List of contents

LIST OF CONTENTS

Declaration
Certificate
Acknowledgement
Abbreviations

Page No.

1. Introduction 1-3

2. Aims and Objectives 4

3. Review of Literature 5-24

3.1 Introduction to malaria

3.2 History of malaria: an ancient disease
3.3 Global distribution of malaria
3.4 Life cycle of Malaria parasite
3.5 Anti malarial drugs and drug resistance:
3.6 Lipid metabolism in Plasmodium
3.6.1 Phospholipids and their synthesis pathways
3.6.1.1 Phosphatidylcholine synthesis pathway
3.6.1.2 CDP-choline (Kennedy) pathway
3.6.1.3 Phosphatidylcholine Biosynthesis from Serine/Ethanolamine in
P. falciparum
3.6.2 Fatty Acid Biosynthesis
3.6.2.1 FAS II Pathway:
3.6.2.2 Fatty acid elongase pathway (FAE)
3.6.3 Lysophospholipase
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4. Material and Methods 25-48

4.1 Material
4.1.1. Plasmodium falciparum strain
4.1.2. Standard markers
4.1.3. Restriction Endonucleases and DNA Modifying Enzymes
4.1.4. Bacterial Strains
4.1.5. Reaction sets (Kits)
4.1.6. Consumables
4.1.7. Immuno-Chemicals
4.1.8. Antibiotics and Substrates
4.1.9. Primers
4.1.10.Standard Media
4.1.11.Reagents and Buffers
4.2 Method
4.2.1. Identification and Phylogenetic analysis of the Pf
Lysophospholipase
4.2.2. Sequence analysis and structural modelling
4.2.3. Gene cloning
4.2.4. Chemically Competent Cells Preparation
4.2.5. Transformation into Chemically Competent E. coli Cells
4.2.6. Mini-preparation of plasmid DNA
4.2.7. Expression and purification of recombinant protein
4.2.8. Generation of antisera against recombinant PfLysoPL in mice and
rabbits
4.2.9. Enzyme-linked immunosorbent assay (ELISA) to determine
antibody responses in BALB/c mice and rabbits against rPfLysoPL 4.2.10.
Polyacrylamide gel electrophoresis (PAGE) of proteins
4.2.11. Western blotting
4.2.12. P. falciparum in vitro culture
4.2.12.1. Thawing of P. falciparum culture frozen stock
4.2.12.2. Sub-culturing and dilution of parasite culture
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4.2.12.3. Synchronization of P. falciparum by sorbitol treatment

4.2.12.4. Isolation of purified parasite from infected blood by saponin lysis
4.2.12.5. Preparation of frozen stocks of parasite culture
4.2.12.6. Isolation of parasite gDNA
4.2.12.7. Transfection of P. falciparum
4.2.12.8. Generation of pfLysoPL-RFA transgenic line
4.2.13. Fluorescence microscopy and indirect immunofluorescence assay
4.2.14. Cryo-immunoelectron microscopy
4.2.15. In vitro parasite growth inhibition assay
4.2.16 Flow cytometry
4.2.17 Live cell imaging and time-lapse microscopy.
4.2.18 Hemozoin extraction and purification
4.2.19 Parasite lipid extraction and analysis

5. Results 49-65
5.1.1 Identification of Plasmodium falciparum Lysophospholipase like
gene: PF3D7_1476700 (PF14_0737)
5.1.2 Sequence analysis of Plasmodium falciparum Lysophospholipase
5.1.3 Phylogenetic analysis of Plasmodium falciparum Lysophospholipase
5.1.4 Homology modelling of Plasmodium Falciparum Lysophospholipase
5.2.1C-terminal tagging of gene and localization of fusion protein in the
transgenic parasites by GFP targeting
5.2.2 TMP Toxicity Assay
5.2.3Generation of PfLysoPL-RFA transgenic line
5.2.4. Confirmation of Genome Integrity at locus
5.2.5 Expression and stabilisation of transiently expressed PfLysoPL--
RFA fusion protein in parasite
5.2.6 Localization of PfLysoPL in different blood stages of the parasite
5.2.6 .1 PfLysoPL is expressed in all blood stages of Plasmodium
falciparum
5.2.6.2 PfLysoPL co localizes with the endoplasmic reticulum (ER) in
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ring and early trophozoite stage

5.2.6.3PfLysoPL localizes with parasitophorous vacuole in young
trophozoite
5.2.6.4 PfLysoPL is targeted to multi-vesicular like structures near food
vacuole.
5.2.6.5 Localization of PfLysoPL by immuno-electron microscopy
5.2.6.6 PfLysoPL associates with TAG lipid bodies in the parasite.
5.3.1 Selective degradation of PfLysoPL inhibits parasite growth
5.3.2 Selective knockdown of PfLysoPL levels disrupts the intra-
erythrocytic parasite cycle.
5.3.3. Reduction in PfLysoPL levels disrupts storage of neutral lipids in
the parasite
5.3.4 Reduction in PfLysoPL levels disrupts hemozoin formation in the
parasite
5.4 Cloning, expression and purification and characterization of PfLysoPL
5.4.1 Purification of r PfLysoPL
5.4.2 Purification of r PfLysoPL-C (Oligo peptidase domain of PfLysoPL)
5.4.3 ELISA titers of sera raised against purified rPfLysoPL-C terminus
fragment
5.4.4 Standardization of activity assay for PfLysopl
5.4.5 Estimation of Kinetic constants

6. Summary 66-68
7. Discussion 69-75
8. Conclusion 76-77
9. References 78-89
10. Publications 90