malaria based proteomics of erythrocyte surface proteins
DESCRIPTION
Malaria based proteomics of erythrocyte surface proteins. Kim Baron University of Pretoria, Department of Pharmacology Supervisor: Dr Duncan Cromarty Co-supervisor: Dr Stoyan Stoychev. Introduction. Few diseases have had same global health & economic impacts of malaria - PowerPoint PPT PresentationTRANSCRIPT
Malaria based proteomics of erythrocyte surface proteins
Kim BaronUniversity of Pretoria, Department of PharmacologySupervisor: Dr Duncan CromartyCo-supervisor: Dr Stoyan Stoychev
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
Introduction
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 erythrocytesA- Recognition C + D- Tight junction formation/movementB- Reorientation E- Internalisation
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
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
Aim
Method Overview
Protein fingerprinting of RBC ghost proteins 2-DE
Protein fingerprinting of RBC ghost proteins SDS-PAGE
RBC ghost sample preparation
Culturing and synchronisation of 3D7 Plasmodium falciparum parasites
PMF RBC membrane proteins involved in P.f invasion by LC-MS/MS
Isolate spots of interest
Comparison: free RBC ghosts/merozoite-bound RBC ghosts/Free merozoites
Protein fingerprinting of merozoite-bound RBC ghosts 2-DE
Free merozoite surface proteins incubated with RBC’s & cross-linked
Merozoite treatment phospholipase C to cleave GPI anchors
Results: SDS-PAGE
2-DE
Band 3??
ASB (amidosulfobetaine)-14 zwitterionic detergent
ASB-14 SDS-PAGE Comparison
Erythrocyte ghosts: absence of ASB-14
Erythrocyte ghosts: sample prep. with ASB-14
* *
ASB-14 utilisation with adjusted voltage settings
Active sample loading
10% T gel instead of 4-20% T
Improvements
Plasmodium falciparum complex & “intelligent” RBC’s are abundant & fragile Avoids damaging RBC Protected from immune system
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
Conclusion