Molecular Interactions Involved In Erythrocyte Invasion By

Malaria Parasite

Thesis Submitted to

Jawaharlal Nehru University

for the Award of the Degree of

Doctor of Philosophy in Molecular Genetics

by

International Centre for Genetic Engineering and Biotechnology

RICCARDO S. GATTA

Introduction

Malaria parasite biology and life cycle,

Expression of binding domain of P.vivax Duffy-binding protein,

Morphology of erythrocyte invasion by Plasmodium spp.,

Malaria parasite - host interactions,

Recombinant PvRII produced as secreted protein in insect cells,

Mouse anti-PvRII antibodies block erythrocyte binding…

Overview:

Work In Brief:

- functionally active,- immunogenic,

{after Sherman, 1998}

Indian medical texts1600 BCE

Hippocrates Lucretius 400 BCE 95BCE

Quinine c 1640 CE

Giovanni Maria Lancisi 1716

Charles Louis Alfonse Laveran 1880

Ronald Ross 1897

Malaria TIMELINE- Introduction

Identification

Chloroquine1934

DDT 1937

WW I I1939-1945

WHO - ERADICATION 1956

DDT resistance 1960’s

WHO - CONTROL 1967

{after Sherman, 1998}

IntroductionMalaria TIMELINE- Control

Artemisinins1979

Gene cloning 1983

Genome sequencing 2002

- Search for new drugs- Vaccine development

{after Sherman, 1998}

IntroductionMalaria TIMELINE- Major Advances

{after WHO, 2000}{after WHO, 2000}

Population at risk40% world-wide

~ 2 billion people

Population infected ~ 200 million people~ 150 million more each year

Research focus ...new drugs ...vaccines

Fatalities ~ 2 million each year~ 3000 children under five die each day

Malaria: a world-wide burden Introduction

{From Hoffman, 1996}

Parasite Life Cycle – Blood StageMalaria

{after Chitnis and Miller, 1994 and Miller and Hoffman, 1998}

The target is:

Vaccines aim to:

1. Sporogonic or Mosquito Stage,2. Exo-erythrocytic or Liver Stage,3. Erythrocytic Stage.

- Vaccine-induced host antibodies (Abs) are taken up with the blood meal, - Block sporozoite development, - Target vector directly, - Abs to sporozoites, - Cellular response: induce both cytotoxic

T-cells and IFN-γ, - Reduce symptoms, - Abs that block merozoite cytoadherance and/or invasion of RBCs, - Abs to antigens on parasitized RBC, - Induce IFN-γ and other cytokines would destroy infected RBCs,

Transmission

BlockingPrevent Infection

and Disease

Reduce Parasitemia

and Disease Invasion – Targets

Malaria Vaccines

MSP family, MAEBL and extended family, AMA-1, and SERA

{From Bannister et al., 2000}

Rhop / RAP complexesDBL-EBP family / PvRBPsSurface molecules Apical organelle localizationBlood Stage – MerozoiteMalaria Parasite

{Caramello, 2002}

Morphology – FiguresMalaria Parasite

Pf

Pv

{Caramello, 2002}{WHO, 1998}

Morphology – ImagesMalaria Parasite

1: Attachment – Reorientation

-PvRBPs,

-MSP-1 complex,

-AMA-1,

-MAEBL...

{From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000}

MorphologyErythrocyte Invasion

2: Irreversible attachment and

junction formation

- micronemes

- rhoptries...

{From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000}{From Cowman and Crabb, 2002, and Chitnis and Blackman, 2000}

{From Dvorak et al., 1975}

3: Parasitophorous vacuole and invasion

MorphologyErythrocyte Invasion

Plasmodium spp. have individual invasion specificities

Erythrocytes / treatment

P.vivax P.knowlesi P. falciparum

Human Duffy +ve + + +

Human Duffy –ve.

Human Neuram.

Rhesus

Rhesus Chymo.

- - +

ND + ND- + -+ + -

Erythrocyte ReceptorsErythrocyte Invasion

Two DBL domains: P. falciparum EBA-175, EBL1, BAEBL, JESEBL, PEBL...

I II III IV V VI

TMSS CYT RII

Single DBL domain:

P. vivax DBP, P. knowlesi DBP (α, β, and γ proteins)…

{after Chitnis and Miller, 1994}

I II III IV V VI

TMSS CYTFIIFI

Erythrocyte Binding ProteinsErythrocyte Invasion

Erythrocyte receptors

{From Tournmouille, 1997}

- ligands can acts as immunogens to induce invasion blocking Abs.

- find parasite ligands,

Parasite Ligands

(Region II)

Erythrocyte Receptors

P. vivax RII ONLY Human Duffy antigenP. knowlesi α-RII Rhesus / human Duffy Ag

P. falciparum F2 Sialic acid / glycophorin A

Sialic acid (rhesus RBC)β-RII

Rhesus RBC (unknown) γ-RII

External

Internal

Duffy Antigen Receptor for Chemokines

(DARC)

Receptor – Ligand InteractionsErythrocyte Invasion

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