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This project investigated the role of PFB0115w in parasite virulence, the identification of its binding partners and the characterisation of its knockout phenotype.

The PFB0115w-GFP interactome

Previously our group localised PFB0115w to the host cell membrane and showed partially co- localisation with KAHRP and RESA, both virulence complex markers of P. falciparum (Fobes 2014; Ooi 2014). However, analysis of the immunoprecipitated complex of solubilized PFB0115w-GFP complex revealed that both KAHRP and RESA were not direct binding partners of solubilized PFB0115w in vivo. Both proteins reside in the virulence complex and these results indicate that the partial co-localisation of both proteins with PFB0115w in the previous immunofluorescence assay (IFA) was a result of the limited microscope resolution and their similar subcellular localisation (G. V. Brown et al. 1985; Pologe et al. 1987).

Mass spectrometric and blue-native page analyses indicate that the PFB0115w does to bind to the exported proteins MESA and PFD80. MESA, the second major interacting partner, was found to solubilize in a similar sized complex as that of PFB0115w, further validating the theory that MESA and PBF0115w are interacting partners in vivo. MESA is known to interact with host

Chapter 3

network (Lustigman et al. 1990; Coppel 1992; Waller et al. 2003). Association of PFB0115w with MESA could suggest that PFB0115w localizes to the 4.1R-actin junction and is involved in the disruption of the cytoskeletal network and associated decrease in deformability. However, this potential contribution to the loss in deformability would still need to be verified through the assessment of deformability in CS2PFB0115 cells.

Selective upregulation of MESA was seen in both paediatric and cerebral malaria as compared to placental malaria and uncomplicated malaria, suggesting that MESA expression is associated more with non-VAR2CSA strains of malaria (Vignali et al. 2011; Bertin et al. 2016). Thus, the association of PFB0115w with MESA would suggest that PFB0115w does not co- express with VAR2CSA, contrary to previous findings (Tuikue Ndam et al. 2005; Francis et al. 2007). However, neither co-expression results nor the current immunoprecipitation results are fully indicative of the association between MESA and PFD0115w, which would require an additional reciprocal pulldown with tagged MESA.

PFD80 (PF3D7_0401800), the second co-precipitate of PFB0115w, is an unannotated PHISTb protein that has been confirmed to localise to the virulence complex on the host cell membrane (Vincensini et al. 2005; Tarr et al. 2014). Although the precise function of PFD80 is not known, disruption of PFD80 led to an associated in vitro growth fitness cost, suggesting it could play an important role in the survival of the parasite in vitro (Zhang, Wang, et al. 2018). Transcriptome studies corroborate the importance of PFD80 in parasite survival in vivo as it has been observed to show increased expression in field isolates (Mackinnon et al. 2009; Tonkin-Hill et al. 2018). While the interaction between PFD80 and PFB0115w was not investigated further in the current project, it would be eventually be necessary to elucidate the role of PFB0115w in pathogenicity and virulence.

Glycophorin binding protein (GBP or PF3D7_1016300) was also present in the complex. Similar to MESA, GBP has been implicated to play a role in the loss of deformability and cytoadherence (Maier et al. 2008). However, GBP does show a high and ubiquitous abundance in the host cytosol (Ravetch et al. 1985) and was not pursued further here in the interest of time. Nevertheless, a further investigation into the function of PFB0115w would need to

confirm the interaction between GBP and PFB0115w through a reciprocal pulldown of tagged GBP in vitro.

Four unique proteins were also identified in the precipitated complex as determined by the Scaffold program. Scaffold identifies proteins as those unique to the precipitated complex regardless of their relative abundance to the bait protein, allowing us to identify potential interacting partners with a low abundance. Of the four proteins, PfHsp70-1 (PF3D7_0818900) was investigated further here to the availability of a GFP-tagged PfHsp70-1 cell line. PfHsp70- 1-GFP was found to be present in a different complex to that of PFB0115w, indicating a lack of an in vivo interaction with PFB0115w. This was not surprising as PfHsp70-1 is an abundant chaperone protein that mainly functions in the parasite cytosol (Foth et al. 2003; Misra & Ramachandran 2009). ‘Ghost' preparation from infected cells requires hypotonic lysis and

thus includes both the host and parasite plasma membrane. The use of ghost preparations would make it possible for parasite proteins that transiently associate with the PPM such as chaperones to be present as artefacts.

Three proteins of human origin were also discovered in the precipitated complex: ankyrin-1, spectrin 1 and catalase. Subsequent western blot analysis revealed that both Ankyrin-1 and spectrin 1 were false positives and did not interact with PFB0115w. The solubilized protein lysate was prepared from ghost cells, whose main protein component is that of cytoskeletal origin. Thus, due to the sheer abundance of spectrin and Ankyrin in the sample, presence in the purified complex might have been either due to residual unbound protein or non-specific background binding to the GFP tag. Catalase, involved in oxidative stress, was also found to be significantly 200-fold more abundant in the precipitated complex. While it is well known that uninfected erythrocytes have high catalase activity and a P. falciparum invasion induces an even higher increase in activity (Atamna & Ginsburg 1993; Clarebout et al. 1998), association of catalase with export proteins has not been observed. However, studies on in vivo tracking with GFP have showed that the tag induces oxidative stress and high catalase activity in cells (Ansari et al. 2016; Ganini et al. 2017), which suggests that the presence of catalase in the PFB0115w complex is due to its ubiquitous presence and increased oxidative stress.

Chapter 3

The interactions of PFB0115w with PDF80w, MESA and GBP remain putative as they need further validation with a reciprocal pulldown and co-immunoprecipitation analysis to be confirmed as in vivo partners.

Functional characterisation of PFB0115w

We also showed here that PFB0115w was successfully knocked out in CS2 wildtype by conventional homologous recombination. During the course of this study, other studies also produced PFB0115w knockouts by SLI knock – sideways, CRISPR-Cas9 and piggyBac mutagenesis (Birnbaum et al. 2017; Chan et al. 2017; Zhang, Wang, et al. 2018). This confirms that PFB0115w is not essential for parasite survival in vitro.

Disruption of PFB0115w had no effect on the surface presentation of VAR2CSA variant PfEMP1 in CS2PFB0115w clones as demonstrated by trypsin cleavage assays. These findings are contrary to those reported by Chan et al. (2017), who observed a cessation of VAR2CSA expression and a drastic reduction in CSA adherence in NF54-CSAPFB0115w parasites (Chan et al. 2017). The variation in phenotypes between the two studies could be a result of the different methodologies employed. The Chan et al. study used an antibody against the variant N-terminus of VAR2CSA and only observed CSA adherence thus not accounting for any potential VAR gene switching post disruption. In our study we used an antibody against ATS, the C-terminus segment conserved among PfEMP1 variants. The anti-ATS antibody indicated that PfEMP1 expression remains consistent in the absence of PFB0115w. Additionally the NF54-CSAPFB0115w parasites in the Chan et al. study also had additional off target gene disruptions in KAHRP and PfEMP3, both of which are required for surface presentation of PfEMP1 (Crabb et al. 1997; Waterkeyn et al. 2001; Rug et al. 2006).

Aside from the expression of VAR2CSA in the absence of PFB0115w, localisation of full length PFB0115w-GFP, in our hands, is seen exclusively at the host cell plasma membrane, which disputes its role as a translation enhancing factor for VAR2CSA as described by Chan et al. (2017).

In conclusion, while prior studies suggested that expression of PFB0115w is linked to VAR2CSA expression, observations made in the current study indicate that PFB0115w is not required for the expression of PfEMP1. Additionally, the PfEMP1 variant stayed the same post-

PFB0115w disruption as the trypsin cleavage product, whose size changes with different variants (Waterkeyn et al. 2000; Rug et al. 2014). PFB0115w otherwise did show any direct association with VAR2CSA. Reciprocal pulldowns of PFD80 and MESA and analysis of PFD80 and MESA distribution in CS2PFB0115w would give further insight into the functional role of the PFB0115w complex. Additionally, characterisation of deformability of CS2PFB0115w clones would give us a better insight into the function of PFB0115w.

Chapter 4

4 Functional characterisation of PFI1780w

Introduction

PFI1780w is a novel export protein that was identified as a component of the virulence complex at the host cell membrane in a mass spectrometric analysis done in our group (Fobes 2014). A two-exon gene, PFI1780w contains a non-canonical PEXEL motif (KSLAE) and a PHIST domain at the C-terminus (Schulze et al. 2015). It is a part of the PHISTc subfamily and has orthologs only in the subgenus of Plasmodium but not in other human Plasmodium species such as P. vivax and P. knowlesi (Sargeant et al. 2006).

A recent NMR (nuclear magnetic resonance) study by Mayer et al. postulated that the PHIST domain of PFI1780w binds to the conserved ATS segment of PfEMP1, the main virulence factor of P. falciparum (Mayer et al. 2012; Oberli et al. 2014). The authors showed that the PHIST domain in PFI1780w has a high affinity to the ATS segment, binding to a conserved epitope on the ATS sequence (Mayer et al. 2012; Oberli et al. 2014). Based on these findings the authors hypothesized that PHIST proteins bind and anchor different PfEMP1 variants to the membrane (Oberli et al. 2014). This was the first instance of an PfEMP1-binding export protein other than KAHRP, a virulence complex protein that is considered the main binding partner of PfEMP1 (Waller et al. 1999). Additionally, the authors could not identify an interaction between KAHRP and PfEMP1. The PHIST-ATS interaction may suggest a new function for the PHIST domain but this interaction is only supported by NMR ex vivo data and requires in vivo validation with analysis of protein-protein interactions.

Additionally, a transcriptome study revealed that PFI1780w shows a significant 2-fold increase in field isolates as compared to laboratory strains (Mackinnon et al. 2009) and is associated with an expression gene cluster in chromosome 9, linked to a cytoadherence phenotype to the CD36 receptor (Shirley et al. 1990; K. I. Barnes et al. 2005; Bourke et al. 1996). Thus, PFI1780w is a worthy candidate for identification of its interacting partners and functional characterisation. Previously we verified the localisation of PFI1780w-GFP to the host cell membrane through immunofluorescence assay (IFA) and where it partially co-localized with KAHRP. PFI1780-GFP did not show co-localisation with PfEMP1 however this may be due to PFI1780w covering the anti-PfEMP1 antibody recognition epitope (Ooi 2014).

In this project, we studied the interacting partners of PFI1780w in vivo, including PfEMP1, through the immunoprecipitation of PFI1780-GFP and also attempted to disrupt PFI1780w for loss of function studies.