PfEMP1 is central to the virulence of P. falciparum parasites (Miller et al., 2002) and the main target of antibody-mediated immunity in symptomatic malaria patients (Chan et al., 2012), but studying these important proteins is challenging. Using SLI, we here generated cell lines predominantly expressing a PfEMP1 of choice and show that this facilitates the study of diverse aspects of PfEMP1 biology, including mutually exclusive expression, trafficking, interactome, and receptor binding. A small epitope tag permits reliable tracking of the SLI-targeted PfEMP1, avoiding issues detecting specific variants or the ATS. In addition, we show that larger tags such as a mDHFR domain or BirA* can be added and used to study transport or obtain the proxiome of functional PfEMP1 from living parasites. This also highlights positions in the PfEMP1 sequence where larger tags are tolerated, including in the external region, although the latter reduced the binding efficiency to some extent. Importantly, SLI ensures expression of the modified locus, which would be difficult with other approaches. We further introduce a second SLI system (SLI2) which permits a convenient further genomic modification while maintaining expression of the desired PfEMP1. This will also be of general usefulness to obtain double genome edited parasites.
The generated lines were capable of switching when G418 was lifted, indicating the system can be used to study switching and mutually exclusive expression of var genes. However, it should be noted that it is not known whether all mechanisms controlling mutually exclusive expression and switching remain intact in parasites with SLI-activated var genes.
Previous work indicated co-activation of genes in a head-to-tail position to the SLI-activated variant gene (Omelianczyk et al., 2020). We here only found evidence of co-activation with the activated var with genes in a head-to-head orientation, suggesting this occurred due to a shared promoter, rather than a general relaxation of silenced chromatin around the active var gene. Similar head-to-head activation had been detected when parasites expressing specific var genes were enriched by panning (Claessens et al., 2012). However, it is unclear if this can be generalized, and it is possible that different var loci respond differently. We also confirmed reduced mutually exclusive expression in a previously published 3D7 cell line (Joergensen et al., 2010) that we here termed 3D7MEED and may be useful to study var silencing mechanisms.
PfEMP1-receptor binding and neutralizing antibody mechanisms are increasingly being understood on a structural level and are relevant to understand malaria pathology and effectivity of the immune response in patients (Rajan Raghavan et al., 2023; Reyes et al., 2024). The straightforward capacity to generate cytoadherent parasite lines uniformly expressing a single PfEMP1 of interest opens up approaches to study receptor-binding as well as antibody-binding and inhibition using native as well as modified PfEMP1. The latter could be done by inserting point mutations, removing, exchanging, or altering domains, for example, by modifications directly in the original SLI plasmid or using CRISPR in the SLI-activated line.
An unexpected finding of this work was that IT4var19-expressing parasites bound ICAM-1 in addition to EPCR as this is considered a PfEMP1 that only binds EPCR (Avril et al., 2012; Nunes-Silva et al., 2015; Adams et al., 2021), although some studies indicated that it may bind additional receptors (Gillrie et al., 2015; Ortolan et al., 2022). Interestingly, selection for EPCR-binding was required to achieve avid EPCR binding of the IT4var19 expressor line. While this binding selection did not change the var expression profile and IT4var19 remained the dominantly expressed PfEMP1, we cannot exclude that this resulted in other changes that could have led to ICAM1 binding. Selection for EPCR-binding was accompanied by higher expression of ptp3 genes previously shown to affect PfEMP1 presentation and cytoadhesion (Maier et al., 2008), suggesting this as a reason why these parasites did not initially bind. As our findings indicate, this was not due to a genome deletion, this raises the possibility of an additional layer controlling surface display through expression of PTP3 as an accessory factor by binding selection. Thus, the combination of uniform var expression and phenotype selection may enable detection of hitherto unrecognized PfEMP1 receptor phenotypes and phenomena controlling PfEMP1 surface display.
In the course of this work, the binding phenotype of the IT4var19 expressor line remained stable over many weeks without further panning. However, given that initial panning had been needed for this particular line, it might be advisable for future studies to monitor the binding phenotype if the line is used for experiments requiring extended periods of cultivation.
Previous work has indicated that mutants of the different proteins involved in PfEMP1 trafficking block its transport at different points on the way to the RBC surface, including at or before passing into the RBC (Cooke et al., 2006; Maier et al., 2007; Rug et al., 2014; Maier et al., 2008). Considering the results here and work on SBP1-disrupted parasites (Blancke Soares et al., 2025), none of these proteins seems to influence PfEMP1 before it reaches the Maurer’s clefts. This aligns with the location of these proteins, which suggests that they function in the host cell. This would mean that the effect of PTEX inactivation on PfEMP1 transport (Beck et al., 2014; Elsworth et al., 2014) is likely direct, as the exported PfEMP1-trafficking proteins (if prevented from reaching the host cell due to the PTEX block) would not influence PfEMP1 before it reached the host cell. Together with the result from the stage-specific block of PTEX in this work, the currently most plausible scenario is that PfEMP1 is transported by PTEX, after which other exported proteins are needed for transport to the surface and correct surface display. Why the mDHFR-fused PfEMP1 was not prevented in transport when WR was added is unclear, but may be due to the long region between the transmembrane domain and mDHFR (Mesén-Ramírez et al., 2016) or due to the lack of GFP which might contribute to the effectivity of folding stabilized mDHFR to prevent translocation.
While our data indicates PfEMP1 uses PTEX to reach the host cell, this could be expected to have resulted in the identification of PTEX components in the PfEMP1 proxiomes, which was not the case. However, as BirA* must be unfolded to pass through PTEX, it likely is unable to biotinylate translocon components unless PfEMP1 is stalled during translocation. For this reason, a lack of PTEX components in the PfEMP1 proxiomes does not necessarily exclude passage through PTEX.
The PfEMP1 proxiome presented here comprised many of the known proteins required for PfEMP1-mediated cytoadhesion. There was a considerable overlap with the Maurer’s clefts proxiome, where many of these proteins are localized. It, however, also included proteins experimentally confirmed to be located at other sites in the host cell, including the host cell membrane. Hence, despite the small number of PfEMP1 molecules displayed at the host cell surface (Sanchez et al., 2019), the proxiomes included hits from that site. A protein notably absent from our PfEMP1 proxiomes was the major knob component KAHRP (Culvenor et al., 1987; Pologe et al., 1987; Rug et al., 2006). While this was surprising in light of the original in vitro binding studies (Oh et al., 2000; Waller et al., 1999; Waller et al., 2000), a newer study was unable to detect an interaction of KAHRP with the ATS but found interaction with PHIST domains (Mayer et al., 2012). These findings match our proxiome data which, particularly with the position 1 construct, detected many PHIST proteins and suggests that PHISTs may be in more direct contact with the ATS than KAHRP. This also agrees with recent BioIDs with KAHRP as a bait that did not efficiently detect PfEMP1 whereas PTP4 as bait did (Davies et al., 2023).
We here report two new proteins needed for PfEMP1-mediated cytoadhesion. As we still detected some surface exposure of PfEMP1, the cytoadhesion defect was either due to reduced transport to the surface or due to incorrect surface display of PfEMP1. One of the identified proteins, TryThrA, was in a recent study with 3D7 found to be dispensable for cytoadhesion (Takano et al., 2019). It is possible that this discrepancy is due to the different P. falciparum strains used. In P. berghei IPIS3, which belongs to the same group of tryptophan-threonine-rich domain proteins, was recently found to be important for sequestration in rodent malaria (Gabelich et al., 2022). Although mouse-infecting malaria parasites do not possess PfEMP1, they do harbor orthologous machinery needed for sequestration, suggesting that virulence factor transport is evolutionary conserved even if the virulence factor is different (De Niz et al., 2016). This raises the possibility that tryptophan-threonine-rich domain proteins belong to the conserved core of this machinery, similar to SBP1 and MAHRP1 (De Niz et al., 2016). PTEF, selected because of its location at the host cell membrane (Birnbaum et al., 2017) and previously linked to VAR2CSA translation (Chan et al., 2017), did not influence cytoadhesion of IT4VAR01.
The SLI system does have limitations for the study of var and PfEMP1 biology. For example, if the targeted exon 2 region is too similar to that of other var genes, the SLI plasmid might insert into an unwanted var gene. This can be solved by providing a codon-changed exon 2 region in the SLI plasmid and shifting the targeting sequence upstream where there is high sequence variation. The feasibility of such an approach was shown here by generating the cell lines to insert BirA* into position 2 and 3 of IT4VAR01. Another limitation is that the discovery of PfEMP1-binding to unknown receptors may be difficult if, as seen with the IT4var19-HAendo parasites, panning for receptor binding is required to select for that binding. However, as most PfEMP1 will bind CD36 or EPCR, pre-selection on these receptors may enable studies of putative receptor interactions. Alternatively, assuming PTP3 expression is causal and the only factor why the IT4var19-HAendo parasites had to be panned, episomal expression of PTP3 could ameliorate this and possibly be used to generally enhance surface display and binding.