Marco Papageorgiou

Characterising New Surface Proteins of Plasmodium falciparum By Marco Papageorgiou Submitted in total or partial fulfilment of the requirements of the degree of Master of Science (Biotechnology) March 2014 The University of Melbourne TABLE OF CONTENTS ABSTRACT………………………………………………………………………………...iv GLOSSARY………………………………………………………………………………...vii 1.0 INTRODUCTION………………………………………………………………………1 1.1 Malaria………………………………………………………………………….1 1.2 Transmission and Lifecycle.....................................................................1 1.3 Erythrocyte Invasion and Development……………………………………..3 2.0 Antigenic Variation and Immune Evasion……………………………………….5 2.1 Background…………………………………………………………………….5 2.2 Cytoadhesion…………………………………………………………………..5 2.3 Variant surface antigens (VSA’s)…………………………………………….6 2.4 Var and stevor genes………………………………………………………....7 3.0 RIFINS………………………………………………………………………………….9 3.1 Background…………………………………………………………………….9 3.2 Topology and Structure……………………………………………………..10 3.3 RIFIN subtypes………………………………………………………………12 3.4 Function in the IE…………………………………………………………….13 3.5 Transport and trafficking…………………………………………………….15 4.0 Transport of VSA’s…………………………………………………………………16 4.1 Maurers Cleft…………………………………………………………………16 4.2 Maurers Cleft proteins……………………………………………………….17 CONCLUSION……………………………………………………………………………18 Page | ii 5.0 AIMS…………………………………………………………………………………...19 6.0 MATERIALS AND METHODS……………………………………………………...20 6.1 Materials…………………………………………………………………………...20 6.1.1 Chemicals……………………………………………………………………...20 6.1.2 Miscellaneous………………………………………………………………….20 6.1.3 Instruments………………………………………………………………….....20 6.1.4 Antibodies………………………………………………………………………21 6.1.5 Ladder Marker………………………………………………………………….21 6.1.6 Culture Media……………………………………………………………….....21 6.1.7 Buffers and Solutions…………………………………………………….…...22 6.1.8 Plasmodium falciparum Strains……………………………………………...23 7.0 CELL BIOLOGY METHODS……………………………………………………......24 7.1 Culture of Plasmodium falciparum…………………………………………....24 7.2 Giemsa Staining and Assessment of Parasitaemia……………………......24 7.3 Freezing and Thawing of P. falciparum……………………………………….25 7.4 Synchronisation with 5% Sorbitol……………………………………………..27 7.5 MACS enrichment of late stage parasites…………………………………...28 7.6 Fractionation of erythrocyte membrane…………………………………......29 7.6.1 Saponin Lysis………………………………………………………………….29 7.6.2 Hypotonic Lysis………………………………………………………………..30 7.7 Extraction of membrane fractions of IE……………………………………...30 7.7.1 Salt Extraction………………………………………………………………....30 Page | iii 7.7.2 Carbonate Extraction………………………………………………………….30 7.7.3 Urea Extraction………………………………………………………………...31 7.7.4 Triton X-100 Extraction………………………………………………….........31 7.7.5 SDS extraction………………………………………………………………....31 7.8 Immunoflourescence Analysis (IFA)…………………………………………..32 7.9 Live IFA……………………………………………………………………………..33 8.0 MOLECULAR BIOLOGY METHODS………………………………………..........34 8.1 Western Blot Analysis……………………………………………………..........34 9.0 RESULTS………………………………………………………………………..........36 9.1 RIFIN protein expression examination with IFA…………………………....36 9.2 Conclusion…………………………………………………………………..........38 9.3 Solubility of RIFIN proteins in membrane and Intra-erythrocyte parasites………………………………………………….....39 9.4 Anti-RIFIN antibodies analysis…………………………………………..........41 9.5 Conclusion………………………………………………………………………...43 10.0 DISCUSSION……………………………………………………………………….44 10.1 Overview………………………………………………………………………...44 10.2 SDS-PAGE/Western blot analysis…………………………………….........45 10.3 IFA of RIFIN expression……………………………………………….……...46 CONCLUSION…………………………………………………………………………....47 Page | iv BIBLIOGRAPHY…………………………………………………………………...........48 APPENDICES…………………………………………………………………………….53 Appendix 1………………………………………………………………………..........53 Appendix 2………………………………………………………………………..........54 Appendix 3………………………………………………………………………..........56 Appendix 4………………………………………………………………………..........57 ACKNOWLEDGMENTS………………………………………………………………...58 Page | v ABSTRACT Plasmodium falciparum has developed several ways to avoid becoming the target of the human naturally acquired immunity. One such tactic utilized by the parasite to evade an antibody response is through P. falciparum’s ability to display small, transmembrane, proteins that undergo constant genetic diversity. The Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP-1), an already well-established surface protein, has allowed for an exploration into repetitive interspersed family (RIFIN) proteins as they are believed to elicit similar actions within infected red blood cells. The putative products of the rif gene family have been shown to be expressed at the surface of a phenotypically modified infected erythrocyte, with a characterised topology structure and sequence information. RIFINS constitute the largest gene family that are believed to account for the antigenic diversity of several Plasmodium falciparum strains, which are also implicated as one of the main interfaces between tissue-specific receptors and malaria infected erythrocytes. RIFIN protein expression and function remains unclear, although research continues to generate some clarity into a highly diverse gene family. This review discusses some introductory information into the most defined strain of malaria, Plasmodium falciparum, which leads into a more focused topic on variant proteins, then specifically explaining the RIFIN proteins and associated transport processes where it finally concludes. Page | vi GLOSSARY Ab antibody bp base pair DAPI 4’,6’-Diamidin-2’-phenylindol-dihydrochlorid DTT dithiothreitol Fig. figure GFP green fluorescent protein HRP horseradish peroxidase IE infected erythrocyte IFA immunofluorescence assay kDa kilodalton kb kilobase MACS magnetic activated cell sorting PBS phosphate buffered saline PEXEL Plasmodium falciparum export element PV parasitophorous vacuole Rif repetitive interspersed family TBS tris buffered saline µl microliter Page | vii MC Maurer’s Cleft MW Molecular Weight RBC Red Blood Cell Page | viii INTRODUCTION 1.0 INTRODUCTION 1.1 Malaria Over 40% of the World’s population are currently at risk of contracting Malaria, which amounts to well over two billion people (Cowman et al 2006, Gardner et al 2002), with the World Health Organisation (WHO) estimating an annual infection rate of over 200 million (Tilley et al 2011). The cause of malaria is from protozoan parasites of the Plasmodium genus, (Tuteja 2007), which has been directly associated with high mortality across the developing World in humans and also in a variety of other vertebrates. Endemic patterns of distribution have been observed in subtropical and tropical regions such as sub-Saharan Africa (Cowman et al 2006) which account for the majority of all malaria cases Worldwide (Tuteja 2007). Infection also comes at a great cost for the most endemic areas of Africa, with an estimated value of US $12 Billion every year this accounts for these areas being some of the most impoverished (UNICEF). 1.2 Transmission and Lifecycle Malaria is transmitted to humans through the bite of a female Anopheles mosquito (Cowman et al 2006). Sporozoites, which represent the infective stage of malaria (Drakeley et al 2006, Tilley et al 2011), are injected into the host during a blood meal from the salivary gland of the female mosquito which then pass into the liver where these sporozoites begin to invade hepatocytes, (Cowman et al 2006), (Fig. 1). Remarkably, each sporozoite undergoes differentiation and mitotic division into thousands of merozoites (Tilley et al 2011) where once released from the liver, begin Page | 1 to invade erythrocytes signalling the end of the asexual process of Erythrocyte invasion. Merozoites can attach at any point along the erythrocyte surface, however their orientation is important and therefore must re-orientate in order to align the apical tip with the erythrocyte, (Fig.2), (Cowman et al 2006). Fig.1 (Enomoto et al 2012) a small dosage, transferred by the female Anopheles, of sporozoites enters the blood stream (liver stage) where they penetrate liver cells and undergo transformation and amplification asexually, producing merozoites. Single merozoites proceed to invade erythrocytes initiating the asexual reproduction whereupon infected RBC’s burst and release between 8-30 merozoites (blood stage) which then indiscriminately invade other uninfected erythrocytes. Page | 2 Fig. 2 (Su et al 2007) Fusion of the gametocytes (male and female) occurs within the midgut of the Anopheles mosquito, further differentiating into a zygote and oocyst and finally injected as sporozoites into the host for further infection. 1.3 Erythrocyte Invasion and Development Once merozoites gain entry into a favourable human erythrocyte, the lifecycle of Plasmodium falciparum is once again repeated with the formation of several intraerythrocyte stages of development (Tuteja 2007), namely, ring trophozoite and schizont stages of development, (Fig. 3). The mature forms of the intra-erythrocyte parasite will distinctively change the cytoskeleton and plasma membrane creating nutrient pathways (Enomoto et al 2012) in order to survive within a RBC host. The asexual phase described above in non-limiting to the parasite, and sexual stage parasites (Drakeley et al 2006) also occur during this process. A relatively small amount of merozoites differentiate into male and female gametocytes, which originate in the host and which occur after numerous cycles of asexual erythrocyte schizogony in the blood stages (Alano 2007, Tuteja 2007) and which are Page | 3 consequently transferred to the midgut of an infective mosquito (Alano 2007). These asexual precursors (Drakeley et al 2006) ultimately fuse together to create a zygote (Alano 2007) which are then taken up by a female Anopheles mosquito during a blood meal. Fig. 3 (Tuteja 2007) three separate images showing (A) ring stage parasites, (B) Trophozoite stage parasites and (C) schizont stage parasites. Image B shows a mature trophozoite with haemozoin towards the top of the IE. Early trophozoite formation is sometimes referred to as late ring stage parasites. Page | 4 ANTIGENIC VARIATION 2.0 Antigenic Variation 2.1 Background Antigenic variation is defined as the ability for a pathogen to alter its surface characteristics in order to evade a host immune response (Craig et al 2001) during parasite infection. In malaria infections, during parasitic development within the erythrocyte, P. falciparum will modify the erythrocyte surface morphology in order to display a series of encoded surface proteins which are suggested to be virulence factors (Mbengue et al 2013). During these progressive stages of malaria infection P. falciparum can switch expression of one gene to the next which leads to unique antigenic differences in surface proteins. The switching of this gene expression ensures that both antigenic and functional properties are modified, affecting the overall infection outcome (Kyes et al 2007). The expression of large multi-gene families is a tightly regulated process (Dzikowski et al 2006) which enables parasites to encode and expose only a single gene (Dzikowski et al 2006) randomly switching expression and further activating silent genes upon immune recognition (Dzikowski et al 2006). 2.2 Cytoadhesion Infected erythrocytes have developed the ability to bind a range of receptors that mediate cytoadhesion (Craig et al 2001). Within the context of malaria infections Cytoadherence is described as the binding of parasite-infected erythrocytes to human host endothelial cells and other adhesion targets (Drakeley et al 2006). P. falciparum must avoid entry into the spleen once in the blood stream and completely Page | 5 ANTIGENIC VARIATION avoid splenic clearance. Cytoadhesion presents a tool for immune evasion and splenic clearing, with the postulation that the mechanism of sequestration whereupon surface expressed proteins adheres to capillary beds (Bull et al 2005) and bind along endothelial cells (Kyes et al 1999) which are implicated in the obstruction of blood flow. 2.3 Variant Surface Antigens (VSA’s) Variant surface Antigen’s (VSA’s) are important proteins in Plasmodium falciparum that allow for a better understanding of immunity and pathogenicity (Newbold 1999) with malarial infections. The var, rif and stevor multigene families have become of significant importance in relation to host-parasite interactions (Niang et al 2009) establishing chronic infections through their respective protein transcripts by initiating malaria specific IgG class antibody responses (Niang et al 2009). VSA’s are all encoded by multigene families that undergo antigenic variation, often referred to as clonal antigenic variation (Bull et al 2005), that lead to alterations in cytoadherent properties (2.2 Cytoadhesion) which are a survival feature, necessary for binding a range of endothelial receptors (Lavstsen et al 2005). RIFIN, STEVOR and PfMC2TM proteins (Fig. 4) are all variant surface antigens that belong to the same two transmembrane superfamily (Bachmann et al 2012) in which the name describes an architecture that forms a ‘loop’ when anchored in an infected erythrocyte membrane. The transcription and expression of the aforementioned variant surface antigens shows that var genes are transcribed initially after RBC invasion, followed by the transcription of RIFIN genes and finally STEVOR genes (Niang et al 2009). Page | 6 ANTIGENIC VARIATION Figure 4 2.4 Var and Stevor genes The var gene family are the most well researched and extensively studied class of VSA’s (Lavstsen et al 2005) expressed on the surface of infected erythrocytes. Var genes encode for a well-established protein product, Plasmodium falciparum Erythrocyte Membrane Protein 1 (PfEMP-1) (Craig et al 2001, McRobert et al 2004) which as the name suggests is a membrane protein that is destined to be transported to the IE surface. PfEMP-1 surface proteins are concentrated on electron-dense knobs along the IE surface (Buffet et al 2010), small protrusions of the IE membrane (Bachmann et al 2009), that are in size ranging from 200-350kDa (Dzikowski et al 2006). Var genes reside in the sub-telomeric regions of all 14 Plasmodium falciparum chromosomes (Craig et al 2001), McRobert et al 2004) along with a smaller gene family, STEVOR, that sit between and are flanked by two groups of var genes (Fig. 5) (Kyes et al 2007). Experimental evidence with Stevor genes has Page | 7 ANTIGENIC VARIATION shown that they are clonally variant and expressed (Niang et al 2009) at schizont stage parasites (Niang et al 2009). Var genes are separated into three main subgroups, A, B, and C (Bachmann et al 2009, Turner et al 2011) which is based on each of their upstream regions, chromosomal locations and orientation (Bachmann et al 2009). The gene structure of PfEMP-1 consists of two exons, a 5’ exon that is relatively large, 4-10kb, (Craig et al 2001) followed by a conserved intron region ending with a highly conserved 3’ exon C-terminal (Craig et al 2001, Dzikowski et al 2006). Fig. 5 (Dzikowski et al 2006) P. falciparum chromosome with the relative locations of the var, rif, stevor and Pfmc-2TM on the sub-telomeric domains. Page | 8 RIFINS 3.0 RIFINS 3.1 Background RIFINS are encoded by the repetitive interspersed family (rif) gene family, that are further sub-divided into two distinct groups based on the presence or absence of a 25 amino acid motif within the semi-conserved domain of the sequence (Mwakalinga et al 2012. As stated previously, Plasmodium falciparum relies on its ability to express and display similar surface variant antigens in order to elicit and maintain infection (Fernandez et al 1999, Petter et al 2007), owing to the pathogenicity of P. falciparum (Fernandez et al 1999, Mwakalinga et al 2012). The location of the rif gene family are situated within the sub-telomeric sites (Petter et al 2007) in the Plasmodium falciparum genome, also in close proximity, and often associated with the var gene family of surface proteins (Kyes et al 1999). Fernandez et al have described the RIFIN putative proteins to be between the sizes 30-40kDa, adding that between 200-500 RIFIN genes exists within the P. falciparum genome (Fernandez et al 1999). Other literature has concluded that there are in fact between 150-200 rif gene copies (Petter et al 2007) in the parasites genome. RIFIN protein expression is limited to late ring and/or early stage parasites (Fig.11) (Craig et al 2001, Fernandez et al 1999), however RIFIN expression has also been attributed to gametocytogenesis (Petter et al 2008) which suggests that RIFIN expression occurs in both sexual and asexual parasites (Petter et al 2008). Page | 9 RIFINS 3.2 Topology and Structure The rif gene, upon transcription and translation consists of two exons (Petter et al 2007) with the first exon encoding a signal peptide followed by a second exon that consists of a semi-conserved N-terminal region (Petter et al 2007). RIFINS are transported proteins and in order to be marked for displacement must possess an export sequence. The rif gene accommodates this transport through a trafficking motif called the Plasmodium Export Element (PEXEL) (Dzikowski et al 2006, Petter et al 2007), which is positioned along the second exon (Fig.6) enabling transport out of the parasite vacuole to the IE surface for presentation (Petter et al 2007). RIFINS are categorised as transmembrane proteins (Lavazec et al 2006, Petter et al 2007) which within the two second, much larger, exon contain two putative transmembrane sequences (Khattab et al) TM1 and TM2 (Petter et al 2007), respectively. Immediately following these two transmembrane regions is a sequence that encodes for a hypervariable loop (Fig.7), which is believed to be responsible for the antigenic diversity (Lavazec et al 2006) driving host immune pressure (Lavazec et al 2006). Page | 10 RIFINS Fig. 6 (Dzikowski et al 2006) shows the sub-telomeric locations of the rif, stevor, PfMC-2TM and var genes. The PEXEL can be seen in the second exon of each gene in image A. The rif and stevor genes sit downstream to the var genes. Fig. 7 (Modified from Lavazec et al 2007) Proposed modelling of the 2TM structure of RIFIN, stevor and PfMC-2TM proteins. The surface exposed loops on each model accounts for the hypervariability of each protein product. Models 2, 3, and 4 show the topology of the proteins with a cystolic c-terminus and an extracellular N-terminus. Page | 11 RIFINS 3.3 RIFIN Subtypes RIFINS are subdivided into two distinct groups, A-type and B-type (Bachann et al 2012, Mwakalinga et al 2012, Petter et al 2008) which as stated earlier is determined by the absence or presence of a short 25 amino acid sequence (Fig. 9) (Bachmann et al 2012, Petter et al 2007) that sits in the conserved region of subtype A. This grouping allows one to understand the proteins intracellular location and sub-cellular localisation patterns (Petter et al 2006) of each subtype, along with understanding their respective functions within an IE (Fig.8). Although postulated to serve varying functions (Petter et al 2007, Petter et al 2008), A-type RIFINS exhibit relocation patterns to the IE erythrocyte surface via the Maurer’s cleft (Bachmann et al 2012, Petter et al 2007, Petter et al 2008) while B-type RIFINS are cytosolic (Bachmann et al 2012) residing within the IE (Bachmann et al 2012, Petter et al 2008). Fig. 9 (Joannin et al 2011) Structure of the RIFIN subtypes A and B with the presence or absence of a 25 amino acid sequence. Page | 12 RIFINS Fig. 8 (Petter et al 2007) Indirect Immunofluorescence Assay (IFA) in asexual stages (ring, trophozoite, schizont), with anti-rif antibodies for both subtypes used to visualise expression patterns in IE. Green fluorescence can be seen across both subtypes representing RIFIN protein expression from transgenically labelled proteins and blue stain which highlights the nuclei. In A type RIFIN (labelled anti-ARIF29) the localisation patterns are towards the IE membrane which suggests trafficking processes are involved. B type RIFINS (labelled as anti-BRIFΔNC and anti-B562) are centralised in contrast with A- type, and are retained in the cytoplasm of the nucleus. 3.4 Function in the IE The biological function of RIFINS is still not fully understood (Petter et al 2008), however it has been suggested that RIFIN expression is linked to host immune evasion through their ability of antigenic variation (Petter et al 2008). The var gene Page | 13 RIFINS protein PfEMP-1 has already been associated to antigenic variation through its recombination events (Kyes et al 2007). Chromosomal locations suggest a common mechanism for recombination and sequence diversity amongst this multi-copy family (Frank et al 2008). The expression of RIFINS suggests that it may lead to host cell binding (Claessens et al 2011) however this is still unclear (Petter et al 2008). Larger gene transcripts, such as rif genes with up to 200 copies per haploid (Joannin et al 2008), introduce even more complexity (Joannin et al 2008) to understanding the exact reason for RIFIN expression and function within IE. Fig. 11 (Winter et al 2005) RIFINS are expressed at late ring stage (Trophozoites) where they move to the surface from the PV by action of Maurer’s Cleft transport proteins. The schizont will release new merozoites once the schizont has burst open and carry out further invasion into new RBC hosts. Page | 14 RIFINS 3.5 Transport and Trafficking Upon invasion into the erythrocyte the malaria parasite carries out a series of morphological changes and alterations within the cytoplasm and surface of the RBC (Tilley et al 2008). These modifications are not only important for the development of the parasite but also contribute to the virulence of P.falciparum, also including cytoadherence and nutrient uptake (Spycher et al 2008). Surface proteins synthesised by P. falciparum are destined for the IE membrane and in order to reach their destination must follow a more complex route (Lanzer et al 2006). These proteins must first pass through the parasites own membrane whereupon they then must pass through the parasitophorous vacuolar membrane (PVM) making their way through the host cytoplasm to span the infected erythrocyte membrane (Lanzer et al 2006). Unlike other eukaryote cells, Red blood cells are not metabolically active and therefore are not equipped with transport structures (Lanzer et al 2006). Due to this lack of trafficking machinery inside the RBC (Tilley et al 2008) the parasite must efficiently synthesise structures and organelles to solve the issue of protein transport. RIFINS have been previously found to be co-transported with the var gene protein PfEMP-1, through the active transport of these proteins by the function of the Maurer’s cleft. Page | 15 TRANSPORT OF VSA’S 4.0 Transport of VSA’s 4.1 Maurer’s Cleft The Maurer’s cleft was discovered in 1902 (Lanzer et al 2006, Tilley et al 2008) within the cytoplasm of erythrocytes that had been infected with Plasmodium falciparum parasite. They have been described as flattened (Cooke et al 2006) vesicular structures (McRobert et al 2004) that are involved in the active transport of parasite-encoded surface proteins to the infected erythrocyte membrane (McRobert et al 2004). Fig. 10 (Maier et al 2009) Electron micrograph of the Maurer’s cleft seen here (MC). The arrows shown indicate the interface of the IE (top) and an endothelial cell (bottom). The knob structures are also seen on the IE which is believed to contain the receptors responsible for host cell binding. Page | 16 TRANSPORT OF VSA’S 4.2 Maurer’s Cleft Proteins Proteins that are associated with the Maurer’s Cleft are the targets of immune resistance by the host defence system (Lanzer et al 2006) which enable and mount an Ig antibody response. A surface bound protein must first exit the parasite into the IE cytoplasm, which is under the direct influence of the presence of the PEXEL sequence located on rif, stevor and Pf-2TM genes (Cooke et al 2006). Next , it is then inserted into the Maurer’s cleft and transported to the surface where it can either anchor into knob structures (Fig.10), in the case for var gene protein products, (Cooke et al 2006), or integrate across the transmembrane, such as rif and stevor protein products. The PfEMP-1, a well-studied membranous protein in P. falciparum has been associated with the Maurer’s cleft (Cooke et al 2006, Lanzer et al 2006, Tilley et al 2008) in which the Maurer’s cleft acts as a chaperone molecule transporting the transcribed gene (Cooke et al 2006) into the IE membrane, facing outwards towards other erythrocytes and the host immune defence. The Maurer’s cleft is also known to be implicated with RIFIN proteins acting much the same way to that of the PfEMP-1 (Petter et al 2007). Page | 17 CONCLUSION CONCLUSION RIFINS are by far the largest multi-copy gene family that currently exist within the haploid genome of Plasmodium falciparum. Although their functions and role within a P. falciparum infected erythrocyte remains unclear, there is some evidence to suggest that these two transmembrane proteins are responsible for driving host immune responses. Understanding more about how, and at what stages, these gene transcripts are expressed within malaria infections along with their defined role and more importantly their overall purpose within infected erythrocytes, will add stronger evidence to the impact of RIFINS on the virulence and infectivity of Plasmodium falciparum. Page | 18 AIMS 5.0 AIMS 5.1 To characterise members of the RIFIN protein family 5.2 To use molecular tools and characterise RIFIN expression, cellular localisation, membrane topology, and function. Page | 19 MATERIALS AND METHODS 6.0 MATERIALS/METHODS 6.1 Materials 6.1.1 Chemicals Albumax Dithiothreitol (DTT) Protease Inhibitor RPMI 1640 Media Triton X-100 6.1.2 Miscellaneous Tissue culture flasks Petri-dishes MACS column Filtration Apparatus 50ml sterile tubes 15mL sterile tubes Western Blot nitrocellulose membrane Wet blot chamber Whatman paper Ice block 6.1.3 Instruments Centrifuge Vario MACS Incubator Olympus microscope Zeiss Microscope Fuji Film developer Page | 20 MATERIALS AND METHODS 6.1.4 Antibodies Primary Antibodies Mouse Anti-HA Mouse Anti-GFP Mouse Anti-Glycophorin A/B Rabbit Anti-Aldolase (1:2000) (1:2000) (1:2000) (1:2000/1:4000) Invitrogen Invitrogen Invitrogen Invitrogen (1:5000/1:10000) (1:5000/1:10000) (1:10000) (1:10000) Invitrogen Invitrogen Invitrogen Invitrogen Secondary Antibodies Anti-mouse HRP Anti-Rabbit HRP Protein G HRP Goat anti-mouse HRP 6.1.5 Ladder Marker Protein Ladder (Appendix 1) Invitrogen 6.1.6 Culture Media TB buffer 10 mM HEPES pH 6.7 15 mM CaCl2 55 mM MnCl2 250 mM KCl Sterile 1.6% NaCl (0.8g NaCl in 100ml AMRESCO 1x PBS) Sterile 0.9% NaCl, 0.2% Glucose (0.1g NaCl, 0.2g Glucose in 100ml AMRESCO 1x PBS) RPMI-1640 medium 500 ml RPMI-1640 10 ml Human serum 23mL NaCl HEPES pH 7.2 Glycerolyte freezing solution 57 % Glycerol 1.6 g Na-lactate 30 mg KCl 1x western blot transfer buffer 20% Methanol 960 mM Glycine 0.187 % SDS 125 mM Tris pH 8.3 Urea Dialysis buffer 0.1M Urea 10mM Tris pH8 1mM EDTA Thawing solution Sterile 12% NaCl (11.2g NaCl in 100ml AMRESCO 1x PBS) Page | 21 MATERIALS AND METHODS 6.1.7 Buffers and Solutions 10x PBS 1.37 M NaCl 26.8 mM KCl 80.6 mM Na2HPO4 14.7 mM KH2PO4 pH 7.4, sterilize by autoclaving MES buffer 50mM MES 50mM Tris 1mM EDTA 0.1% SDS pH 7.3 10x TBS 500mM Tris 1.5 M NaCl Sterilize by autoclaving pH 7.5-8.0 Ponceau S Solution solution 0.1% Ponceau solution 5% Acetic Acid Salt extraction buffer 10 mM HEPES pH 7.2 600mM KCl 3 mM MgCl2 5mM DTT 1x Protease buffer Inhibitor before use Carbonate extraction buffer 100mM NaHCO3/Na2CO3 pH 11 1 mM EDTA 1x Protease buffer Inhibitor before use 2x Protein loading buffer 100mM Tris 100mM DTT 4% SDS 20% Glycerol 0.02% Bromophenol Blue Urea extraction buffer 8M Urea 10mM Tris pH8 1mM EDTA 1x Protease buffer Inhibitor before use 5% Milk in TBS-Tween 5g skim milk powder 100mL 1xTBS-Tween Page | 22 MATERIALS AND METHODS 6.1.8 Plasmodium falciparum strains 3D7 Plasmodium falciparum genome clone pARL 3 parasite line (Appendix 3) pARL 6 Parasite line (Appendix 3) Page | 23 CELL BIOLOGY METHODS 7.0 CELL BIOLOGY METHODS 7.1 Culture of P. falciparum Culture conditions of Plasmodium falciparum were kept at within the following parameters: 10mL and 25mL petri dishes were used to grow Plasmodium falciparum infected human RBS’s that were kept at 37 degrees Celsius, with 1-2% CO2. The petri dishes were stored in Perspex boxes to allow parasites to invade added red blood cells. Parasites were grown to a parasitaemia between 5-8% before any further experimentation was carried out, i.e. MACS purification. For a lower parasitaemia, P. falciparum parasites were transferred to a 10mL petri dish and maintained under normal culture conditions. 7.2 Giemsa Staining/Assessment of Parasitaemia 3-6µl of parasites/blood was prepared onto a microscopy slide. Thin blood smears were achieved by using a second slide as a spreader. The spreader slide was placed at a 45 degree angle next to the blood droplet in a manner that allowed the blood to be evenly distributed along the spreader slide edge. The smear was left to dry for a short time whereupon the smear was fixed onto the slide by 100% methanol for 1 minute. The slide was then washed gently with deionised water and stained in Giemsa stain for 20 minutes. The slide was then washed with deionised water and blot dried with paper towels and then further dried with a hair dryer to remove any Page | 24 CELL BIOLOGY METHODS last traces of residue. The fixed and stained smear was then analysed under the light microscope (Fig.12) and the parasitaemia was calculated. A B Fig. 12 (A) Giemsa stained P. falciparum culture showing ring and trophozoite infected RBCs. (B) P. falciparum petri dishes were kept under normal incubation conditions in a transparent Perspex box. 7.3 Freezing and thawing of P. falciparum Freezing Parasites were grown at early ring stage at a parasitaemia of 5-8% before freezing procedure was carried out. A 10mL petri dish of parasites was spun down at 1500RPM for 5 minutes. The volume packed RBC’s was calculated by observing the volume of the blood pellet and adding 2x the amount of this RBC volume with glycerolyte (which was stored at RT), i.e. for a1mL pellet, 2mL of glycerolyte was added. Firstly 1/3 of the volume of glycerolyte was added drop wise and left to stand for 3-5 minutes. Next, the Page | 25 CELL BIOLOGY METHODS remaining volume of glycerolyte was added drop wise with gentle shaking in between each drop and then 300-600µl of this final volume was transferred into cryotubes (the number of cryotubes was determined by the partitioning of 300-600µl per cryotube). The cryotubes were then placed into an isopropanol freeing container in -80 degrees for 24 hours and then transferred into liquid nitrogen for long term storage. Thawing A cryotube was removed from the cold storage and thawed at 37 degrees for 1-2 minutes. The contents in the cryotube(s) were transferred into a 15mL sterile tube with a sterile pipette. Next, descending concentrations of NaCl were added slowly to the to the 15mL tube with first 0.1 x Volume of 12% NaCl with constant agitation, and the tube was left to stand for 5 minutes. Then 10 X Volume of 1.6% NaCl was added drop wise with constant shaking. The tube was then centrifuged at 1500RPM at 20 degrees for 5 minutes. The supernatant was removed from the pellet and 10 x Volume of 0.9% NaCl, 0.2% Glucose was added slowly with gentle shaking. The tube was centrifuged at 1500RPM at 20 degrees for 5 minutes and the supernatant removed. The blood cell pellet was then resuspended in fresh media, transferred to a 10mL petri dish, and then grown by normal culturing conditions. Page | 26 CELL BIOLOGY METHODS 7.4 Synchronisation with 5% sorbitol Synchronisation with sorbitol allows for a high level of synchrony of only ring stage parasites (Fig.13). This meant that a high parasitaemia of ring stage parasites should be present in vitro culture before sorbitol treatment. 5% Sorbitol solution and RPMI-1640 media was pre-warmed in a water bath for 25 minutes. The parasitaemia of 25mL petri dish parasites were determined by giemsa staining and light microscopy observation. I.e. Sorbitol synchronisation was carried out only when a high parasitaemia of late stage parasites were present. The parasites were then spun down at 500g for 5 minutes and the SN was discarded. 10mL of 5% sorbitol solution was then transferred to the tube(s), mixed and incubated in a 37 degree water bath for 7-10 minutes. The tube(s) were then spun down at 1500g for 5 minutes and SN discarded. The pellet was then resuspended in fresh RPMI-1640 media and transferred to a sterile 10mL petri dish with fresh blood added drop wise. The dish was then incubated under normal culture conditions to promote parasitic growth. Fig.13. (Toyama et al 2012) Sorbitol Synchronised parasites shows only ring stage parasites stained with Giemsa when viewed under the light microscope. Obtaining highly synchronous parasites was vital before any further analysis. Page | 27 CELL BIOLOGY METHODS 7.5 MACS enrichment of late stage parasites MACS selection for late stage parasites from cultured P. falciparum parasites was carried out at a calculated parasitaemia of 5-8% after staining with Giemsa stain and cell counting under the Ziess light microscope. The Vario MACS apparatus (Fig.14) was setup by firstly attaching a MACS column with a three-way valve to the apparatus. A 25mL syringe was then attached to the valve along with a 0.5mL needle which was connected at the base of the three-way valve. 1x PBS was then flushed through the column via the 20mL syringe for calibration, this was repeated 3 times. The PBS was then let to drip through the needle where the flow was corrected to drop by drop speed. The parasites were pooled together and 30mL batches of mature parasites were added to the column until all parasites had run though the column. 1x PBS was again washed through the MACS column, repeated 3 times or until the eluent went from a red to a clear colour. The valve was closed, turned upside-down and the MACS enriched parasites were flushed into a new 50mL tube(s). The enriched IE Parasitaemia was then estimated by diluting into 1x PBS and placing a small volume in a Neubauer chamber for cell counting. A B Fig. 14 (Miltenyl Biotec) Vario MACS setup with (A) MACS column to which the 3-way valve is connected, (B) Magnetic compartment and (C) effluent collector. The Vario MACS was setup and operated under sterile conditions C Page | 28 CELL BIOLOGY METHODS 7.6 Fractionation of erythrocyte membrane Parasites that were grown to a high parasitaemia were analysed for the solubility of RIFINS, firstly by lysing the cells and then applying the different fractionation protocols. A series of sequential fractionations were carried out in order to study the solubility of RIFINS and these fractions were directly mixed with 2x SDS Loading buffer for western blot analysis, along with IFA analysis. The outline of fractions that were obtained from the protocols is shown in Table 1 in the Results section. 7.6.1 Saponin lysis Saponin is a reagent that lyses the RBC membrane and the PVM. The only remaining content after saponin treatment within an IE is the parasite membrane (Fig. 15). The MACS purified parasites were lysed with 0.075% saponin with 1x PBS at a concentration of 1x106IE/µl and then placed on ice for 10-15 minutes. The action of saponin could be noticed as the tubes were inverted and mixed and the media went from a thick red colour to a light almost transparent mixture, which indicated that the cells had been lysed. The cells were then centrifuged at 1200 x g for 10 minutes at room temperature and the SN was transferred into a vial(s) for subsequent western blot analysis. Erythrocyte membrane Parasite Fig.15 Saponin treatment disrupts the RBC membrane whilst leaving the parasite membrane intact. Other organelles such as the MC membrane and and also the PVM. PVM Page | 29 CELL BIOLOGY METHODS 7.6.2 Hypotonic lysis Hypotonic lysis buffer was added to parasites as a concentration of 1 x 10 6 IE/µl or at a concentration that was adjusted based on the parasitaemia of cultured and purified parasites. The hypotonic solution was then frozen and thawed out three times in Liquid nitrogen (LN2) and spun for 10 minutes at 20 000x g, 4 degrees Celsius. The supernatant was removed from the tube and transferred into a separate vial (Hypotonic soluble contents). The pellet was then washed with three rounds of cold PBS, spun each time and SN discarded. 7.7 Extraction of membrane fractions of IE 7.7.1 Salt extraction Salt extraction buffer was added to the cell pellet at a concentration of 4x10 6 IE/µl, resuspended several times by inversion and incubated for 30 minutes on ice. After this incubation time, the extract was spun at 20, 000g for 10 minutes at 4 degrees Celsius. The supernatant was then transferred to a new vial and stored at -20 degrees Celsius. 7.7.2 Carbonate extraction Carbonate extraction buffer was added to the cell pellet at a concentration of 4x106 IE/µl, resuspended and incubated on ice for 30 minutes on ice. After 30 minutes the solution was spun at 20,000g for 10 minutes at 4 degrees Celsius Page | 30 CELL BIOLOGY METHODS and the supernatant transferred to a new vial (containing carbonate extracted proteins) and stored at -20 degrees Celsius. 7.7.3 Urea extraction Urea extraction buffer was added to a the cell pellet at a concentration of 4x106 IE/µl, resuspended and incubated for 1 hour at room temperature. After 1 hour the extract was centrifuged at 20,000x g for 10 minutes at 4 degrees Celsius and then the supernatant was transferred carefully into a new vial. The vial was then dialysed against dialysis buffer over night at room temperature. 7.7.4 Triton X-100 extraction Cells were resuspended in 4x106 IE/µl in 1% Triton X-100 + PBS with 1x Proteasae inhibitor. The final concentration was then spun at 20,000x g for 10 minutes at 4 degrees Celsius and the supernatant was then transferred to a vial (this vial contained Triton X-100 extractable proteins). The remaining pellet was resuspended in 2x SDS loading buffer (LB) at a concentration of 4x106 IE/µl, incubated for 5 minutes at 95 degrees and spun down at 20,000g at 4 degrees for 10 minutes. The supernatant was then transferred to a new vial and the pellet was discarded. 7.7.5 SDS extraction The cell pellet was treated with 2x loading buffer at a concentration of 1x10 6 IE/µl, and the 1.3mL sample tubes boiled at 95 degrees Celsius for 5 minutes. Page | 31 CELL BIOLOGY METHODS The tube(s) were then centrifuged at 20 000x g for 10 minutes at 4 degrees Celsius and the SN was carefully transferred into a new vial avoiding uptake of the pellet, and the remaining pellet was discarded. 7.8 Immunofluorescence Analysis (IFA) Smears were prepared of cultured parasites and air dried overnight. The slides were fixed in a cold methanol/acetone mixture for 5 minutes and then air dried. A silicon pen was used to mark small squares onto the glass slide and left to dry for 5 minutes. The slide(s) was rehydrated in 1x PBS for 10 minutes and the excess fluid was shaken off. The primary antibody was added to each field (Fig. 16) which was diluted in 1x PBS before addition. The slide(s) were then incubated at room temperature for 2 hours in a small container which contained a water soaked tissue paper at the base of the container. The slide(s) were washed 3 times for 5 minutes in 1x PBS, excess fluid carefully shaken off and secondary antibody added which was used in conjunction with DAPI and 1x PBS solution at a concentration of 1:1000. The slide(s) were incubated at room temperature for 2 hours in the small container. The slide(s) were then washed in 3 washes of 1x PBS for 5 minutes each, excess fluid shaken off carefully and a coverslip was added. The slide(s) were transferred into a box and left in the dark in a dry area overnight. The slide(s) were analysed under a 100x objectives oil immersion. Page | 32 CELL BIOLOGY METHODS Square 1: 1st Ab mouse α-GFP Square 1 Square 2 Square 3 Square 4 Square 2: 1st Ab Rabbit α-HA Square 3: 1st Ab mouse α-HA Square 4: 1st Ab Rabbit α-aldolase Fig.16. Smaller fields were marked with a DAKO marker in order to distinguish which primary (right) and secondary Ab was used in each field. Primary and secondary Ab’s were added by pipette in a volume that covered an entire inner square which avoided cross contamination of conflicting Ab. 7.9 Live IFA Microscopy slides were labelled with a DAKO marker to outline squares that were to contain the DAPI solution. DAPI was added in a concentration of 1:1000 in 10x pellet volume of 1x PBS. The pellet was then washed 3 times with PBS, resuspended and 6ul of cell suspension was added to the slides. A coverslip was applied and the slide was analysed under the Olympus microscope. Page | 33 MOLECULAR BIOLOGY METHODS 8.0 MOLECULAR BIOLOGY METHODS 8.1 Western Blot Analysis MACS purified and fractionated parasites were run onto a pre- made 4-12% bis/tris 10-15 well SDS-PAGE gel and then transferred to a nitrocellulose membrane in 1x TBS. the ‘sandwich’ was set-up (Fig.17) and the nitrocellulose membrane was transferred to a chamber and run for 1 h 30 min at 400mA/cm 2. Successful transfer of proteins from the nitrocellulose membrane was observed via efficient transfer into Ponceau stain solution for 2 minutes and then washing immediately after the protein visualising. The membrane was then washed 3 times with 1x TBS with agitation and then blocked with 5% milk powder in TBS for 30 minutes at room temperature with constant agitation. The membrane was then transferred into a 50mL tube and primary antibodies were then added at a concentration of 1:2000. The tube was left to incubate overnight on a laboratory rocker at 4 degrees Celsius. After the membrane was washed 3 times with TBS for 15-20 minutes, the membrane(s) were then blocked with 5% Milk powder In TBS for 20 minutes. Secondary antibodies coupled with HRP were then added to the membrane(s) at a concentration of 1:2000 and incubated at room temperature for 2 hours. The membrane(s) were then washed 3 times with TBS each for 20 minutes and the blots were placed onto a cassette where the substrate (1mL) was added and left to incubate for between 5-10 minutes. The signal was then visualised by exposure of X-ray film and development by the Fuji developer processes in the dark room (Level 3 RMH). Exposures of 1 and 5 minutes, or 1 hour were carried out for each X-ray film. Page | 34 MOLECULAR BIOLOGY METHODS Clear top (+ve) Sponge 2x whatman paper Membrane Gel 2x whatman paper Sponge Black plastic holder (-ve) Fig.17 The arrangement of the ‘sandwich’ set-up configuration. The electrophoresis gel was covered with the nitrocellulose membrane and flanked from both ends by a series of whatman papers and sponges. C A B Fig.18. (Biorad) the Western blot transfer apparatus taken from biorad. Showing (A) transfer electrodes, (B) cassette which contains membrane/gel configuration and (C) transfer tank which runs at 400mA. 1x MES buffer was transferred to the chamber to allow the added proteins in each well to migrate through the gel. Transfer of the protein of interest to the nitrocellulose membrane was done with 1x TBS buffer. Page | 35 RESULTS 9.0 RESULTS 9.1 RIFIN protein expression examination (IFA) RIFINS have been visualised via means of IFA methods in order to understand their localisation and expression patterns (Petter et al 2007) in IE. Parasites were analysed at a high level of synchrony from treatment of 5% sorbitol which eliminated late stage parasites (Toyama et al 2012) and allowed only early stage parasites to continue division in order to achieve a homogenous culture of trophozoites that had a higher chance of containing the RIFIN proteins. Images 1, 2 and 3 were taken from the application of live IFA to parasites under conditions outlined in the Methods (4.10 Live IFA) section. DAPI stain was added to each parasite sample in order to closely understand where exactly the RIFIN’s were expressed. Since all samples were to be lysed for western blot analysis, the IFA, both fixed with antibodies and Live, were used as a confirmation analysis that ensured the presence or absence of RIFINS before membrane fractionation was executed. Page | 36 RESULTS Image 1 Visualisation of the RIFIN protein GFP Late stage mature P. falciparum parasites have been stained with DAPI (DNA stain) and can be seen in the centre of view. The darker green (GFP) stained areas in the IE possibly indicates rifin expression. Image 2 DAPI stained P. falciparum IE A B P. falciparum DNA has been successfully stained with DAPI (B), however the lack of GFP fluorescence may suggest that RIFIN expression is not present or perhaps the GFP sequence has been displaced from the RIFIN sequence during culturing conditions. Cells in the background (A) represent those RBCs that remained unstained and therefore uninfected by P. falciparum during culturing conditions. Page | 37 RESULTS A Image 3 B Schizont stage parasite stained with DAPI RIFINS are thought to be expressed from early trophozoite stage all the way to schizont stage parasites (Fig. 5). Image 3 (A) shows a DAPI stained schizont with several parasites within the IE ready to be released as merozoites for re-invasion. The image on the right (B) is also a schizont stage parasite (filtered only for green light) and shows green fluorescence which may suggest GFP expression, and hence RIFIN expression. 9.2 Conclusion RIFIN expression was monitored by fixed IFA and Live IFA with a higher parasitaemia (>8%) yielded for each parasite 3D7 line during MACS purification and isolation. The results from the IFA analysis indicate that there was indeed a signal of GFP within the IE but the extent to which was attributed to RIFIN expression remains unclear since this may well be natural fluorescence from cellular components. DAPI stained nuclei were successful (seen in Image 1 and 2) which does indicate the presence of the P. falciparum parasite within erythrocytes. DAPI stain visualisation Page | 38 RESULTS under the Olympus microscope was an indication that RBCs were infected with the parasite strain and could be easily distinguished to those RBCs that were uninfected (see Image 2). Fixed IFA involved the use of primary and secondary antibodies to provide a much more sensitive fluorescence from the binding of primary antibodies to the GFP sequence conjugated into the RIFIN sequence (Appendix 2 (A)) for tagging. The images above do not represent an accurate portrayal of RIFIN localisation and further analyses must be accomplished in the order of culturing parasites to a healthy parasitaemia, synchronising parasites to maintain strict stagespecific behaviour and observing the staining patterns of IE from early, and late stage parasites. 9.3 Solubility of RIFIN proteins in membrane and intra-erythrocyte parasites. Membrane fractionations was carried out on trophozoite stage parasites that were raised at a parasitaemia of either 5-8% or up to 10% in order to yield a greater number of IE that may have been lost during MACS purification. The western blot experiments provided some information relating to the solubility of RIFINS during their harvesting and isolation; however no formal conclusions could be made based on the expression of RIFINS. Page | 39 RESULTS Table 1 Fractionation Method Hypotonic Lysis Saponin Lysis Total Cell lysate Extraction of pellet 1. Supernatant Western blot analysis 1. Salt extraction Western blot Analysis 2. Carbonate extraction 3. Urea extraction 4. TX-100 extraction 5. SDS extraction Tabulated representation of fractionations used for western blot analysis. The sequential extraction of the cell pellet from the hypotonic lysate was continued (steps 1-5). The Saponin and total cell lysate were extracted in solution and SN analysed on WB. Page | 40 RESULTS 9.4 Anti- RIFIN antibodies analysis Primary antibodies were used to detect sequences associated with the HA- or GFPtag that were transfected into RIFINS during transformation. RIFIN expression was difficult to observe under IFA (5.1.1) and many control samples were screened on western blots in order to troubleshoot SDS and WB procedures. The following subsequent images represent the control fractions that were utilised for later comparison with RIFIN isolates. The primary and secondary antibodies used on these tagged proteins were also used on further western blot analyses on purified mature stage parasites to identify the RIFIN proteins. 1 Image 4 Left: Western blot of pARL 6 parasite line showing bands around the 98kDa (1), and another thick band that is within the range of 49-38kDa (2). Right: Bands are resolved at positions analogous to the WB shown on the left. Both WB were probed with the same primary antibody (Rabbit αaldolase) and bands were visualised by incubation with Goat α-rabbit HRP secondary antibody. The sample sizes loaded were read against the protein ladder contained in Appendix 1. 2 Page | 41 RESULTS Image 5 Western blot of fractionated and lysate contents taken from a control sample of P. falciparum parasites containing the SBP-1 membrane protein seen here as bands within the SDS (lane 3), Triton X-100 SDS (Lane 7), and, lysate (lane 8). The bands correspond to a size of 49kDa. Thinner bands could be resolved around the 49kDa size in carbonate and urea fractions, (lanes 1 and 2, respectively). The presence of SBP-1 was confirmation that this membrane associated protein was contained within the MC, and could be used as a control for further western blot studies against the RIFINS. Protein ladder was marked for MW size (middle). Summarised Table 2 Table 2 Lane Fraction Band(s) kDa 1 Carbonate 188, 20 2 Urea 20 3 SDS 188, 98, 55, 20 4 Ladder N/A 5 Hypotonic Lysis 49 6 TritonX-100 - 7 Triton X-100 (SDS soluble proteins) Lysate 49, 20, 55 8 49, 20, 55 Page | 42 RESULTS Image 6 Rabbit α-aldolase primary antibodies (1:2000) were probed onto cell lysates and visualised with Goat α-rabbit HRP used as a secondary antibody. Thick protein bands (line) can be seen around the 42kDa mark which indicated the presence of aldolase. Fainter bands are represented in the total lysate (lane 8) which were not analysed. 42kD a 9.5 Conclusion Several bands were observed throughout the western blot analyses when each fraction and lysate was probed and exposed on X-ray film. Although no RIFINS were formally concluded from these experiments there was however substantial evidence to show the presence of the Maurer’s cleft membrane protein SBP-1 (Image 5). The strongest protein signals came from the anti aldolase antibodies seen in Image 6. Page | 43 DISCUSSION 10.0 DISCUSSION 10.1 Overview Variant surface antigens are believed to serve the main purpose in a P. falciparum IE to elicit an immune response triggering an antibody response (Bull et al 2005) and also by completely avoiding entry into the spleen (Bull et al 2005) by latching onto blood vessel walls (Kyes et al 1999). The PfEMP-1 surface antigen has been described as a variant antigen (Craig & Scherf 2001) that exposes the majority of its protein sequence on the extracellular side of IE, while the postulated 2TM variant antigens such as rif and stevor genes possess a loop region (Lavazec et al 2006) that accounts for antigenic diversity (Lavazec et al 2006) against the host immune system response. Expression of the RIFIN products does not occur by chance or accident and since their genomic data is present within the telomeric regions of the P. falciparum chromosomes (Petter et al 2007) in conjunction with the var and stevor genes (Dzikowski et al 2006) there is much more evidence to suggest a role in immune evasion (Petter et al 2008). The results gathered from this report cannot clearly conclude the RIFINS were in fact peripherally expressed and subsequently if they were involved with immune evasion and/or cytoadhesion, however the results do conclude that molecular and biological techniques employed in this research were valid for the isolation and IFA visualisation of proposed membrane associated proteins and other cellular components such as Plasmodium falciparum DNA. Page | 44 DISCUSSION 10.2 SDS-PAGE/Western Blot Analysis P. falciparum 3D7 cultures were harvested and purified to increase the cell number of late stage parasites. Upon western blot experimentation several protein bands were observed along nitrocellulose membranes that were derived from the successful transfer of protein from 6-12% SDS-PAGE bis/tris gels. 10-15 well lanes were used to load samples that included membrane associated proteins from the sequential fractionation of the IE membrane (4.0 Cell Biology Methods, 4.8 Extraction of membrane fractions of infected erythrocytes). Samples were loaded and run against a high to low molecular weight protein marker from each fractionation step and separate Western Blot analysis were setup for saponin-RBC lysates which excluded red blood cells from the overall parasitaemia allowing only free parasites to remain in culture. Controls were analysed to provide a guide regarding the location of intra-erythrocyte proteins associated with the MC which could provide some theory into the expression of RIFINS. The protein associated with the Maurer’s cleft (SBP-1 membrane protein) was visualised at a position of 49kDa in the carbonate, urea, SDS, Triton X-100 SDS and total cell lysate, which provided some information relating to the lysis and solubility of these transport proteins. If for example, the RIFINS were expressed and associated with the MC protein SBP-1, then it could be suggested that for each of the fractions analysed, (carbonate, urea, SDS, Triton X100 SDS and total cell lysate) these were in fact compatible with the solubility of RIFINS and via MC as a transport protein, could be possibly identified with WB. However, from the lack of expression of the RIFINS this could not be established and SBP-1 would only provide a comparable size marker. Table 2 describes the Page | 45 DISCUSSION bands that were visualised from the western blot and from this information, conclusions can be reached with the effectiveness of the WB procedure, the addition of the substrate to the HRP conjugated secondary antibody and the film exposure. Table 2 also indicates a series of much smaller unresolved protein bands that were not analysed in this study as the protein of interest from this WB analysis was the SBP-1. Adding to this, anti-aldolase antibodies were utilised to identify an aldolase protein (42kDa) that was to be a control protein for the further western blot experiments to pin point the RIFINS from the sequential extraction methods. 10.3 IFA of RIFIN expression The IFA showed the presence of green fluorescence from the GFP tagged RIFIN protein (9.0 Results, image 1). Whether or not this was due to auto-fluorescence from the IE rather than the actual expression of RIFINS remains unknown. It could be that during cell culturing, after transformation of the rif genes into a plasmid, the RIFIN protein products were displaced from the RIFIN sequence (ref) or perhaps natural fluorescence (auto-fluorescence) had occurred which may have contaminated the GFP sequence with endogenous cellular contents from the IE and consequently alter imaging of the RIFINS (Image 1 and Image 2). Possible RIFIN expression was observed in schizont stage parasites which indicated a stronger signal to the DAPI stain, as some signal from the activation of GFP can be seen from Image 3, however failed to provide a clear insight into the localisation patterns of a subset of RIFIN proteins. Page | 46 CONCLUSION CONCLUSION The literature covering expression, topology and localisation of the RIFIN proteins states that plasmodium falciparum transcribes two classes of RIFIN sub-types, each that have been previously shown to serve specific purposes inside an IE. 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JEM 201 (11):- Page | 52 APPENDICES APPENDIX 1 Page | 53 APPENDICES APPENDIX 2 (A) Protein Sequence MLLCLFLLNKLLLLLLLLPLLLSCNFQSNGYISPHTRSVTKITTSRTLSEFDKYKNNYDD DPEMKELMKRFNERTAVRLKEYDEQKKKKQKIYKEKKDKDIKEIIVKDKIQKQLTKQLSK LEKVTDTDDLFKGKNEKKVATKGKKGRKKNKKTLGQTLSEWNILPNIDMYEWIPFSSKEA KVDCNIKNIKQALTRVGYIGPNKFVTLGSKDGKDAIDMDNLFSSIMSSSNALYQKYIDKD DTSNGSSFKKQSGFLSFALYALWEIFEHIVLPVVTSMLLNGNDSESQVSGVDGHGHEVAH GVSVLYESFIALYTIASILLILYYILKYYRKIRMEKKEKYMKILQD Sequence Length: 346 aa (B) Coding sequence ATGTTATTATGTTTATTTTTATTGAATAAATTATTGTTATTATTATTATTGTTACCATTA TTATTATCATGCAATTTTCAAAGCAACGGTTACATTTCACCACATACAAGAAGCGTCACA AAAATAACTACATCGAGAACATTAAGCGAATTTGACAAATATAAAAATAATTATGATGAT GATCCTGAGATGAAAGAATTAATGAAAAGATTTAATGAGCGAACAGCAGTACGATTGAAA GAATATGATGAACAAAAAAAGAAGAAGCAAAAAATATATAAAGAAAAAAAAGATAAAGAT ATAAAAGAAATTATTGTAAAAGATAAAATACAAAAACAACTAACAAAACAATTATCAAAG TTAGAAAAAGTAACTGATACGGACGATTTATTTAAAGGTAAAAATGAAAAAAAGGTAGCA ACTAAAGGGAAAAAAGGTCGTAAAAAAAATAAGAAAACATTAGGTCAAACATTATCAGAA TGGAACATTTTACCTAATATTGATATGTATGAATGGATACCGTTTTCTTCTAAGGAAGCT AAAGTTGATTGTAATATAAAAAATATAAAACAAGCTCTTACTAGAGTTGGTTATATAGGT CCTAATAAATTTGTTACTCTTGGTAGTAAAGATGGAAAAGATGCTATTGATATGGATAAC TTATTTAGTTCTATTATGAGTTCTTCTAATGCTTTATATCAAAAATATATAGATAAAGAT GATACATCTAATGGTAGTAGTTTTAAAAAACAAAGTGGTTTTCTCTCTTTTGCTCTTTAT GCGTTATGGGAGATTTTTGAACATATTGTACTCCCCGTTGTCACTTCTATGTTGTTGAAT GGTAACGATTCTGAGAGTCAAGTTTCTGGGGTTGATGGTCATGGACATGAAGTTGCTCAT GGTGTTAGTGTACTTTATGAATCCTTTATTGCATTATATACTATAGCATCAATTCTATTA ATTCTTTATTACATTTTAAAATATTATAGAAAAATAAGAATGGAAAAAAAAGAGAAATAC ATGAAAATATTACAAGATTAG Sequence Length: 1041 bp Page | 54 APPENDICES (C) GFP Protein sequence MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGR PVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDL LSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRY QDIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF Page | 55 APPENDICES APPENDIX 3 pARL3 HA-GFP tag pARL6 HA-GFP tag HA-GFP tags were positioned in different locations along the RIFIN protein. pARL3 and pARL 6 were similar in arrangement and were used for the majority of the experimental work looking into their expression and imaging pattern Page | 56 APPENDICES Appendix 4 Page | 57 ACKNOWLEDGMENTS ACKNOWLEDGEMENTS I wish to express sincere appreciation to Dr. Michael Duffy and Dr. Michaela Petter for their assistance with my laboratory work, and for their patience and hospitality in making me feel welcoming into the Duffy Group. In addition, special thanks to the laboratory members who gratefully assisted me throughout my research and gave me their support. A special thanks to Mum and Dad, Anna, and Roxane and also to all of my friends who gave me amazing support throughout 2013. Lastly, a very big thankyou to Nurul Ashiqin Abidin Zainal for her unconditional love and support, and for being the inspiration throughout my life. Page | 58 End of Thesis Page | 59