Exported proteins at the host cell periphery

Most characterised proteins that localise to the host cell periphery are also found in detergent resistant domain fractions, indicating they preferentially localise to lipid microdomains on the membrane (Nagao et al. 2002; Murphy et al. 2007). At the host cell periphery, proteins can

either remain in Maurer’s clefts or localise to the membrane-associated complex, which appears as electron dense knobs on the surface of the infected cell (Aikawa et al. 1983; Gruenberg et al. 1983).

The knob complex, which is physically tethered to Maurer’s clefts and will be jointly referred to as the virulence complex from hereafter, houses several proteins essential for P. falciparum

component or are involved in trafficking and presentation of PfEMP1 (de Koning-Ward et al. 2016).

KAHRP: The characteristic protein of the virulence complex is the knob-associated histidine rich protein (KAHRP or PF3D7_0202000). KAHRP binds to actin, the ⍺-chain of spectrin network and ankyrin R to cause an increase in rigidification of the host cell (Kilejian et al. 1991; Pei et al. 2005; Weng et al. 2014). KAHRP is essential for knob formation as the disruption of KAHRP disrupts knob formation, associated rigidification and reduces PfEMP1 epithelial receptor binding (Crabb et al. 1997; Horrocks et al. 2005; Rug et al. 2006). KAHRP is also thought to bind and anchor to the ATS segment of PfEMP1 since it shows evidence of ATS binding domains (Waller et al. 1999; Waller et al. 2002; Oh et al. 2000). Studies on ‘knobless’

clones indicate that although KAHRP is not essential in vitro, it is required for cytoadherence and spleen evasion, which are both essential for parasite survival in vivo (Raventos-Suarez et al. 1985).

PfEMP3: P. falciparum erythrocyte membrane protein 3 (PfEMP3 or PF3D7_0201900) is an exported protein is required for the surface presentation of PfEMP1 (Pasloske et al. 1993; Waterkeyn et al. 2000). Similar to KAHRP, PfEMP3 shows evidence of interacting with both spectrin and F-actin, as shown by binding studies performed in a heterologous system (Waller et al. 2007).

RESA: Ring-infected erythrocyte surface antigen (RESA or PF3D7_0102200) is an immunogenic DnaJ protein, expressed primarily in the early ring, late schizont and merozoite stages (Aikawa et al. 1990; Rug et al. 2004). RESA prevents the dissociation of spectrin β-chains from mechanical and heat stress (Foley et al. 1991; Da Silva et al. 1994; Pei et al. 2007) and consequently contributes towards the decrease in deformability (Mills et al. 2007; Diez-Silva et al. 2012). Gene knockout studies suggest that RESA plays a protective role against febrile heat stress that occurs with the rupture of schizonts (Silva et al. 2005). Furthermore, studies on merozoite invasion indicate RESA expression is necessary to prevent re-invasion in infected cells (Pei et al. 2007). The protein is dispensable in vitro but contributes to the increase in rigidity of the infected erythrocyte and modulates its cytoadherence (Silva et al. 2005). Field

Introduction

studies suggest that RESA is necessary in vivo as it consistently expressed in a conserved form in field isolates (Perlmann et al. 1987).

Figure 1-7 Structure of the virulence complex, in infected erythrocytes. The virulence complex is

linked to the host cytoskeleton through the binding of several host cytoskeletal proteins. Maurer’s clefts are attached the ‘knob’ component of the complex through protein and actin filament tethers.

MESA: Another component of the complex is the mature parasite infected erythrocyte antigen (MESA or PF3D7_0500800), expressed primarily in trophozoite and schizont stages. It competitively binds to the host junction protein 4.1R and disrupts the normal interaction between 4.1R and p55 (Coppel et al. 1988; Coppel 1992; Waller et al. 2003). 4.1R forms a ternary complex with p55 and glycophorin and connects the cytoskeleton to the membrane (Marfatia et al. 1994). The ternary complex acts a junction to link spectrin chains and is vital for the mechanical stability of the cell (Salomao et al. 2008). Association of MESA with 4.1R is also accompanied with increased phosphorylation of 4.1R mediated by host protein casein

kinase (Lustigman et al. 1990; Magowan et al. 1998). Phosphorylation of 4.1R has been previously shown to reduce its affinity to spectrin resulting in reduced mechanical stability of the cell (Eder et al. 2002; Gauthier et al. 2011). While the contribution of MESA to deformability has not been directly studied, reduced stability would indicate an overall change in deformability. Gene disruption analysis of MESA in normal and 4.1R deficient erythrocytes

in vitro has shown that it is important for parasite viability in the presence of 4.1R protein, indicating the 4.1R and MESA linkage is required for parasite survival in vivo (Magowan et al. 1995).

Other exported proteins: The proteins mentioned above are the more extensively characterized proteins and are a small fraction of the parasite exportome. Recent advances in genetic manipulation in Plasmodium has led to an increase in characterisation of exported proteins. A loss-of-function screen for more than 80 predicted exported proteins expanded the annotated protein repertoire and revealed several more proteins with a phenotypic effect on host cell modification (Maier et al. 2008). PFD1170c (PF3D7_0424600) showed a similar phenotype to KAHRP where disruption of the gene led to alteration of knobs on the surface, reduced deformability and cytoadherence (Maier et al. 2008). PFE60 (PF3D7_0501200), an integral MC protein, was found to affect deformability in its absence. Later studies revealed that PFE60 was essential for the transport of Pf332, a large membrane bound protein involved in membrane deformability (Zhang, Faou, et al. 2018).

Although research in the characterisation of export proteins is extensive, there is still a large number of proteins without any assigned function. The composition of the virulence complex and mechanisms behind its assembly are still not completely understood as well. Furthermore, there is the possibility of other complexes on the membrane, given the magnitude of proteins exported and the discovery of proteins localized between knob complexes (Proellocks et al. 2014). Thus, characterisation of additional export genes, their localisation and phenotypic effect on cytoadherence and deformability, is necessary to understand and define these processes. However, there is also the issue that several of these unannotated export proteins can have functionally redundant roles or have functions that cannot be resolved by an in vitro

Introduction

Subsequent studies would require an additional filtering process to select novel export proteins that have an implied role in host cell modifications. In this project, this filtering process was the mass spectrometric analysis of complex-enriched membranes that was previously performed in our group (Fobes 2014). Trophozoites were enriched with the virulence complex through sucrose density gradient centrifugation and then analysed by low chromatography mass spectrometry (LC/MS) for both annotated and unannotated proteins. The study revealed 14 unannotated proteins with several of them containing a PHIST domain (Fobes 2014). Among the 14 proteins however was an unannotated protein without an export motif, PFB0115w (PF3D7_0202400). Proteomic comparison of cell lines without ‘knobs’ and

without MC trafficking protein PfSBP1, which is necessary for the surface presentation of PfEMP1, revealed that PFB0115w was membrane-associated and relied on the presence of knobs to localize to the host cell membrane.

Additionally, PFB0115w was also found upregulated in several transcriptomic studies of placental malaria isolates (Tuikue Ndam et al. 2005; Francis et al. 2007; Vignali et al. 2011; Bertin et al. 2013). This was supported by an in vitro study that observed its upregulation in VAR2CSA, the PfEMP1 variant specific for CSA, specific strain that also showed increased rosette formation (Mok et al. 2007). This suggested that PFB0115w plays a role in VAR2CSA presentation and thus contributed towards the virulence phenotype. Thus, the identification of a potential new PNEP and a possible PfEMP1 regulator prompted the further investigation into PFB0115w in this study. PFB0115w was characterised here for its function in PfEMP1 presentation and overall parasite virulence through both interaction studies and loss-of- function phenotype studies.

The other protein investigated here was PFI1780w. A recent NMR (nuclear magnetic resonance) ex vivo study revealed that PFI1780w, a PHISTc protein, had a high binding affinity for the ATS segment of PfEMP1 and more so than KAHRP, which is considered the binding partner for PfEMP1 (Waller et al. 1999; Mayer et al. 2012) . This study indicated the first example of a protein, other than KAHRP, binding to PfEMP1 and the authors later postulated that the PHIST domains serves as an interacting domain for PfEMP1 (Mayer et al. 2012; Oberli et al. 2014). Additionally, PFI1780w is expressed in both severe and uncomplicated malaria (Mackinnon et al. 2009; Tonkin-Hill et al. 2018), which suggests that the protein is important

for parasite survival in vivo. Thus, PFI1780w was investigated here to validate and elaborate on the PFI1780w-PfEMP1 interaction at in vivo conditions and identify other interacting partners.

The main aims of this thesis were the functional characterisation of PFB0115w and PFI1780w in context of the virulence complex. In order to quantify the phenotypic effect of both proteins, both cytoadherence and deformability of gene disrupted studies need to be evaluated. However, as mentioned before current techniques in deformability fall short in measuring changes in membrane deformability at a high-throughput level. Therefore, the third aim of this project was to develop a deformability technique that can resolve changes in membrane deformability over a population of cells.

Chapter 2