups at periphery,
  transcriptionally silent
• Active site associated with small region of
  euchromatin
• Silent genes in regions of deacetylation SIR2
• Mutually exclusive expression mediated by paired
  promoters from intron (sterile transcripts)

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Summary Malaria Antigenic Variation

• Antigenic variation mediated by family of VAR
  genes encoding PfEMP1
• 60 genes in genome
• 1 expressed per cell
• High switch rate
• PfEMP1 have dual role in adhesion (multiple
  adhesive domains) and antigenic variation
• Multiple VARs transcribed early on, but
  controlled to allow only one VAR per cell
• Transcriptional repression involves chromatin
  modification, positioning, and paired intron
  transcripts.