Download Malaria based proteomics of erythrocyte surface proteins

Kim Baron
University of Pretoria, Department of Pharmacology
Supervisor: Dr Duncan Cromarty
Co-supervisor: Dr Stoyan Stoychev
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Few diseases have had same global health &
economic impacts of malaria
300 million people affected globally each year &
0.7-2.7 million annual deaths
Caused by protozoan parasite Plasmodium
(falciparum) most severe form humans
Discovered 100 years ago  stage specific
differential protein expression
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Control of malaria uses several approaches
◦ Improve diagnosis
◦ Improve prophylactic chemotherapy
◦ Integrated vector control
Prevalence malaria evident worldwide
◦ Drug-resistant parasites
◦ Insecticide-resistant vectors
New malaria drugs are expensive & limited in
supply
Safe, effective & affordable treatment  critical
global public health priority
The life cycle of P.
falciparum in the
human host & the
Anopheles mosquito
vector
Merozoite invasion of erythrocytes
A- Recognition C + D- Tight junction formation/movement
B- Reorientation E- Internalisation
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Merozoite invasion of RBC’s is highly complex 
cascade of specific molecular interactions
Complete receptor-ligand interactions unknown
Invasion is central & essential in development of
malaria  speed & efficiency contributed to
evolutionary success & parasitemia
Inhibition of parasite invasion before manifestation
of clinical symptoms  Advantage
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To identify which erythrocyte surface proteins act
as receptors during the initial recognition and
binding of the Plasmodium falciparum merozoite to
the erythrocyte during the invasion process of the
malaria parasite
Culturing and synchronisation of 3D7 Plasmodium
falciparum parasites
RBC ghost sample preparation
Protein fingerprinting of RBC ghost proteins SDS-PAGE
Protein fingerprinting of RBC ghost proteins 2-DE
Merozoite treatment  phospholipase C to cleave GPI
anchors
Free merozoite surface proteins incubated with RBC’s &
cross-linked
Protein fingerprinting of merozoite-bound RBC ghosts 2DE
Comparison: free RBC ghosts/merozoite-bound RBC
ghosts/Free merozoites
Isolate spots of interest
PMF RBC membrane proteins involved in P.f invasion by
LC-MS/MS
Band 3??
ASB (amidosulfobetaine)-14  zwitterionic
detergent
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Erythrocyte ghosts:
absence of ASB-14
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Erythrocyte ghosts:
sample prep. with ASB-14
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ASB-14 utilisation with adjusted voltage settings
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Active sample loading
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10% T gel instead of 4-20% T
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Plasmodium falciparum  complex & “intelligent”
 RBC’s are abundant & fragile
 Avoids damaging RBC
 Protected from immune system
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To potentially stop deadly parasite  new
innovative therapies
Determining exact R-L during invasion  discovery
of new key targets for drug & vaccine based
antimalarial strategies