The Cytoplasmic Region of Plasmodium falciparum SURFIN4.2 Is Required for Transport from Maurer’s Clefts to the Red Blood Cell Surface

Background: Plasmodium, the causative agent of malaria, exports many proteins to the surface of the infected red blood cell (iRBC) in order to modify it toward a structure more suitable for parasite development and survival. One such exported protein, SURFIN_4.2, from the parasite of human malignant malaria, P. falciparum, was identified in the trypsin-cleaved protein fraction from the iRBC surface, and is thereby inferred to be exposed on the iRBC surface. SURFIN_4.2 also localize to Maurer’s clefts—parasite-derived membranous structures established in the RBC cytoplasm and tethered to the RBC membrane—and their role in trafficking suggests that they are a pathway for SURFIN_4.2 transport to the iRBC surface. It has not been determined the participation of protein domains and motifs within SURFIN_4.2 in transport from Maurer’s clefts to the iRBC surface; and herein we examined if the SURFIN_4.2 intracellular region containing tryptophan-rich (WR) domain is required for its exposure on the iRBC surface. Results: We generated two transgenic parasite lines which express modified SURFIN_4.2, with or without a part of the intracellular region. Both recombinant SURFIN_4.2 proteins were exported to Maurer’s clefts. However, only SURFIN_4.2 possessing the intracellular region was efficiently cleaved by surface treatment of iRBC with proteinase K. Conclusions: These results indicate that SURFIN_4.2 is exposed on the iRBC surface and that the intracellular region containing WR domain plays a role on the transport from Maurer’s clefts to the iRBC membrane.     

Intracellular structures of normal and aberrant Plasmodium falciparum malaria parasites imaged by soft x-ray microscopy

Soft x-ray microscopy is a novel approach for investigation of intracellular organisms and subcellular structures with high spatial resolution. We used x-ray microscopy to investigate structural development of Plasmodium falciparum malaria parasites in normal and genetically abnormal erythrocytes and in infected erythrocytes treated with cysteine protease inhibitors. Investigations in normal red blood cells enabled us to recognize anomalies in parasite structures resulting from growth under unfavorable conditions. X-ray microscopy facilitated detection of newly elaborated structures in the cytosol of fixed, unstained, intact erythrocytes, redistribution of mass (carbon) in infected erythrocytes, and aberrant parasite morphology. In cysteine protease inhibitor-treated, infected erythrocytes, high concentrations of material were detected in abnormal digestive vacuoles and aggregated at the parasite plasma membrane. We have demonstrated that an abnormal host erythrocyte skeleton affects structural development of parasites and that this aberrant development can be detected in the following generation when parasites from protein 4.1-deficient red blood cells infect normal erythrocytes. This work extends our current understanding of the relationship between the host erythrocyte membrane and the intraerythrocytic malaria parasite by demonstrating for the first time that constituents of the erythrocyte membrane play a role in normal parasite structural development.     

Complement receptor 1 is the host erythrocyte receptor for Plasmodium falciparum PfRh4 invasion ligand

Plasmodium falciparum is responsible for the most severe form of malaria disease in humans, causing more than 1 million deaths each year. As an obligate intracellular parasite, P. falciparum’s ability to invade erythrocytes is essential for its survival within the human host. P. falciparum invades erythrocytes using multiple host receptor–parasite ligand interactions known as invasion pathways. Here we show that CR1 is the host erythrocyte receptor for PfRh4, a major P. falciparum ligand essential for sialic acid–independent invasion. PfRh4 and CR1 interact directly, with a K_d of 2.9 μM. PfRh4 binding is strongly correlated with the CR1 level on the erythrocyte surface. Parasite invasion via sialic acid–independent pathways is reduced in low-CR1 erythrocytes due to limited availability of this receptor on the surface. Furthermore, soluble CR1 can competitively block binding of PfRh4 to the erythrocyte surface and specifically inhibit sialic acid–independent parasite invasion. These results demonstrate that CR1 is an erythrocyte receptor used by the parasite ligand PfRh4 for P. falciparum invasion.     

Plasmodium falciparum var gene expression is modified by host immunity

Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a potentially important family of immune targets, which play a central role in the host–parasite interaction by binding to various host molecules. They are encoded by a diverse family of genes called var, of which there are ≈60 copies in each parasite genome. In sub-Saharan Africa, although P. falciparum infection occurs throughout life, severe malarial disease tends to occur only in childhood. This could potentially be explained if (i) PfEMP1 variants differ in their capacity to support pathogenesis of severe malaria and (ii) this capacity is linked to the likelihood of each molecule being recognized and cleared by naturally acquired antibodies. Here, in a study of 217 Kenyan children with malaria, we show that expression of a group of var genes “cys2,” containing a distinct pattern of cysteine residues, is associated with low host immunity. Expression of cys2 genes was associated with parasites from young children, those with severe malaria, and those with a poorly developed antibody response to parasite-infected erythrocyte surface antigens. Cys-2 var genes form a minor component of all genomic var repertoires analyzed to date. Therefore, the results are compatible with the hypothesis that the genomic var gene repertoire is organized such that PfEMP1 molecules that confer the most virulence to the parasite tend also to be those that are most susceptible to the development of host immunity. This may help the parasite to adapt effectively to the development of host antibodies through modification of the host–parasite relationship.     

Malaria PCR Detection in Cambodian Low-Transmission Settings: Dried Blood Spots versus Venous Blood Samples

In the context of malaria elimination, novel strategies for detecting very low malaria parasite densities in asymptomatic individuals are needed. One of the major limitations of the malaria parasite detection methods is the volume of blood samples being analyzed. The objective of the study was to compare the diagnostic accuracy of a malaria polymerase chain reaction assay, from dried blood spots (DBS, 5 μL) and different volumes of venous blood (50 μL, 200 μL, and 1 mL). The limit of detection of the polymerase chain reaction assay, using calibrated Plasmodium falciparum blood dilutions, showed that venous blood samples (50 μL, 200 μL, 1 mL) combined with Qiagen extraction methods gave a similar threshold of 100 parasites/mL, ∼100-fold lower than 5 μL DBS/Instagene method. On a set of 521 field samples, collected in two different transmission areas in northern Cambodia, no significant difference in the proportion of parasite carriers, regardless of the methods used was found. The 5 μL DBS method missed 27% of the samples detected by the 1 mL venous blood method, but most of the missed parasites carriers were infected by Plasmodium vivax (84%). The remaining missed P. falciparum parasite carriers (N = 3) were only detected in high-transmission areas.     

Targeting the ERAD pathway via inhibition of signal peptide peptidase for antiparasitic therapeutic design

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein’s ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.     

African apes as reservoirs of Plasmodium falciparum and the origin and diversification of the Laverania subgenus

We investigated two mitochondrial genes (cytb and cox1), one plastid gene (tufA), and one nuclear gene (ldh) in blood samples from 12 chimpanzees and two gorillas from Cameroon and one lemur from Madagascar. One gorilla sample is related to Plasmodium falciparum, thus confirming the recently reported presence in gorillas of this parasite. The second gorilla sample is more similar to the recently defined Plasmodium gaboni than to the P. falciparum–Plasmodium reichenowi clade, but distinct from both. Two chimpanzee samples are P. falciparum. A third sample is P. reichenowi and two others are P. gaboni. The other chimpanzee samples are different from those in the ape clade: two are Plasmodium ovale, and one is Plasmodium malariae. That is, we have found three human Plasmodium parasites in chimpanzees. Four chimpanzee samples were mixed: one species was P. reichenowi; the other species was P. gaboni in three samples and P. ovale in the fourth sample. The lemur sample, provisionally named Plasmodium malagasi, is a sister lineage to the large cluster of primate parasites that does not include P. falciparum or ape parasites, suggesting that the falciparum + ape parasite cluster (Laverania clade) may have evolved from a parasite present in hosts not ancestral to the primates. If malignant malaria were eradicated from human populations, chimpanzees, in addition to gorillas, might serve as a reservoir for P. falciparum.     

Single amino acid substitution in Plasmodium yoelii erythrocyte ligand determines its localization and controls parasite virulence

The major virulence determinant of the rodent malaria parasite, Plasmodium yoelii, has remained unresolved since the discovery of the lethal line in the 1970s. Because virulence in this parasite correlates with the ability to invade different types of erythrocytes, we evaluated the potential role of the parasite erythrocyte binding ligand, PyEBL. We found 1 amino acid substitution in a domain responsible for intracellular trafficking between the lethal and nonlethal parasite lines and, furthermore, that the intracellular localization of PyEBL was distinct between these lines. Genetic modification showed that this substitution was responsible not only for PyEBL localization but also the erythrocyte-type invasion preference of the parasite and subsequently its virulence in mice. This previously unrecognized mechanism for altering an invasion phenotype indicates that subtle alterations of a malaria parasite ligand can dramatically affect host–pathogen interactions and malaria virulence.     

From the Cover: Structural basis for the inhibition of the essential Plasmodium falciparum M1 neutral aminopeptidase

Plasmodium falciparum parasites are responsible for the major global disease malaria, which results in >2 million deaths each year. With the rise of drug-resistant malarial parasites, novel drug targets and lead compounds are urgently required for the development of new therapeutic strategies. Here, we address this important problem by targeting the malarial neutral aminopeptidases that are involved in the terminal stages of hemoglobin digestion and essential for the provision of amino acids used for parasite growth and development within the erythrocyte. We characterize the structure and substrate specificity of one such aminopeptidase, PfA-M1, a validated drug target. The X-ray crystal structure of PfA-M1 alone and in complex with the generic inhibitor, bestatin, and a phosphinate dipeptide analogue with potent in vitro and in vivo antimalarial activity, hPheP[CH₂]Phe, reveals features within the protease active site that are critical to its function as an aminopeptidase and can be exploited for drug development. These results set the groundwork for the development of antimalarial therapeutics that target the neutral aminopeptidases of the parasite.     

RH5–Basigin interaction plays a major role in the host tropism of Plasmodium falciparum

Plasmodium falciparum, the cause of almost all human malaria mortality, is a member of the Laverania subgenus which infects African great apes. Interestingly, Laverania parasites exhibit strict host specificity in their natural environment: P. reichenowi, P. billcollinsi, and P. gaboni infect only chimpanzees; P. praefalciparum, P. blacklocki, and P. adleri are restricted to gorillas, and P. falciparum is pandemic in humans. The molecular mechanism(s) responsible for these host restrictions are not understood, although the interaction between the parasite blood-stage invasion ligand EBA175 and the host erythrocyte receptor Glycophorin-A (GYPA) has been implicated previously. We reexamined the role of the EBA175–GYPA interaction in host tropism using recombinant proteins and biophysical assays and found that EBA175 orthologs from the chimpanzee-restricted parasites P. reichenowi and P. billcollinsi both bound to human GYPA with affinities similar to that of P. falciparum, suggesting that the EBA175–GYPA interaction is unlikely to be the sole determinant of Laverania host specificity. We next investigated the contribution of the recently discovered Reticulocyte-binding protein Homolog 5 (RH5)–Basigin (BSG) interaction in host-species selectivity and found that P. falciparum RH5 bound chimpanzee BSG with a significantly lower affinity than human BSG and did not bind gorilla BSG, mirroring the known host tropism of P. falciparum. Using site-directed mutagenesis, we identified residues in BSG that are responsible for the species specificity of PfRH5 binding. Consistent with the essential role of the PfRH5–BSG interaction in erythrocyte invasion, we conclude that species-specific differences in the BSG receptor provide a molecular explanation for the restriction of P. falciparum to its human host.The most deadly of the malaria parasites, Plasmodium falciparum, is highly divergent from the other species of Plasmodium known to infect humans (1–3), with its closest relatives comprising a group of chimpanzee and gorilla parasites from the subgenus Laverania (3–7). Despite the origin of P. falciparum as a zoonosis, and the continuing coexistence of humans and apes in West and Central Africa, extensive field studies have failed to detect P. falciparum in wild-living chimpanzees and gorillas (5). Although there are reports of P. falciparum infecting chimpanzees either in certain captive settings (2) or following splenectomy and deliberate transfer of P. falciparum-infected human blood (8–10), the resulting infections have low parasitemia and are not known to result in malignant tertian malaria, suggesting a host-specific barrier for replete infection. The existence of host-specific barriers within the Laverania subgenus is supported further by the strict host specificity exhibited by ape Laverania parasites in the wild: Plasmodium reichenowi, Plasmodium billcollinsi, and Plasmodium gaboni infect only chimpanzees, and Plasmodium praefalciparum, Plasmodium blacklocki, and Plasmodium adleri are restricted to gorillas (3–6).The molecular basis for the host tropism of P. falciparum and other Laverania parasites is not currently understood and is difficult to investigate experimentally, because no ape Laverania parasites have been adapted to in vitro culture, and because ethical considerations clearly preclude the use of endangered African apes for in vivo experiments (3). In principle, the species specificity of known host–parasite interactions could be examined using recombinant proteins, but the technical challenges associated with expressing functional Plasmodium proteins in a recombinant form have made this approach difficult thus far (11). There also are multiple points in the complex Plasmodium life cycle that could represent restriction points, and some evidence suggests that transmission of Laverania to anthropophilic Anopheles species, for example, may be restricted (12). Although several restriction points are possible, the blood stage of Plasmodium infection, which is initiated when host erythrocytes are invaded by the parasite, is of particular interest. Erythrocyte invasion is an obligate stage in the parasite life cycle (13), and the inability of P. falciparum to produce infections of high parasitemia in chimpanzees by transfer of infected blood, despite contrived permissive experimental conditions, suggests that a host-specific barrier for infection exists at the blood stage. Moreover, erythrocyte invasion involves extracellular interactions between several different receptor–ligand pairs which are coevolving rapidly, driven by strong immune selection pressure and functional constraints (14, 15). This rapid coevolution means that interactions between parasite blood-stage ligands and erythrocyte receptors quickly could become host-specific, isolating a certain parasite species within a single host. Given these findings, erythrocyte invasion therefore is likely to represent a significant restriction point in determining host tropism.P. falciparum erythrocyte invasion depends on a partially redundant set of parasite ligands and erythrocyte receptors (14, 16–18), and there is experimental support for the suggestion that at least two of these interactions may be involved in P. falciparum host tropism. The interaction between Plasmodium falciparum Erythrocyte Binding Antigen-175 (PfEBA175) and the erythrocyte receptor Glycophorin-A (GYPA) is important for invasion and is known to require sialic acid residues displayed on GYPA (19, 20). The sialic acid content of human GYPA differs from that of other apes because humans lack a functional cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) enzyme. Consequently, human GYPA contains only N-Acetylneuraminic acid (Neu5Ac) sialic acids, whereas chimpanzees and gorillas, both of which have an active CMAH gene, contain a mixture of both Neu5Ac and its hydroxylated derivative, N-Glycolylneuraminic acid (Neu5Gc), with Neu5Gc being more abundant (21). This difference in GYPA sialic acid content led Martin et al. (10) to propose that the restriction of P. falciparum to humans results from the sialic acid-binding specificity in the PfEBA175–GYPA interaction. The cocrystal structure of the glycan-binding regions of P. falciparum EBA175 in complex with a Neu5Ac-containing glycan derivative (22) followed by modeling of the equivalent EBA175 sequence from the chimpanzee-restricted parasite, P. reichenowi (23), suggested a structural basis for this binding specificity. However, this hypothesis is inconsistent with a much earlier study by Orlandi et al. (24) in which the binding of native PfEBA175 to human erythrocytes was observed to be potently inhibited by soluble Neu5Gc and oligosaccharides containing Neu5Acα2–3Gal but not by Neu5Ac as a monosaccharide. This glycan-binding specificity was confirmed in a recent biochemical investigation using a recombinant protein consisting of the entire ectodomain of PfEBA175 (25). Furthermore, the sialic acid specificity of PfEBA175 orthologs cannot explain the restriction of ape Laverania parasites to their respective chimpanzee and gorilla hosts, both of which carry a functional CMAH gene (5). Therefore there is reasonable doubt about the importance of the EBA175–GYPA interaction in Laverania host tropism. A second and unrelated invasion ligand, Plasmodium falciparum Reticulocyte-binding protein homolog 5 (PfRH5) has also been implicated in host tropism by governing the ability of P. falciparum to infect New World Aotus monkeys (26, 27). The erythrocyte cell-surface receptor of PfRH5, Basigin (BSG), was identified only recently (28), and so the contribution of the PfRH5–BSG interaction toward determining host-species selectivity in P. falciparum is unknown.We recently have developed technology that enables the recombinant expression of P. falciparum cell-surface and secreted proteins in a functional form using a mammalian expression system (29). We have used this method to identify novel host–parasite interactions (28, 30), but here we apply it to investigate the relative roles of the EBA175–GYPA and PfRH5–BSG interactions as determinants of P. falciparum host tropism.     

Epidemiological aspects of vivax and falciparum malaria: global spectrum

Malaria, a mosquito-borne disease, is caused by the infection of apicomplexan parasites belonging to the genus Plasmodium, five species of which [Plasmodium vivax, Plasmodium falciparum (P. falciparum), Plasmodium ovale, Plasmodium malariae and Plasmodium knowlesi] account for all forms of human malaria. P. falciparum is responsible for the highest degree of complications (severe malarial anaemia and cerebral malaria) and mortality in the tropics and subtropics of the world. Despite the large burden of vivax malaria, it is overlooked and left in the shadow of severity of falciparum malaria in the globe, but current reports provide evidence of severe vivax malaria symptoms similar to P. falciparum infection. The major challenging factor is the emergence of multidrug resistant Plasmodium strains to the conventionally used antimalarials over the last two decades, and, more recently, to artemisinins. The WHO recommended artemisinin based combination therapies (ACTs). The non-ACT regimens are also found to be effective, safe, and affordable compared to ACTs. However, current successful antimalarial interventions are under threat from the ability of the parasite and its mosquito vector to develop resistance to medicines and insecticides, respectively. Hence, with widespread use of effective drugs and vector control with insecticide-treated bed nets and indoor residual spraying, an ideal malaria vaccine would be the actual means of malaria prevention. This review represents the current evidence, based upon the search of SCI-and non-SCI journal, on epidemiological aspects of two forms (vivax and falciparum) of human malaria, which is still a great global concern.     

Membrane specific mapping and colocalization of malarial and host skeletal proteins in the Plasmodium falciparum infected erythrocyte by dual-color near-field scanning optical microscopy

Accurate localization of proteins within the substructure of cells and cellular organelles enables better understanding of structure–function relationships, including elucidation of protein–protein interactions. We describe the use of a near-field scanning optical microscope (NSOM) to simultaneously map and detect colocalized proteins within a cell, with superresolution. The system we elected to study was that of human red blood cells invaded by the human malaria parasite Plasmodium falciparum. During intraerythrocytic growth, the parasite expresses proteins that are transported to the erythrocyte cell membrane. Association of parasite proteins with host skeletal proteins leads to modification of the erythrocyte membrane. We report on colocalization studies of parasite proteins with an erythrocyte skeletal protein. Host and parasite proteins were selectively labeled in indirect immunofluorescence antibody assays. Simultaneous dual-color excitation and detection with NSOM provided fluorescence maps together with topography of the cell membrane with subwavelength (100 nm) resolution. Colocalization studies with laser scanning confocal microscopy provided lower resolution (310 nm) fluorescence maps of cross sections through the cell. Because the two excitation colors shared the exact same near-field aperture, the two fluorescence images were acquired in perfect, pixel-by-pixel registry, free from chromatic aberrations, which contaminate laser scanning confocal microscopy measurements. Colocalization studies of the protein pairs of mature parasite-infected erythrocyte surface antigen (MESA)(parasite)/protein4.1(host) and P. falciparum histidine rich protein (PfHRP1)(parasite)/protein4.1(host) showed good real-space correlation for the MESA/protein4.1 pair, but relatively poor correlation for the PfHRP1/protein4.1 pair. These data imply that NSOM provides high resolution information on in situ interactions between proteins in biological membranes. This method of detecting colocalization of proteins in cellular structures may have general applicability in many areas of current biological research.     

Evaluation of the NOW Malaria Immunochromatographic Test for Quantitative Diagnosis of Falciparum and Vivax Malaria Parasite Density

The NOW® Malaria Test, an immunochromatographic test (ICT), was evaluated to determine its ability to quantitatively detect malaria parasites using 100 blood samples from Thailand, including 50 Plasmodium falciparum (Pf) infections and 50 P. vivax (Pv) infections. Intensities of the thickness of the visible bands of the positive ICT were compared with the parasite densities. In cases of Pf infection, the intensities of both HRP-2 bands (T1 bands: Pf specific bands) and aldolase bands (T2 bands: pan-Plasmodium bands) correlated with the parasite densities. The intensities of T2 bands in Pf positive samples showed better correlation with the parasite densities than the T1 bands. In the cases of Pv infection, the intensities of T2 bands were also well correlated with parasite density. These results suggest that the ICT is useful not only for rapid detection of malaria parasites but also for estimating parasite density.     

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR–thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.     

Plasmodium evasion of mosquito immunity and global malaria transmission: The lock-and-key theory

Plasmodium falciparum malaria originated in Africa and became global as humans migrated to other continents. During this journey, parasites encountered new mosquito species, some of them evolutionarily distant from African vectors. We have previously shown that the Pfs47 protein allows the parasite to evade the mosquito immune system of Anopheles gambiae mosquitoes. Here, we investigated the role of Pfs47-mediated immune evasion in the adaptation of P. falciparum to evolutionarily distant mosquito species. We found that P. falciparum isolates from Africa, Asia, or the Americas have low compatibility to malaria vectors from a different continent, an effect that is mediated by the mosquito immune system. We identified 42 different haplotypes of Pfs47 that have a strong geographic population structure and much lower haplotype diversity outside Africa. Replacement of the Pfs47 haplotypes in a P. falciparum isolate is sufficient to make it compatible to a different mosquito species. Those parasites that express a Pfs47 haplotype compatible with a given vector evade antiplasmodial immunity and survive. We propose that Pfs47-mediated immune evasion has been critical for the globalization of P. falciparum malaria as parasites adapted to new vector species. Our findings predict that this ongoing selective force by the mosquito immune system could influence the dispersal of Plasmodium genetic traits and point to Pfs47 as a potential target to block malaria transmission. A new model, the “lock-and-key theory” of P. falciparum globalization, is proposed, and its implications are discussed.The most deadly form of malaria in humans is caused by Plasmodium falciparum parasites. Malaria originated in Africa (1, 2) and is transmitted by anopheline mosquitoes. The disease became global as humans migrated to other continents and parasites encountered different mosquito species that were sometimes evolutionarily distant from African vectors (3). For example, anophelines of the subgenus Nyssorhynchus (malaria vectors in Central and South America, such as Anopheles albimanus) diverged from the subgenus Cellia (malaria vectors in Africa, India, and South Asia) about 100 Mya (4). P. falciparum parasites are transmitted by more than 70 different anopheline species worldwide (3), but compatibilities differ between specific vector–parasite combinations (5). For example, P. falciparum NF54 (Pf NF54), of putative African origin, effectively infects Anopheles gambiae, the main malaria vector in sub-Saharan Africa; but A. albimanus is highly refractory to this strain (6–8); whereas Asian P. falciparum isolates infect Anopheles stephensi (Nijmegen strain), a major vector in India, more effectively than A. gambiae (9). Similar differences in compatibility have been reported between Plasmodium vivax and different anopheline species (10, 11). The A. gambiae immune system can mount effective antiplasmodial responses mediated by the complement-like system that limit infection (12). We have previously shown that some P. falciparum lines can avoid detection by the A. gambiae immune system (13) and identified Pfs47 as the gene that mediated immune evasion (14). Here, we present direct evidence of selection of P. falciparum by the mosquito immune system and show that providing P. falciparum with a Pfs47 haplotype compatible for a given anopheline mosquito is sufficient for the parasite to evade mosquito immunity. The implications of P. falciparum selection by mosquitoes for global malaria transmission are discussed.     

Plasmodium gallinaceum preferentially invades vesicular ATPase-expressing cells in Aedes aegypti midgut

Penetration of the mosquito midgut epithelium is obligatory for the further development of Plasmodium parasites. Therefore, blocking the parasite from invading the midgut wall disrupts the transmission of malaria. Despite such a pivotal role in malaria transmission, the cellular and molecular interactions that occur during the invasion are not understood. Here, we demonstrate that the ookinetes of Plasmodium gallinaceum, which is related closely to the human malaria parasite Plasmodium falciparum, selectively invade a cell type in the Aedes aegypti midgut. These cells, unlike the majority of the cells in the midgut, do not stain with a basophilic dye (toluidine blue) and are less osmiophilic. In addition, they contain minimal endoplasmic reticulum, lack secretory granules, and have few microvilli. Instead, these cells are highly vacuolated and express large amounts of vesicular ATPase. The enzyme is associated with the apical plasma membrane, cytoplasmic vesicles, and tubular extensions of the basal membrane of the invaded cells. The high cost of insecticide use in endemic areas and the emergence of drug resistant malaria parasites call for alternative approaches such as modifying the mosquito to block the transmission of malaria. One of the targets for such modification is the parasite receptor on midgut cells. A step toward the identification of this receptor is the realization that malaria parasites invade a special cell type in the mosquito midgut.     

Small-molecule histone methyltransferase inhibitors display rapid antimalarial activity against all blood stage forms in Plasmodium falciparum

Epigenetic factors such as histone methylation control the developmental progression of malaria parasites during the complex life cycle in the human host. We investigated Plasmodium falciparum histone lysine methyltransferases as a potential target class for the development of novel antimalarials. We synthesized a compound library based upon a known specific inhibitor (BIX-01294) of the human G9a histone methyltransferase. Two compounds, BIX-01294 and its derivative TM2-115, inhibited P. falciparum 3D7 parasites in culture with IC₅₀ values of ∼100 nM, values at least 22-fold more potent than their apparent IC₅₀ toward two human cell lines and one mouse cell line. These compounds irreversibly arrested parasite growth at all stages of the intraerythrocytic life cycle. Decrease in parasite viability (>40%) was seen after a 3-h incubation with 1 µM BIX-01294 and resulted in complete parasite killing after a 12-h incubation. Additionally, mice with patent Plasmodium berghei ANKA strain infection treated with a single dose (40 mg/kg) of TM2-115 had 18-fold reduced parasitemia the following day. Importantly, treatment of P. falciparum parasites in culture with BIX-01294 or TM2-115 resulted in significant reductions in histone H3K4me3 levels in a concentration-dependent and exposure time-dependent manner. Together, these results suggest that BIX-01294 and TM2-115 inhibit malaria parasite histone methyltransferases, resulting in rapid and irreversible parasite death. Our data position histone lysine methyltransferases as a previously unrecognized target class, and BIX-01294 as a promising lead compound, in a presently unexploited avenue for antimalarial drug discovery targeting multiple life-cycle stages.     

Reversible host cell remodeling underpins deformability changes in malaria parasite sexual blood stages

The sexual blood stage of the human malaria parasite Plasmodium falciparum undergoes remarkable biophysical changes as it prepares for transmission to mosquitoes. During maturation, midstage gametocytes show low deformability and sequester in the bone marrow and spleen cords, thus avoiding clearance during passage through splenic sinuses. Mature gametocytes exhibit increased deformability and reappear in the peripheral circulation, allowing uptake by mosquitoes. Here we define the reversible changes in erythrocyte membrane organization that underpin this biomechanical transformation. Atomic force microscopy reveals that the length of the spectrin cross-members and the size of the skeletal meshwork increase in developing gametocytes, then decrease in mature-stage gametocytes. These changes are accompanied by relocation of actin from the erythrocyte membrane to the Maurer’s clefts. Fluorescence recovery after photobleaching reveals reversible changes in the level of coupling between the membrane skeleton and the plasma membrane. Treatment of midstage gametocytes with cytochalasin D decreases the vertical coupling and increases their filterability. A computationally efficient coarse-grained model of the erythrocyte membrane reveals that restructuring and constraining the spectrin meshwork can fully account for the observed changes in deformability.The most virulent of the human malaria parasites, Plasmodium falciparum causes ∼440,000 deaths annually (1). Pathology is associated with asexual multiplication within red blood cells (RBCs). The trophozoite (growing) and schizont (dividing) stages (∼24–48 h after invasion) sequester in deep tissue using adhesive proteins presented on platform-like structures called “knobs” at the infected RBC surface. Cytoadhesion enables the parasite to avoid passage through the splenic sinuses and thus mechanical clearance from the circulation. Unfortunately, complications associated with sequestration of infected RBCs in the brain are responsible for much of the malaria-related mortality and morbidity.After a period of asexual cycling, a proportion of blood-stage parasites commit to sexual development (gametocytogenesis). The intraerythrocytic gametocyte develops through five distinct stages (I–V) over a period of 10–12 d, eventually adopting the characteristic crescent (falciform) shape that gives P. falciparum its name. Elongation is driven by assembly of a sheath of microtubules, attached to an inner membrane complex, underneath the parasite plasma membrane. From stage II to IV, gametocytes disappear from the circulation (2, 3); however, the mechanism of sequestration is not well understood. Upon maturation, the microtubule cytoskeleton is disassembled, and stage V gametocytes re-enter the circulation (2, 3). Ingestion of mature gametocytes by an Anopheles mosquito triggers release from the RBCs, followed by sexual recombination in the insect gut, and eventual transmission.Efforts to control malaria are often thwarted by the presence of gametocytes in asymptomatic individuals. These infected individuals serve as a reservoir during the low transmission season, ready to retransmit disease when mosquito numbers increase. As a consequence, there is intense interest in understanding gametocyte cell biology with the aim of interfering with this developmental stage.Of particular interest are the molecular and biomechanical changes that accompany the sequestration and release of gametocytes. Developing gametocytes (stages II–IV) have significantly reduced cellular deformability (2, 4, 5). This increased rigidity may enable gametocytes to become mechanically trapped in the bone marrow and splenic cords. In contrast, stage V gametocytes exhibit increased deformability (4–6), which may help them survive in the circulation, where they can be picked up by mosquitoes.Survival in the circulation requires RBCs to undergo deformation without fragmentation, as they transit through the 1.5- to 2-μm interendothelial slits in the spleen. The remarkable deformability properties of RBCs are thought to derive from their submembranous protein skeleton (7, 8). The skeleton is composed of a regular hexagonal array of spectrin heterodimers that self-associate head-to-head to form tetramers. The tails of the spectrin heterodimers are linked into junctional complexes containing actin oligomers (each with 14–16 protomers), protein 4.1R, adducin, and accessory proteins (9). Flexible linkages between the triple-helical segments of spectrin heterodimers, as well as tetramer dissociation, and breakable linkages into the junction points, are assumed to accommodate the distortions imposed by shear forces in the circulation.Vertical interactions connect the skeletal meshwork to the plasma membrane. A subpopulation of band-3 dimers connects to spectrin via ankyrin (9). Band-3 dimers also participate in a second linkage complex that involves glycophorin C. This complex is linked to the membrane skeleton via glycophorin C/protein 4.1 interactions as well as band-3/adducin interactions.The molecular determinants of the increased rigidity of mature asexual parasite-infected RBCs are beginning to be elucidated. Atomic force microscopy (AFM) has revealed reorganization and expansion of the spectrin network of the host cell membrane (10, 11), while cryo-electron tomography suggests that this reorganization occurs as a result of mining of the actin junctions in order to generate actin filaments that connect parasite-derived organelles known as Maurer''s clefts to the knobs (12). A parasite-encoded protein called the Knob-Associated Histidine-Rich Protein (KAHRP) is thought to be a major contributor to RBC rigidification (8, 13). KAHRP binds spectrin and self-assembles into a structure that distorts the RBC membrane with surface protrusions. Recent modeling suggests that composite strengthening and strain hardening of the infected RBC membrane result from modified lateral and vertical interactions within the membrane skeleton and deposition of rigidifying knob structures (8).In contrast, relatively little is known about host RBC remodeling in gametocytes. There are no knobs on gametocytes, and very limited (if any) surface expression of adhesins (14). In this work, we used AFM to investigate the membrane skeleton structure in gametocyte-infected RBCs and probed the interactions between RBC integral membrane proteins and the membrane skeleton using fluorescence photobleaching. In stage III gametocytes, we observed relocation of actin to Maurer’s clefts, accompanied by expansion of the spectrin skeleton and enhanced coupling of the membrane skeleton to the plasma membrane. These changes are reversed in stage V gametocytes. The actin depolymerizing agent cytochalasin D modulates the properties of stage III gametocytes, consistent with reversible actin remodeling. Coarse-grained molecular dynamics (CGMD) modeling reveals that enhanced lateral interactions and constraints on the spectrin motions can fully account for the compromised biomechanical properties of stage III gametocytes.     

The malarial parasite Plasmodium falciparum imports the human protein peroxiredoxin 2 for peroxide detoxification

Coevolution of the malarial parasite and its human host has resulted in a complex network of interactions contributing to the homeodynamics of the host-parasite unit. As a rapidly growing and multiplying organism, Plasmodium falciparum depends on an adequate antioxidant defense system that is efficient despite the absence of genuine catalase and glutathione peroxidase. Using different experimental approaches, we demonstrate that P. falciparum imports the human redox-active protein peroxiredoxin 2 (hPrx-2, hTPx1) into its cytosol. As shown by confocal microscopy and immunogold electron microscopy, hPrx-2 is also present in the Maurer''s clefts, organelles that are described as being involved in parasite protein export. Enzyme kinetic analyses prove that hPrx-2 accepts Plasmodium cytosolic thioredoxin 1 as a reducing substrate. hPrx-2 accounts for roughly 50% of thioredoxin peroxidase activity in parasite extracts, thus indicating a functional role of hPrx-2 as an enzymatic scavenger of peroxides in the parasite. Under chloroquine treatment, a drug promoting oxidative stress, the abundance of hPrx-2 in the parasite increases significantly. P. falciparum has adapted to adopt the hPrx-2, thereby using the host protein for its own purposes.