Malaria parasites secrete proteins across the vacuolar membrane into the erythrocyte, inducing modifications linked to disease and parasite survival. We identified an 11-amino acid signal required for the secretion of proteins from the Plasmodium falciparum vacuole to the human erythrocyte. Bioinformatics predicted a secretome of >320 proteins and conservation of the signal across parasite species. Functional studies indicated the predictive value of the signal and its role in targeting virulence proteins to the erythrocyte and implicated its recognition by a receptor/transporter. Erythrocyte modification by the parasite may involve plasmodial heat shock proteins and be vastly more complex than hitherto realized.

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Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Conditional gene regulation approaches are required to study the function of gene products critical for growth and erythrocyte invasion of blood stage parasites. Here we employ CRISPR/Cas9 to facilitate use of the dimerisable Cre-recombinase (DiCre) that is frequently used to mediate the excision and loss of loxP-flanked DNA sequences in a rapamycin controlled manner. We describe novel CRISPR/Cas9 transfection plasmids and approaches for the speedy, stable and marker-free introduction of transgenes encoding the DiCre recombinase into genomic loci dispensable for blood stage development. Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. falciparum lines in any genetic background. Furthermore, the newly developed 3D7-derived parasite lines, constitutively and stably expressing DiCre, generated using this toolkit will prove useful for the analysis of gene products. Lastly, we introduce an improved treatment protocol that uses a lower rapamycin concentration and shorter treatment times, leading to loxP-guided recombination with close to 100% efficiency within the same replication cycle.

The malaria agent Plasmodium falciparum is predicted to export a “secretome” of several hundred proteins to remodel the host erythrocyte. Prediction of protein export is based on the presence of an ER-type signal sequence and a downstream Host-Targeting (HT) motif (which is similar to, but distinct from, the closely related Plasmodium Export Element [PEXEL]). Previous attempts to determine the entire secretome, using either the HT-motif or the PEXEL, have yielded large sets of proteins, which have not been comprehensively tested. We present here an expanded secretome that is optimized for both P. falciparum signal sequences and the HT-motif. From the most conservative of these three secretome predictions, we identify 11 proteins that are preserved across human- and rodent-infecting Plasmodium species. The conservation of these proteins likely indicates that they perform important functions in the interaction with and remodeling of the host erythrocyte important for all Plasmodium parasites. Using the piggyBac transposition system, we validate their export and find a positive prediction rate of ∼70%. Even for proteins identified by all secretomes, the positive prediction rate is not likely to exceed ∼75%. Attempted deletions of the genes encoding the conserved exported proteins were not successful, but additional functional analyses revealed the first conserved secretome function. This gave new insight into mechanisms for the assembly of the parasite-induced tubovesicular network needed for import of nutrients into the infected erythrocyte. Thus, genomic screens combined with functional assays provide unexpected and fundamental insights into host remodeling by this major human pathogen.

In the asexual blood stages of malarial infection, merozoites invade erythrocytes and replicate within a parasitophorous vacuole to form daughter cells that eventually exit (egress) by sequential rupture of the vacuole and erythrocyte membranes. The current model is that PKG, a malarial cGMP-dependent protein kinase, triggers egress, activating malarial proteases and other effectors. Using selective inhibitors of either PKG or cysteine proteases to separately inhibit the sequential steps in membrane perforation, combined with video microscopy, electron tomography, electron energy loss spectroscopy, and soft X-ray tomography of mature intracellular Plasmodium falciparum parasites, we resolve intermediate steps in egress. We show that the parasitophorous vacuole membrane (PVM) is permeabilized 10-30 min before its PKG-triggered breakdown into multilayered vesicles. Just before PVM breakdown, the host red cell undergoes an abrupt, dramatic shape change due to the sudden breakdown of the erythrocyte cytoskeleton, before permeabilization and eventual rupture of the erythrocyte membrane to release the parasites. In contrast to the previous view of PKG-triggered initiation of egress and a gradual dismantling of the host erythrocyte cytoskeleton over the course of schizont development, our findings identify an initial step in egress and show that host cell cytoskeleton breakdown is restricted to a narrow time window within the final stages of egress. malaria | egress | electron tomography | soft X-ray microscopy | electron energy loss spectroscopy T he major cause of severe human malaria is Plasmodium falciparum, and its asexual blood cycle is the source of all clinical disease (1). Egress is an important step in the blood life cycle, as it allows daughter merozoites produced by intracellular parasite replication to escape and invade new erythrocytes, thereby continuing and amplifying the infection. Merozoites develop within a parasitophorous vacuole (PV), a membrane-bound compartment that forms during invasion (2-4), so the daughter parasites have two compartments to escape (5, 6).Blood-stage malaria parasites replicate by schizogony, in which several rounds of nuclear division form a multinucleated syncytium called a schizont. Individual merozoites are then produced by an unusual form of cytokinesis called budding or segmentation, which involves invagination of the single plasma membrane of the schizont. Minutes before egress, the segmented schizont suddenly transforms from an irregular to a relatively symmetrical structure with the merozoites arranged around the central digestive vacuole (5). This process, referred to as "flower formation" or rounding up, is usually accompanied by noticeable swelling of the PV and apparent shrinkage of the host cell (4, 5, 7-9). The first membrane to rupture at egress is the parasitophorous vacuole membrane (PVM) (5,6,8). When the PV does not occupy the entire infected cell, the individual merozoites can be seen to be expelled into the blood cell cytosol seconds before they escape fr...