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