Genetic Evidence for Erythrocyte Receptor Glycophorin B Expression Levels Defining a Dominant Plasmodium falciparum Invasion Pathway into Human Erythrocytes.

Plasmodium falciparum, the parasite that causes the deadliest form of malaria, has evolved multiple proteins known as invasion ligands that bind to specific erythrocyte receptors to facilitate invasion of human erythrocytes. The EBA-175/glycophorin A (GPA) and Rh5/basigin ligand-receptor interactions, referred to as invasion pathways, have been the subject of intense study. In this study, we focused on the less-characterized sialic acid-containing receptors glycophorin B (GPB) and glycophorin C (GPC). Through bioinformatic analysis, we identified extensive variation in glycophorin B (GYPB) transcript levels in individuals from Benin, suggesting selection from malaria pressure. To elucidate the importance of the GPB and GPC receptors relative to the well-described EBA-175/GPA invasion pathway, we used an ex vivo erythrocyte culture system to decrease expression of GPA, GPB, or GPC via lentiviral short hairpin RNA transduction of erythroid progenitor cells, with global surface proteomic profiling. We assessed the efficiency of parasite invasion into knockdown cells using a panel of wild-type P. falciparum laboratory strains and invasion ligand knockout lines, as well as P. falciparum Senegalese clinical isolates and a short-term-culture-adapted strain. For this, we optimized an invasion assay suitable for use with small numbers of erythrocytes. We found that all laboratory strains and the majority of field strains tested were dependent on GPB expression level for invasion. The collective data suggest that the GPA and GPB receptors are of greater importance than the GPC receptor, supporting a hierarchy of erythrocyte receptor usage in P. falciparum

Uncovering Common Principles in Protein Export of Malaria Parasites

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Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in <i>Plasmodium falciparum</i> Protein Export

<div><p>Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite <i>Plasmodium falciparum</i>. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.</p></div

N-termini of new PNEPs without a signal peptide promote export of a reporter with a PNEP TM.

<p>(A) Parasites expressing the 20 first amino acids of PF07_0007 (top two rows, wild type and scrambled version of N-termini) or PFF0090w (bottom rows, wild type and scrambled version of N-termini) fused N-terminally to R^REX2-TM (truncated mTRAP-GFP containing the REX2TM). For the scrambled PF07_0007 N-terminus the most typical phenotype (mixed) is shown. For the PFF0090w scrambled N-terminus a non-exported and a mixed phenotype is shown. Red bar, PNEP domain; striated bar, randomly scrambled version; grey bar, mTRAP part; green bar, GFP. Export levels are shown to the right for each cell line and were assessed by counting (blinded) the number of cells showing export only (export), export together with parasite periphery and/or internal fluorescence (mixed), or parasite periphery and/or internal fluorescence only (no export). Graphs represent counting of at least 50 cells on three different occasions; error bars represent SD. (B) As in A but parasites expressing R^REX2-TM with the first 20 amino acids of PF11_0505 (yellow bar). Nuclei were stained with DAPI. Size bars: 5 µm.</p

Transcription profile based screen for new PNEPs.

<p>(A) Venn diagram of selection of PNEPs based on a transcription profile similar to genes encoding known PNEPs and ETRAMPs. (B, C) Fluorescence pattern of non-exported (B) or exported (C) GFP fusion proteins. Protein structure of each candidate is indicated before each panel (red bars with hydrophobic regions indicated in black, not to scale). For PF08_0003-GFP two panels showing a trophozoite stage (troph) and a ring stage (ring) parasite are shown to demonstrate the different localisations in these stages. For PF14_0045-GFP two panels are shown to demonstrate cells with (yellow arrows) and without additional foci of fluorescence in the host cell (ratio indicated in %, at least 50 cells were analysed on 3 occasions, standard deviation in brackets). Nuclei were stained with DAPI. Size bars: 5 µm. (D) The fluorescence in the host cell of PF13_0194-GFP represents full length soluble protein as determined by Western blot using anti-GFP antibodies with extracts from saponin lysed infected RBCs separated into pellet (P) and supernatant (SN). Parasite cytosolic aldolase was used to control for parasite integrity; REX3 (found soluble in infected RBCs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat.1003546-Spielmann2" target="_blank">[17]</a>) was used as a control for release of infected host cell cytosol.</p

Genomic position based screen for new PNEPs.

<p>(A) Venn diagram of selection of PNEPs based on a subtelomeric gene location (<200 kb from chromosome end). (B, C) Fluorescence pattern of non-exported (B) or exported (C) GFP fusion proteins. Protein structure of each candidate is indicated to the left of each panel as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1</a>. Nuclei were stained with DAPI. Size bars: 5 µm. (D) The fluorescence in the host cell of PF08_0005-GFP represents full length soluble protein as determined by Western blot as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1D</a>, except that GAPDH was used as the parasite internal control.</p

Soluble PNEPs need to be unfolded to reach the host cell.

<p>(A) Double transgenic parasites expressing MSRP6-GFP as well as MSRP6 fused to mDHFR and mCherry (constructs indicated on top) grown either with (+wr) or without (control) WR99210. The DIC image with DAPI stained nuclei (DIC/DAPI) and the merged GFP and mCherry signal (merge) are also presented. (B) As in (A) but with parasites expressing MSRP7-GFP and MSRP7-mDHFR-mCherry. The fluorescence internal to the parasite represents re-internalised protein in the food vacuole. Size bars: 5 µm. (C) Western blots of Percoll enriched double transgenic parasites shown in (A) and grown in the absence (control, exported) or presence (+wr, fluorescence remaining in parasite periphery) of WR99210 that were treated with saponin and separated into supernatant (SN) and pellet (P). SERP is found soluble in the PV and was used to demonstrate the action of saponin. GAPDH is the parasite internal control. The intensity of the mCherry, GFP and SERP signals in the extracts of parasites treated with wr is less than that of parasites not treated with wr, which may reflect either a somewhat slower growth cycle or slight differences in the stage composition of the parasite population after sample preparation. MSRP7-mDHFR-mCherry in the PV may also be more efficiently re-internalised and degraded in the food vacuole, reducing its amount in the blocked compared to the unblocked state.</p

Further PNEPs encoded by genes at the <i>msp7</i> gene locus.

<p>(A) Fluorescence pattern of PF13_0192-GFP. (B) Co-localisation IFA of PF13_0192-GFP with SBP1. (C) Fluorescence pattern of PF13_0191-GFP. Two panels are shown to demonstrate cells with (yellow arrows) and without additional foci of fluorescence in the host cell (ratio indicated in %, at least 50 cells were analysed on 3 occasions, standard deviation in brackets). (D) Bodipy-TR-C₅-ceramide (Bodipy) stained parasites expressing PF13_0191-GFP. Protein structure in A and C indicated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003546#ppat-1003546-g001" target="_blank">Figure 1</a>. Nuclei were stained with DAPI. Size bars: 5 µm.</p

MSRP6 is a Maurer's clefts protein expressed in trophozoites and schizonts.

<p>(A) Western blot with parental 3D7 (3D7), MSRP6-GFP expressing parasites and MSRP6 knock out parasites (Δ<i>msrp6</i>) demonstrates specificity of the antibodies. Molecular weight standards are indicated in kDa. Parasite GAPDH was used as a loading control. (B) Western blot with specific MSRP6 serum and extracts from stage specific parasites (hours post invasion indicated above each lane). GAPDH was used as a loading control. (C) Co-localisation IFA using anti-REX1 and anti-MSRP6 antibodies with MSRP6 knock out parasites (top) and parental 3D7 parasites (bottom). Size bar: 5 µm. (D) Pre-embedding immuno-EM of infected RBC, where the host cell cytosol has been released with tetanolysin, reacted with anti-MSRP6 antisera. Small panels show overviews with boxes highlighting individual Maurer's clefts for which enlargements are shown. Gold label is indicated with arrowheads. Size bars: 1 µm.</p