Talking to each other to initiate sexual differentiation.

During the blood stage of their life cycle, malaria parasites invade and replicate within host erythrocytes. Some parasites differentiate to form male and female gametocytes, enabling transmission to the insect vector. Two studies by Regev-Rudzki et al. and Mantel et al. reveal an unexpected role for cell-cell communication using extracellular vesicles in triggering the commitment of malaria parasites toward sexual differentiation.

Associations between prenatal malaria exposure, maternal antibodies at birth, and malaria susceptibility during the first year of life in Burkina Faso.

In this study, we investigated how different categories of prenatal malaria exposure (PME) influence levels of maternal antibodies in cord blood samples and the subsequent risk of malaria in early childhood in a birth cohort study (N = 661) nested within the COSMIC clinical trial (NCT01941264) in Burkina Faso. Plasmodium falciparum infections during pregnancy and infants' clinical malaria episodes detected during the first year of life were recorded. The levels of maternal IgG and IgG1-4 to 15 P. falciparum antigens were measured in cord blood by quantitative suspension array technology. Results showed a significant variation in the magnitude of maternal antibody levels in cord blood, depending on the PME category, with past placental malaria (PM) more frequently associated with significant increases of IgG and/or subclass levels across three groups of antigens defined as pre-erythrocytic, erythrocytic, and markers of PM, as compared to those from the cord of non-exposed control infants. High levels of antibodies to certain erythrocytic antigens (i.e., IgG to EBA140 and EBA175, IgG1 to EBA175 and MSP142, and IgG3 to EBA140 and MSP5) were independent predictors of protection from clinical malaria during the first year of life. By contrast, high levels of IgG, IgG1, and IgG2 to the VAR2CSA DBL1-2 and IgG4 to DBL3-4 were significantly associated with an increased risk of clinical malaria. These findings indicate that PME categories have different effects on the levels of maternal-derived antibodies to malaria antigens in children at birth, and this might drive heterogeneity to clinical malaria susceptibility in early childhood.

Naturally acquired antibody kinetics against Plasmodium vivax antigens in people from a low malaria transmission region in western Thailand.

BACKGROUND: Plasmodium vivax (P. vivax) is the dominant Plasmodium spp. causing the disease malaria in low-transmission regions outside of Africa. These regions often feature high proportions of asymptomatic patients with sub-microscopic parasitaemia and relapses. Naturally acquired antibody responses are induced after Plasmodium infection, providing partial protection against high parasitaemia and clinical episodes. However, previous work has failed to address the presence and maintenance of such antibody responses to P. vivax particularly in low-transmission regions. METHODS: We followed 34 patients in western Thailand after symptomatic P. vivax infections to monitor antibody kinetics over 9 months, during which no recurrent infections occurred. We assessed total IgG, IgG subclass and IgM levels to up to 52 P. vivax proteins every 2-4 weeks using a multiplexed Luminex® assay and identified protein-specific variation in antibody longevity. Mathematical modelling was used to generate the estimated half-life of antibodies, long-, and short-lived antibody-secreting cells. RESULTS: Generally, an increase in antibody level was observed within 1-week post symptomatic infection, followed by an exponential decay of different rates. We observed mostly IgG1 dominance and IgG3 sub-dominance in this population. IgM responses followed similar kinetic patterns to IgG, with some proteins unexpectedly inducing long-lived IgM responses. We also monitored antibody responses against 27 IgG-immunogenic antigens in 30 asymptomatic individuals from a similar region. Our results demonstrate that most antigens induced robust and long-lived total IgG responses following asymptomatic infections in the absence of (detected) boosting infections. CONCLUSIONS: Our work provides new insights into the development and maintenance of naturally acquired immunity to P. vivax and will guide the potential use of serology to indicate immune status and/or identify populations at risk.

Role of a patatin-like phospholipase in Plasmodium falciparum gametogenesis and malaria transmission.

Transmission of Plasmodium falciparum involves a complex process that starts with the ingestion of gametocytes by female Anopheles mosquitoes during a blood meal. Activation of gametocytes in the mosquito midgut triggers "rounding up" followed by egress of both male and female gametes. Egress requires secretion of a perforin-like protein, PfPLP2, from intracellular vesicles to the periphery, which leads to destabilization of peripheral membranes. Male gametes also develop flagella, which assist in binding female gametes for fertilization. This process of gametogenesis, which is key to malaria transmission, involves extensive membrane remodeling as well as vesicular discharge. Phospholipase A2 enzymes (PLA2) are known to mediate membrane remodeling and vesicle secretion in diverse organisms. Here, we show that a P. falciparum patatin-like phospholipase (PfPATPL1) with PLA2 activity plays a key role in gametogenesis. Conditional deletion of the gene encoding PfPATPL1 does not affect P. falciparum blood stage growth or gametocyte development but reduces efficiency of rounding up, egress, and exflagellation of gametocytes following activation. Interestingly, deletion of the PfPATPL1 gene inhibits secretion of PfPLP2, reducing the efficiency of gamete egress. Deletion of PfPATPL1 also reduces the efficiency of oocyst formation in mosquitoes. These studies demonstrate that PfPATPL1 plays a role in gametogenesis, thereby identifying PLA2 phospholipases such as PfPATPL1 as potential targets for the development of drugs to block malaria transmission.

Blood cytokine, chemokine and growth factor profiling in a cohort of pregnant women from tropical countries.

The immune status of women changes during and after pregnancy, differs between blood compartments at delivery and is affected by environmental factors particularly in tropical areas endemic for multiple infections. We quantified the plasma concentration of a set of thirty-one TH1, TH2, TH17 and regulatory cytokines, pro-inflammatory and anti-inflammatory cytokines and chemokines, and growth factors (altogether biomarkers), in a cohort of 540 pregnant women from five malaria-endemic tropical countries. Samples were collected at recruitment (first antenatal visit), delivery (periphery, cord and placenta) and postpartum, allowing a longitudinal analysis. We found the lowest concentration of biomarkers at recruitment and the highest at postpartum, with few exceptions. Among them, IL-6, HGF and TGF-ß had the highest levels at delivery, and even higher concentrations in the placenta compared to peripheral blood. Placental concentrations were generally higher than peripheral, except for eotaxin that was lower. We also compared plasma biomarker concentrations between the tropical cohort and a control group from Spain at delivery, presenting overall higher biomarker levels the tropical cohort, particularly pro-inflammatory cytokines and growth factors. Only IL-6 presented lower levels in the tropical group. Moreover, a principal component analysis of biomarker concentrations at delivery showed that women from Spain grouped more homogenously, and that IL-6 and IL-8 clustered together in the tropical cohort but not in the Spanish one. Plasma cytokine concentrations correlated with Plasmodium antibody levels at postpartum but not during pregnancy. This basal profiling of immune mediators over gestation and in different compartments at delivery is important to subsequently understand response to infections and clinical outcomes in mothers and infants in tropical areas.

Building momentum for malaria vaccine research and development: key considerations.

To maintain momentum towards improved malaria control and elimination, a vaccine would be a key addition to the intervention toolkit. Two approaches are recommended: (1) promote the development and short to medium term deployment of first generation vaccine candidates and (2) support innovation and discovery to identify and develop highly effective, long-lasting and affordable next generation malaria vaccines.

Calcium-dependent phosphorylation of Plasmodium falciparum serine repeat antigen 5 triggers merozoite egress.

The human malaria parasite Plasmodium falciparum proliferates in red blood cells following repeated cycles of invasion, multiplication, and egress. P. falciparum serine repeat antigen 5 (PfSERA5), a putative serine protease, plays an important role in merozoite egress. However, regulation of its activity leading to merozoite egress is poorly understood. In this study, we show that PfSERA5 undergoes phosphorylation prior to merozoite egress. Immunoprecipitation of parasite lysates using anti-PfSERA5 serum followed by MS analysis identified calcium-dependent protein kinase 1 (PfCDPK1) as an interacting kinase. Association of PfSERA5 with PfCDPK1 was corroborated by co-sedimentation, co-immunoprecipitation, and co-immunolocalization analyses. Interestingly, PfCDPK1 phosphorylated PfSERA5 in vitro in the presence of Ca2+ and enhanced its proteolytic activity. A PfCDPK1 inhibitor, purfalcamine, blocked the phosphorylation and activation of PfSERA5 both in vitroas well as in schizonts, which, in turn, blocked merozoite egress. Together, these results suggest that phosphorylation of PfSERA5 by PfCDPK1 following a rise in cytosolic Ca2+ levels activates its proteolytic activity to trigger merozoite egress.

VAR2CSA Serology to Detect Plasmodium falciparum Transmission Patterns in Pregnancy.

Pregnant women constitute a promising sentinel group for continuous monitoring of malaria transmission. To identify antibody signatures of recent Plasmodium falciparum exposure during pregnancy, we dissected IgG responses against VAR2CSA, the parasite antigen that mediates placental sequestration. We used a multiplex peptide-based suspension array in 2,354 samples from pregnant women from Mozambique, Benin, Kenya, Gabon, Tanzania, and Spain. Two VAR2CSA peptides of limited polymorphism were immunogenic and targeted by IgG responses readily boosted during infection and with estimated half-lives of <2 years. Seroprevalence against these peptides reflected declines and rebounds of transmission in southern Mozambique during 2004-2012, reduced exposure associated with use of preventive measures during pregnancy, and local clusters of transmission that were missed by detection of P. falciparum infections. These data suggest that VAR2CSA serology can provide a useful adjunct for the fine-scale estimation of the malaria burden among pregnant women over time and space.

Identifying Immune Correlates of Protection Against Plasmodium falciparum Through a Novel Approach to Account for Heterogeneity in Malaria Exposure.

Background: A main criterion to identify malaria vaccine candidates is the proof that acquired immunity against them is associated with protection from disease. The age of the studied individuals, heterogeneous malaria exposure, and assumption of the maintenance of a baseline immune response can confound these associations. Methods: Immunoglobulin G/immunoglobulin M (IgG/ IgM) levels were measured by Luminex® in Mozambican children monitored for clinical malaria from birth until 3 years of age, together with functional antibodies. Studied candidates were pre-erythrocytic and erythrocytic antigens, including EBAs/PfRhs, MSPs, DBLs, and novel antigens merely or not previously studied in malaria-exposed populations. Cox regression models were estimated at 9 and 24 months of age, accounting for heterogeneous malaria exposure or limiting follow-up according to the antibody's decay. Results: Associations of antibody responses with higher clinical malaria risk were avoided when accounting for heterogeneous malaria exposure or when limiting the follow-up time in the analyses. Associations with reduced risk of clinical malaria were found only at 24 months old, but not younger children, for IgG breadth and levels of IgG targeting EBA140III-V, CyRPA, DBL5ε and DBL3x, together with C1q-fixation activity by antibodies targeting MSP119. Conclusions: Malaria protection correlates were identified, only in children aged 24 months old when accounting for heterogeneous malaria exposure. These results highlight the relevance of considering age and malaria exposure, as well as the importance of not assuming the maintenance of a baseline immune response throughout the follow-up. Results may be misleading if these factors are not considered.

Cytoadhesion to gC1qR through Plasmodium falciparum Erythrocyte Membrane Protein 1 in Severe Malaria.

Cytoadhesion of Plasmodium falciparum infected erythrocytes to gC1qR has been associated with severe malaria, but the parasite ligand involved is currently unknown. To assess if binding to gC1qR is mediated through the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family, we analyzed by static binding assays and qPCR the cytoadhesion and var gene transcriptional profile of 86 P. falciparum isolates from Mozambican children with severe and uncomplicated malaria, as well as of a P. falciparum 3D7 line selected for binding to gC1qR (Pf3D7gC1qR). Transcript levels of DC8 correlated positively with cytoadhesion to gC1qR (rho = 0.287, P = 0.007), were higher in isolates from children with severe anemia than with uncomplicated malaria, as well as in isolates from Europeans presenting a first episode of malaria (n = 21) than Mozambican adults (n = 25), and were associated with an increased IgG recognition of infected erythrocytes by flow cytometry. Pf3D7gC1qR overexpressed the DC8 type PFD0020c (5.3-fold transcript levels relative to Seryl-tRNA-synthetase gene) compared to the unselected line (0.001-fold). DBLß12 from PFD0020c bound to gC1qR in ELISA-based binding assays and polyclonal antibodies against this domain were able to inhibit binding to gC1qR of Pf3D7gC1qR and four Mozambican P. falciparum isolates by 50%. Our results show that DC8-type PfEMP1s mediate binding to gC1qR through conserved surface epitopes in DBLß12 domain which can be inhibited by strain-transcending functional antibodies. This study supports a key role for gC1qR in malaria-associated endovascular pathogenesis and suggests the feasibility of designing interventions against severe malaria targeting this specific interaction.

IgM and IgG against Plasmodium falciparum lysate as surrogates of malaria exposure and protection during pregnancy.

BACKGROUND: Difficulties to disentangle the protective versus exposure role of anti-malarial antibodies hamper the identification of clinically-relevant immune targets. Here, factors affecting maternal IgG and IgMs against Plasmodium falciparum antigens, as well as their relationship with parasite infection and clinical outcomes, were assessed in mothers and their children. Antibody responses among 207 Mozambican pregnant women at delivery against MSP119, EBA175, AMA1, DBLα and parasite lysate (3D7, R29 and E8B parasite lines), as well as the surface of infected erythrocytes, were assessed by enzyme-linked immunosorbent assay and flow cytometry. The relationship between antibody levels, maternal infection and clinical outcomes was assessed by multivariate regression analysis. RESULTS: Placental infection was associated with an increase in maternal levels of IgGs and IgMs against a broad range of parasite antigens. The multivariate analysis including IgGs and IgMs showed that the newborn weight increased with increasing IgG levels against a parasite lysate, whereas the opposite association was found with IgMs. IgGs are markers of protection against poor pregnancy outcomes and IgMs of parasite exposure. CONCLUSIONS: Adjusting the analysis for the simultaneous effect of IgMs and IgGs can contribute to account for heterogeneous exposure to P. falciparum when assessing immune responses effective against malaria in pregnancy.

Molecular mechanisms that mediate invasion and egress of malaria parasites from red blood cells.

PURPOSE OF REVIEW: Malaria parasites invade and multiply in diverse host cells during their complex life cycle. Some blood stage parasites transform into male and female gametocytes that are transmitted by female anopheline mosquitoes. The gametocytes are activated in the mosquito midgut to form male and female gametes, which egress from RBCs to mate and form a zygote. Here, we will review our current understanding of the molecular mechanisms that mediate invasion and egress by malaria parasites at different life cycle stages. RECENT FINDINGS: A number of key effector molecules such as parasite protein ligands for receptor-engagement during invasion as well as proteases and perforin-like proteins that mediate egress have been identified. Interestingly, these parasite-encoded effectors are located in internal, vesicular organelles and are secreted in a highly regulated manner during invasion and egress. Here, we will review our current understanding of the functional roles of these effectors as well as the signaling pathways that regulate their timely secretion with accurate spatiotemporal coordinates. SUMMARY: Understanding the molecular basis of key processes such as host cell invasion and egress by malaria parasites could provide novel targets for development of inhibitors to block parasite growth and transmission.

Production of recombinant PvDBPII, receptor binding domain of Plasmodium vivax Duffy binding protein, and evaluation of immunogenicity to identify an adjuvant formulation for vaccine development.

Plasmodium vivax is dependent on interaction with the Duffy antigen receptor for chemokines (DARC) for invasion of human erythrocytes. The P. vivax Duffy binding protein (PvDBP) mediates interaction of P. vivax merozoites with DARC. The DARC receptor-binding domain lies in a conserved N-terminal cysteine-rich region of PvDBP referred to as region II (PvDBPII). PvDBPII is an attractive vaccine candidate since antibodies raised against PvDBPII block erythrocyte invasion by P. vivax. Here, we describe methods to produce recombinant PvDBPII in its correctly folded conformation. A synthetic gene optimized for expression of PvDBPII in Escherichia coli and fed batch fermentation process based on exponential feeding strategy was used to achieve high levels of expression of recombinant PvDBPII. Recombinant PvDBPII was isolated from inclusion bodies, refolded by rapid dilution and purified by ion exchange chromatography. Purified recombinant PvDBPII was characterized for identity, purity and functional activity using standardized release assays. Recombinant PvDBPII formulated with various human compatible adjuvants including glycosylpyranosyl lipid A-stable emulsion (GLA-SE) and alhydrogel was used for immunogenicity studies in small animals to downselect a suitable formulation for clinical development. Sera collected from immunized animals were tested for recognition of PvDBPII and inhibition of PvDBPII-DARC binding. GLA-SE formulations of PvDBPII yielded higher ELISA and binding inhibition titres compared to PvDBPII formulated with alhydrogel. These data support further development of a recombinant vaccine for P. vivax based on PvDBPII formulated with GLA-SE.

Proinflammatory responses and higher IL-10 production by T cells correlate with protection against malaria during pregnancy and delivery outcomes.

Pregnancy triggers immunological changes aimed to tolerate the fetus. However, it has not been properly addressed whether similar changes occur in tropical areas with high infection pressure and whether these changes render women more susceptible to infectious diseases. We compared the frequencies of T cell subsets, including regulatory T cells, in pregnant and nonpregnant women from Papua New Guinea, a high malaria transmission area, and from Spain, a malaria-free country. We also assessed the relationship among these cellular subsets, malaria infection, and delivery outcomes. CD4(+)FOXP3(+)CD127(low) T cells (Tregs) were decreased in pregnant women in both countries but were not associated with malaria infection or poor delivery outcomes. An expansion of IFN-γ-producing cells and intracytoplasmic IFN-γ levels was found in pregnant compared with nonpregnant women only in Papua New Guinea. Increased CD4(+)IL-10(+)IFN-γ(+) frequencies and Treg-IFN-γ production were found in women with current Plasmodium falciparum infection. Higher CD4(+)IL-10(-)IFN-γ(+) T cells frequencies and production of proinflammatory cytokines (including TNF and IL-2) at recruitment (first antenatal visit) had a protective association with birth weight and future (delivery) P. falciparum infection, respectively. Higher intracellular IL-10 levels in T cells had a protective association with future P. falciparum infection and hemoglobin levels at delivery. The protective associations were found also with nonmalaria-specific T cell responses. Treg frequencies positively correlated with plasma eotaxin concentrations, but this subset did not express eotaxin receptor CCR3. Thus, an activated immune system during pregnancy might contribute to protection against malaria during pregnancy and poor delivery outcomes.

The central role of cAMP in regulating Plasmodium falciparum merozoite invasion of human erythrocytes.

All pathogenesis and death associated with Plasmodium falciparum malaria is due to parasite-infected erythrocytes. Invasion of erythrocytes by P. falciparum merozoites requires specific interactions between host receptors and parasite ligands that are localized in apical organelles called micronemes. Here, we identify cAMP as a key regulator that triggers the timely secretion of microneme proteins enabling receptor-engagement and invasion. We demonstrate that exposure of merozoites to a low K+ environment, typical of blood plasma, activates a bicarbonate-sensitive cytoplasmic adenylyl cyclase to raise cytosolic cAMP levels and activate protein kinase A, which regulates microneme secretion. We also show that cAMP regulates merozoite cytosolic Ca2+ levels via induction of an Epac pathway and demonstrate that increases in both cAMP and Ca2+ are essential to trigger microneme secretion. Our identification of the different elements in cAMP-dependent signaling pathways that regulate microneme secretion during invasion provides novel targets to inhibit blood stage parasite growth and prevent malaria.

Pregnancy and malaria exposure are associated with changes in the B cell pool and in plasma eotaxin levels.

Pregnancy triggers immunological changes aimed to tolerate the fetus, but its impact on B lymphocytes is poorly understood. In addition, exposure to the Plasmodium parasite is associated with altered distribution of peripheral memory B cell (MBC) subsets. To study the combined impact of high malaria exposure and pregnancy in B cell subpopulations, we analyzed PBMCs from pregnant and nonpregnant individuals from a malaria-nonendemic country (Spain) and from a high malaria-endemic country (Papua New Guinea). In the malaria-naive cohorts, pregnancy was associated with a significant expansion of all switched (IgD(-)) MBC and a decrease of naive B cells. Malaria-exposed women had more atypical MBC and fewer marginal zone-like MBC, and their levels correlated with both Plasmodium vivax- and Plasmodium falciparum-specific plasma IgG levels. Classical but not atypical MBC were increased in P. falciparum infections. Moreover, active atypical MBC positively correlated with proinflammatory cytokine plasma concentrations and had lower surface IgG levels than the average. Decreased plasma eotaxin (CCL11) levels were associated with pregnancy and malaria exposure and also correlated with B cell subset frequencies. Additionally, active atypical and active classical MBC expressed higher levels of eotaxin receptor CCR3 than the other B cell subsets, suggesting a chemotactic effect of eotaxin on these B cell subsets. These findings are important to understand immunity to infections like malaria that result in negative outcomes for both the mother and the newborn and may have important implications on vaccine development.

Malaria and HIV infection in Mozambican pregnant women are associated with reduced transfer of antimalarial antibodies to their newborns.

BACKGROUND: Malaria and human immunodeficiency virus (HIV) infection during pregnancy affect the transplacental transfer of antibodies against several pathogens from mother to fetus, although the effect of malaria and HIV infection on the transfer of antimalarial antibodies remains unclear. METHODS: Levels of total immunoglobulin G (IgG), immunoglobulin M (IgM), and IgG subtypes against the following Plasmodium falciparum antigens were measured in 187 pairs of mother-cord plasma specimens from Mozambique: 19-kDa fragment of merozoite surface protein 1 (MSP119), erythrocyte binding antigen 175 (EBA175), apical membrane antigen 1 (AMA1), and parasite lysate. Placental antibody transfer was defined as the cord-to-mother ratio (CMR) of antibody levels. RESULTS: Maternal malaria was associated with reduced CMR of EBA175 IgG (P = .014) and IgG1 (P = .029), AMA1 IgG (P = .002), lysate IgG1 (P = .001), and MSP1 IgG3 (P = .01). Maternal HIV was associated with reduced CMR of MSP1 IgG1 (P = .022) and IgG3 (P = .023), lysate IgG1 (P = .027) and IgG3 (P = .025), AMA1 IgG1 (P = .001), and EBA175 IgG3 (P = .001). Decreased CMR was not associated with increased adverse pregnancy outcomes or augmented risk of malaria in the infant during the first year of life. CONCLUSIONS: Placental transfer of antimalarial antibodies is reduced in pregnant women with malaria and HIV infection. However, this decrease does not contribute to an increased risk of malaria-associated morbidity during infancy.

Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP) and the Duffy Antigen Receptor for Chemokines (DARC) and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s) lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

Role of calcineurin and actin dynamics in regulated secretion of microneme proteins in Plasmodium falciparum merozoites during erythrocyte invasion.

Plasmodium falciparum invades host erythrocytes by multiple invasion pathways. The invasion of erythrocytes by P. falciparum merozoites is a complex process that requires multiple interactions between host receptors and parasite ligands. A number of parasite proteins that mediate interaction with host receptors during invasion are localized to membrane-bound apical organelles referred to as micronemes and rhoptries. The timely release of these proteins to the merozoite surface is crucial for receptor engagement and invasion. It has been demonstrated previously that exposure of merozoites to a low potassium (K(+)) ionic environment as found in blood plasma leads to a rise in cytosolic calcium (Ca(2+)), which triggers microneme secretion. The signalling pathways that regulate microneme discharge in response to rise in cytosolic Ca(2+) are not completely understood. Here, we show that a P. falciparum Ca(2+)-dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca(2+)-dependent microneme exocytosis. An increase in PfCN activity was observed in merozoites following exposure to a low K(+) environment. Treatment of merozoites with calcineurin inhibitors such as FK506 and cyclosporin A prior to transfer to a low K(+) environment resulted in inhibition of secretion of microneme protein apical merozoite antigen-1 (PfAMA-1). Inhibition of PfCN was shown to result in reduced dephosphorylation and depolymerization of apical actin, which appears to be criticalfor microneme secretion. PfCN thus serves as an effector of Ca(2+)-dependent microneme exocytosis by regulating depolymerization of apical actin. Inhibitors that target PfCN block microneme exocytosis and limit growth of P. falciparum blood-stage parasites providing a novel approach towards development of new therapeutic strategies against malaria.

Perforin-like protein PPLP2 permeabilizes the red blood cell membrane during egress of Plasmodium falciparum gametocytes.

Egress of malaria parasites from the host cell requires the concerted rupture of its enveloping membranes. Hence, we investigated the role of the plasmodial perforin-like protein PPLP2 in the egress of Plasmodium falciparum from erythrocytes. PPLP2 is expressed in blood stage schizonts and mature gametocytes. The protein localizes in vesicular structures, which in activated gametocytes discharge PPLP2 in a calcium-dependent manner. PPLP2 comprises a MACPF domain and recombinant PPLP2 has haemolytic activities towards erythrocytes. PPLP2-deficient [PPLP2(-)] merozoites show normal egress dynamics during the erythrocytic replication cycle, but activated PPLP2(-) gametocytes were unable to leave erythrocytes and stayed trapped within these cells. While the parasitophorous vacuole membrane ruptured normally, the activated PPLP2(-) gametocytes were unable to permeabilize the erythrocyte membrane and to release the erythrocyte cytoplasm. In consequence, transmission of PPLP2(-) parasites to the Anopheles vector was reduced. Pore-forming equinatoxin II rescued both PPLP2(-) gametocyte exflagellation and parasite transmission. The pore sealant Tetronic 90R4, on the other hand, caused trapping of activated wild-type gametocytes within the enveloping erythrocytes, thus mimicking the PPLP2(-) loss-of-function phenotype. We propose that the haemolytic activity of PPLP2 is essential for gametocyte egress due to permeabilization of the erythrocyte membrane and depletion of the erythrocyte cytoplasm.