Background

The most virulent form of malaria is caused by the protozoan parasite Plasmodium falciparum, killing over 600,000 people annually (Weiss et al., 2019). During the asexual blood stage, the parasite invades the red blood cell (RBC) and feeds on intracellular hemoglobin; however, while circulating in the host bloodstream, the infected RBC is at risk of elimination if it transits the host’s splenic sinuses. To avoid splenic clearance, the parasite remodels the host cell by exporting proteins into the RBC cytoplasm (Marti et al., 2004; Sargeant et al., 2006; Boddey et al., 2013; Heiber et al., 2013). During this process, a key modification is the presentation of the variant antigen P. falciparum erythrocyte membrane protein 1 (EMP1) at the surface of infected RBCs (Smith et al., 1995). The antigen EMP1 is encoded by the var gene family where each parasite expressing one of approximately 60 variants at any time. These variants act as adhesins to a range of cellular ligands, expressed on a range of endothelial cells in the capillaries throughout the body, allowing the infected RBC to sequester within the host’s vasculature. The cytoadhesion of infected RBCs can lead to fatal complications associated with cerebral and placental malaria (Storm and Craig, 2014; Jensen et al., 2020; Sahu et al., 2021). EMP1 transport to the surface remains poorly understood as the parasite, with the parasite building its own de novo trafficking system, which is highly divergent from classical eukaryotic trafficking machinery. For these reasons, EMP1 trafficking is of high interest from both clinical and basic biology perspectives.

In our recent studies, we identified parasite proteins that are exported into the host cell and affect the trafficking and presentation of the antigen EMP1 (McHugh et al., 2020; Carmo et al., 2022). The majority of EMP1 is trafficked to the host cell surface 16–20 h post invasion, and mid-trophozoite stage-infected RBCs (20–32 h post invasion) are used to study EMP1 presentation at the host cell surface (Kriek et al., 2003). To determine if EMP1 is present at the surface of infected RBCs, we employ two complementary assays. These include: 1) trypsin cleavage assay, where surface-exposed EMP1 is shaved from the surface by trypsin and the membrane-embedded domain is then detected by immunoblotting (Cooke et al., 2006; Carmo et al., 2022); and 2) binding assays, in which the infected RBCs are passed through a channel coated with ligand and the number of cells adhering is quantified (McHugh et al., 2015 and 2020; Carmo et al., 2022). These assays are useful; however, they have their limitations. For example, the trypsin assay is semi-quantitative at best, while defects in adhesion underflow can be multifactorial (not due to EMP1 surface presentation alone).

In an alternative approach outlined here, antibodies specific to the ectodomain of EMP1 are used to label live cells, which are then quantitated by flow cytometry, to measure the number of labeled cells and their relative intensity of surface-exposed EMP1 (first established in Smith et al., 1995; Beeson et al., 2004; Elliott et al., 2005) (Figure 1). Of the techniques assaying EMP1 surface presentation published to date, this is the most time- and resource-efficient method. In addition, the assay is implemented in a 96-well plate format, making it easily scalable. This technique can also be adjusted to quantitate surface presentation of surface proteins in other cellular contexts where an antibody is available for the ectodomain of the protein of interest.

Figure 1. Cell labeling and analysis flowchart. (A) Live cell labeling of the ectodomain of the VAR2CSA, an EMP1 variant available on the surface of P. falciparum infected red blood cells (RBCs). The VAR2CSA variant is associated with adhesion to chondroitin sulphate A in the placenta, causing placental malaria. A zoom-in window depicts the VAR2CSA antigen (blue) with a list of the live cell labeling steps: incubations with three antisera, the final of which is conjugated to an Alexa Fluor 488, and finally a DNA stain. (B) The labeled cells are analyzed by flow cytometry. (C) Major populations can be separated by plotting the Pacific blue height (cells with DNA) against FITC height (cells labeled with Alexa Fluor 488), where each dot is a single cell and high densities of cells are indicated by blue through to red hues. Schematics of RBCs indicate the type of cell gated in each quartile. Uninfected RBCs do not have DNA; therefore, Q4 represents uninfected cells. Q1 represents infected cells that do not have VAR2CSA ectodomains available to the antisera. Q2 indicates infected cells that have VAR2CSA ectodomains available to the antisera. These data are used to compare the Q2 populations between cell lines to determine if mutations to P. falciparum cell lines affect VAR2CSA translocation to the surface of the infected RBC.