BackgroundTo avoid spleen-dependent killing mechanisms parasite-infected erythrocytes (IE) of Plasmodium falciparum malaria patients have the capacity to bind to endothelial receptors. This binding also known as sequestration, is mediated by parasite proteins, which are targeted to the erythrocyte surface. Candidate proteins are those encoded by P. falciparum multicopy gene families, such as var, rif, stevor or PfMC-2TM. However, a direct in vivo proof of IE sequestration and expression of multicopy gene families is still lacking. Here, we report on the analysis of IE from a black African immigrant, who received the diagnosis of a malignant lymphoproliferative disorder and subsequently underwent splenectomy. Three weeks after surgery, the patient experienced clinical falciparum malaria with high parasitemia and circulating developmental parasite stages usually sequestered to the vascular endothelium such as late trophozoites, schizonts or immature gametocytes.Methodology/Principal FindingsInitially, when isolated from the patient, the infected erythrocytes were incapable to bind to various endothelial receptors in vitro. Moreover, the parasites failed to express the multicopy gene families var, A-type rif and stevor but expression of B-type rif and PfMC-2TM genes were detected. In the course of in vitro cultivation, the parasites started to express all investigated multicopy gene families and concomitantly developed the ability to adhere to endothelial receptors such as CD36 and ICAM-1, respectively.Conclusion/SignificanceThis case strongly supports the hypothesis that parasite surface proteins such as PfEMP1, A-type RIFIN or STEVOR are involved in interactions of infected erythrocytes with endothelial receptors mediating sequestration of mature asexual and immature sexual stages of P. falciparum. In contrast, multicopy gene families coding for B-type RIFIN and PfMC-2TM proteins may not be involved in sequestration, as these genes were transcribed in infected but not sequestered erythrocytes.
Virulence of the most deadly malaria parasite Plasmodium falciparum is linked to the variant surface antigen PfEMP1, which is encoded by about 60 var genes per parasite genome. Although the expression of particular variants has been associated with different clinical outcomes, little is known about var gene expression at the onset of infection. By analyzing controlled human malaria infections via quantitative real-time PCR, we show that parasite populations from 18 volunteers expressed virtually identical transcript patterns that were dominated by the subtelomeric var gene group B and, to a lesser extent, group A. Furthermore, major changes in composition and frequency of var gene transcripts were detected between the parental parasite culture that was used to infect mosquitoes and Plasmodia recovered from infected volunteers, suggesting that P. falciparum resets its var gene expression during mosquito passage and starts with the broad expression of a specific subset of var genes when entering the human blood phase.
BackgroundVariant surface antigens (VSA) exposed on the membrane of Plasmodium falciparum infected erythrocytes mediate immune evasion and are important pathogenicity factors in malaria disease. In addition to the well-studied PfEMP1, the small VSA families RIFIN, STEVOR and PfMC-2TM are assumed to play a role in this process.MethodsThis study presents a detailed comparative characterization of the localization, membrane topology and extraction profile across the life cycle of various members of these protein families employing confocal microscopy, immunoelectron microscopy and immunoblots.ResultsThe presented data reveal a clear association of variants of the RIFIN, STEVOR and PfMC-2TM proteins with the host cell membrane and topological studies indicate that the semi-conserved N-terminal region of RIFINs and some STEVOR proteins is exposed at the erythrocyte surface. At the Maurer’s clefts, the semi-conserved N-terminal region as well as the variable stretch of RIFINs appears to point to the lumen away from the erythrocyte cytoplasm. These results challenge the previously proposed two transmembrane topology model for the RIFIN and STEVOR protein families and suggest that only one hydrophobic region spans the membrane. In contrast, PfMC-2TM proteins indeed seem to be anchored by two hydrophobic stretches in the host cell membrane exposing just a few, variable amino acids at the surface of the host cell.ConclusionTogether, the host cell surface exposure and topology of RIFIN and STEVOR proteins suggests members of these protein families may indeed be involved in immune evasion of the infected erythrocyte, whereas members of the PfMC-2TM family seem to bear different functions in parasite biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0784-2) contains supplementary material, which is available to authorized users.
During its intraerythrocytic development, the malaria parasite Plasmodium falciparum exposes variant surface antigens (VSAs) on infected erythrocytes to establish and maintain an infection. One family of small VSAs is the polymorphic STEVOR proteins, which are marked for export to the host cell surface through their PEXEL signal peptide. Interestingly, some STEVORs have also been reported to localize to the parasite plasma membrane and apical organelles, pointing toward a putative function in host cell egress or invasion. Using deep RNA sequencing analysis, we characterized P. falciparum stevor gene expression across the intraerythrocytic development cycle, including free merozoites, in detail and used the resulting stevor expression profiles for hierarchical clustering. We found that most stevor genes show biphasic expression oscillation, with maximum expression during trophozoite stages and a second peak in late schizonts. We selected four STEVOR variants, confirmed the expected export of these proteins to the host cell membrane, and tracked them to a secondary location, either to the parasite plasma membrane or the secretory organelles of merozoites in late schizont stages. We investigated the function of a particular STEVOR that showed rhoptry localization and demonstrated its role at the parasite-host interface during host cell invasion by specific antisera and targeted gene disruption. Experimentally determined membrane topology of this STEVOR revealed a single transmembrane domain exposing the semiconserved as well as variable protein regions to the cell surface. IMPORTANCE Malaria claims about half a million lives each year. Plasmodium falciparum, the causative agent of the most severe form of the disease, uses proteins that are translocated to the surface of infected erythrocytes for immune evasion. To circumvent the detection of these gene products by the immune system, the parasite evolved a complex strategy that includes gene duplications and elaborate sequence polymorphism. STEVORs are one family of these variant surface antigens and are encoded by about 40 genes. Using deep RNA sequencing of blood-stage parasites, including free merozoites, we first established stevor expression of the cultured isolate and compared it with published transcriptomes. We reveal a biphasic expression of most stevor genes and confirm this for individual STEVORs at the protein level. The membrane topology of a rhoptry-associated variant was experimentally elucidated and linked to host cell invasion, underlining the importance of this multifunctional protein family for parasite proliferation.
Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.
Sequestration of Plasmodium falciparum-infected erythrocytes to host endothelium through the parasite-derived PfEMP1 adhesion proteins is central to the development of malaria pathogenesis. PfEMP1 proteins have diversified and expanded to encompass many sequence variants conferring each parasite a similar array of human endothelial receptor binding phenotypes. Here, we analyzed RNA-seq profiles of parasites isolated from 32 P. falciparum infected adult travelers returning to Germany. Patients were categorized into either malaria naïve (n=15) or pre-exposed (n=17), and into severe (n=8) or non-severe (n=24) cases. For differential expression analysis of PfEMP1-encoding var gene transcripts were de novo assembled from RNA-seq data and, in parallel, var expressed sequence tags were analyzed and used to predict the encoded domain composition of the transcripts. Both approaches showed in concordance that severe malaria was associated with PfEMP1 containing the endothelial protein C receptor (EPCR)-binding CIDRα1 domain, whereas CD36-binding PfEMP1 was linked to non-severe malaria outcomes. First-time infected adults were more likely to develop severe symptoms and tended to be infected for a longer period. Thus, parasites with more pathogenic PfEMP1 variants are more common in patients with a naïve immune status and/or adverse inflammatory host responses to first infections favors growth of EPCR-binding parasites.
The pathogenesis of Plasmodium falciparum malaria is linked to the variant surface antigen Pf EMP1, which mediates tethering of infected erythrocytes to the host endothelium and is encoded by approximately 60 var genes per parasite genome. Repeated episodes of malaria infection result in the gradual acquisition of protective antibodies against Pf EMP1 variants. The antibody repertoire is believed to provide a selective pressure driving the clonal expansion of parasites expressing unrecognized Pf EMP1 variants, however, due to the lack of experimental in vivo models there is only limited experimental evidence in support of this concept. To get insight into the impact of naturally acquired immunity on the expressed var gene repertoire early during infection we performed controlled human malaria infections of 20 adult African volunteers with life-long malaria exposure using aseptic, purified, cryopreserved P . falciparum sporozoites (Sanaria PfSPZ Challenge) and correlated serological data with var gene expression patterns from ex vivo parasites. Among the 10 African volunteers who developed patent infections, individuals with low antibody levels showed a steep rise in parasitemia accompanied by broad activation of multiple, predominantly subtelomeric var genes, similar to what we previously observed in naïve volunteers. In contrast, individuals with intermediate antibody levels developed asymptomatic infections and the ex vivo parasite populations expressed only few var gene variants, indicative of clonal selection. Importantly, in contrast to parasites from naïve volunteers, expression of var genes coding for endothelial protein C receptor (EPCR)-binding Pf EMP1 that are associated with severe childhood malaria was rarely detected in semi-immune adult African volunteers. Moreover, we followed var gene expression for up to six parasite replication cycles and demonstrated for the first time in vivo a shift in the dominant var gene variant. In conclusion, our data suggest that P . falciparum activates multiple subtelomeric var genes at the onset of blood stage infection facilitating rapid expansion of parasite clones which express Pf EMP1 variants unrecognized by the host’s immune system, thus promoting overall parasite survival in the face of host immunity.
Key Points• P falciparum STEVORs interact with the erythrocyte cytoskeletal ankyrin complex.• Infected erythrocyte deformability is regulated by PKA-mediated phosphorylation of STEVOR cytoplasmic domain.Deformability of Plasmodium falciparum gametocyte-infected erythrocytes (GIEs) allows them to persist for several days in blood circulation and to ensure transmission to mosquitoes. Here, we investigate the mechanism by which the parasite proteins STEVOR (SubTElomeric Variable Open Reading frame) exert changes on GIE deformability. Using the microsphiltration method, immunoprecipitation, and mass spectrometry, we produce evidence that GIE stiffness is dependent on the cytoplasmic domain of STEVOR that interacts with ankyrin complex at the erythrocyte skeleton. Moreover, we show that GIE deformability is regulated by protein kinase A (PKA)-mediated phosphorylation of the STEVOR C-terminal domain at a specific serine residue (S 324 ). Finally, we show that the increase of GIE stiffness induced by sildenafil (Viagra) is dependent on STEVOR phosphorylation status and on another independent mechanism. These data provide new insights into mechanisms by which phosphodiesterase inhibitors may block malaria parasite transmission. (Blood. 2016;127(24):e42-e53)
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